Molecular Mechanism of Steroid Hormone Action: Recent Advances 9783110885026, 9783110101188

Table of contents :
Structure and Molecular Organization Molecular organization of the estrogen receptor system
Structure, properties and subcellular localization of the chick oviduct progesterone receptor
Studies of the subunit composition of the 8.5S rabbit uterine progestin receptor
Alterations in mouse glucocorticoid receptor structure: Effects of various hydrolytic enzymes
Affinity labeling steroids as biologically active probes of glucocorticoid receptor structure and function
The physiological significance of the structure of glucocorticoid and progesterone receptors
Activation/Transformation
An allosteric regulatory mechanism for estrogen receptor activation
Specific effects of monovalent cations and of adenine nucleotides on glucocorticoid receptor activation, as studied by aqueous two-phase partitioning
Low-molecular-weight and macro molecular translocation modulators affecting the binding of activated receptor-glucocorticoid complex to nuclei, chromatin and DNA
The important role of cytoplasmic modulators in the pathway for steroid receptor to be converted to the biologically active form
Phosphorylation
Phosphorylation on tyrosine of the 17B-estradiol receptor
Phosphorylation of progesterone receptor
Purification, activation and phosphorylation of the glucocorticoid receptor
Interaction of nucleotides with steroid hormone receptors
Regulation and Biological Responses
Steroid hormone receptor dynamics: The key to tissue responsiveness
An endogenous ligand for type II binding sites in normal and neoplastic tissues
Regulation of mammary responsiveness to estrogen: An analysis of differences between mammary gland and the uterus
Progesterone regulation of nuclear estrogen receptors: Evidence for a receptor regulatory factor
Receptors and biological responses of estrogens, antiestrogens and progesterone in the fetal and newborn uterus
Nuclear Components and Gene Expression Steroid receptor-DNA interactions
The rat pituitary estrogen receptor: Role of the nuclear receptor in the regulation of transcription of the prolactin gene and the nuclear localization of the unoccupied receptor
Characterization of different forms of the androgen receptor and their interaction with constituents of cell nuclei
Differential sensitivity of specific genes in mouse kidney to androgens and antiandrogens
Pharmacology and Clinical Correlations
Molecular pharmacology of tamoxifen; an antiestrogen with antitumor properties in animals and man
Studies on glucocorticoid receptors in normal and neoplastic rodent and human leukocytes: Structure, degradation, kinetics of formation and activation
Progestin treatment, progesterone receptors, and breast cancer
Steroid hormones, receptors and neurotransmitters
New Systems, Techniques The steroid receptor of Achlya ambisexualis
The receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: Similarities and dissimilarities with steroid hormone receptors
Separation and characterization of isoforms of steroid hormone receptors using high performance liquid chromatography
Author Index
Subject Index

Citation preview

Molecular Mechanism of Steroid Hormone Action Recent Advances

Molecular Mechanism of Steroid Hormone Action Recent Advances Editor V Κ Moudgil

W DE

G

Walter de Gruyter · Berlin · New York 1985

Editor Virinder Κ. Moudgil, Ph. D. Department of Biological Sciences Oakland University Rochester, Michigan 48063 U.S.A.

Library of Congress Cataloging in Publication Data

Molecular mechanism of steroid hormone action. Includes bibliographies and indexes. 1. Steroid hormones-Receptors. 2. Hormones, Sex-Receptors. 3. Molecular biology. I. Moudgil, Virinder K., 1945QP572.S7M65 1985 599'.0142 85-6787 ISBN 0-89925-032-7 (U.S.)

CIP-Kurztitelaufnahme der Deutschen

Bibliothek

Molecular mechanism of steroid hormone action : recent advances / ed. V K. Moudgil. - Berlin ; New York : de Gruyter, 1985. ISBN 3-11-010118-1 (Berlin) ISBN 0-89925-032-7 (New York) NE: Moudgil, Virinder K. [Hrsg.]

3110101181 Walter de Gruyter • Berlin • New York 0-89925-032-7 Walter de Gruyter, Inc., New York

Copyright © 1985 by Walter de Gruyter & Co., Berlin 30 All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nortranslated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. Binding: Dieter Mikolai, Berlin. Printed in Germany.

To dearest Bauji

FOREWORD

During the last two decades, progress in research on steroid hormone receptors has resulted in the development of new approaches for contraception and the diagnosis and treatment of endocrine-related disorders and cancers. Significant advancements also have been recorded in the purification, characterization and immunochemistry of steroid hormone receptors. However, despite such progress, understanding of the precise molecular mechanism of steroid hormone action has remained obscure. It is in the spirit of seeking a better understanding of the mode of action of steroid hormones that the idea of compiling such information was conceived. The chapters in this book represent invited contributions from the leading investigators engaged in research in the general area of steroid hormone receptors. The effort of comprising this volume has brought together the most significant and current work of prominent, world reknowned scientists. Each chapter has been written to provide the reader with an adequate background of the subject while, at the same time, introducing the state of the art and most current contributions of the investigator(s). Although many excellent books, monographs and comprehensive reviews on the subject already exist, limitation of such works has often times rested in the inadequate interpretation and discussion of the information reviewed. This book provides a first-hand interaction with the various approaches and directions adopted by the authors in their quest for knowledge of the molecular mechanism of steroid hormone action. The need for a discussion on this subject cannot be overemphasized since, even after great strides and significant achievements in the molecular aspects of steroid receptors, the native structure of the receptor molecule still alludes investigators. It is hoped that this book will provide a much needed and timely background for the cell, molecular and developmental biologist. Although the book was written with the scientist in mind, it should also provide an invaluable reference source for graduate students in biology and medicine.

VIII I am indebted to Dr. David Toft, Mayo Clinic, for introducing me to this most exciting subject of steroid hormone receptors, and most certainly for his continued guidance and encouragement. I thank Ms. Cynthia Winston for her secretarial assistance and Walter de Gruyter for the rapid publication of this volume.

The effort invested in the organization and preparation of this book is dedicated to the living memory of my father, Pandit Harbhagwan Moudgil.

Rochester, January 1985

The Editor

CONTENTS Structure and Molecular Organization Molecular organization of the estrogen receptor system AKIRA MURAYAMA Structure, properties and subcellular localization of the chick oviduct progesterone receptor JAN MESTER, JEAN-MARIE GASC, THIERRY BUCHOU, JACK-MICHEL RENOIR, IRENE JOAB, CHRISTINE RADANYI, NADINE BINART, MARIA-GRAZIA CATELLI and ETIENNE-EMILE BAULIEU Studies of the subunit composition of the 8.5S rabbit uterine progestin receptor LEE E. FABER, PINE-K., K. TAI, YOSHIAKI MAEDA and JOHN E. MYERS Alterations in mouse glucocorticoid receptor structure: Effects of various hydrolytic enzymes WAYNE V. VEDECKIS, BRANKA KOVACIC-MILIVOJEVIC, MARGOT C. La Pointe and CHERYL E. REKER Affinity labeling steroids as biologically active probes of glucocorticoid receptor structure and function S. STONEY SIMONS, JR The physiological significance of the structure of glucocorticoid and progesterone receptors CORIANNE M. SILVA and JOHN A. CIDLOWSKI Activation/Transformation An allosteric regulatory mechanism for estrogen receptor activation ANGELO C. NOTIDES, SHLOMO SASSON and STEVE CALLISON . Specific e f f e c t s of monovalent cations and of adenine nucleotides on glucocorticoid receptor activation, as studied by aqueous two-phase partitioning P.A. ANDREASEN and K. JUNKER

·

χ Low-molecular-weight and macromolecular translocation modulators affecting the binding of activated receptor-glucocorticoid complex to nuclei, chromatin and DNA FUMIHIDE BOHASHI and Y U K I Y A SAKAMOTO

225

The important role of cytoplasmic modulators in the pathway for steroid receptor to be converted to the biologically active form BUNZO SATO, YASUKO NISHIZAWA, KEIZO NOMA, M A K A T O NAKAO, SUSUMU KISHIMOTO and KEISHI MATSUMOTO

249

Phosphorylation Phosphorylation on tyrosine of the 17B-estradiol receptor FERDINANDO AURICCHIO, ANTIMO MIGLIACCIO, ANDREA ROTONDI and GABRIELLA CASTORIA

279

Phosphorylation of progesterone receptor JOHN J. DOUGHERTY

299

Purification, activation and phosphorylation of the glucocorticoid receptor THOMAS J. SCHMIDT and GERALD LITWACK

309

Interaction of nucleotides with steroid hormone receptors VIRINDER K. MOUDGIL

351

Regulation and Biological Responses Steroid hormone receptor dynamics: The key to tissue responsiveness THOMAS G. MULDOON

377

An endogenous ligand for type II binding sites in normal and neoplastic tissues B A R R Y M. MARKAVERICH and JAMES H. CLARK

399

Regulation of mammary responsiveness to estrogen: An analysis of differences between mammary gland and the uterus GOPALAN SHYAMALA

413

Progesterone regulation of nuclear estrogen receptors: Evidence for a receptor regulatory factor WENDELL W. LEAVITT

437

XI Receptors and biological responses of estrogens, antiestrogens and progesterone in the fetal and newborn uterus CHARLOTTE SUMIDA and JORGE RAUL PASQUALINI

471

Nuclear Components and Gene Expression Steroid receptor-DNA interactions S. A N A N D K U M A R a n d H E R B E R T W. D I C K E R M A N

505

The rat pituitary estrogen receptor: Role of the nuclear receptor in the regulation of transcription of the prolactin gene and the nuclear localization of the unoccupied receptor JAMES D. SHULL, WADE V. WELSHONS, MARA E. LIEBERMAN a n d J A C K GORSKI

539

Characterization of different forms of the androgen receptor and their interaction with constituents of cell nuclei EPPO MULDER and ALBERT 0 . BRINKMAN

563

Differential sensitivity of specific genes in mouse kidney to androgens and antiandrogens JAMES F. CATTERALL, CHERYL S. WATSON, KIMMO K. KONTULA, OLLI A . J A N N E and C. W A Y N E B A R D I N

587

Pharmacology and Clinical Correlations Molecular pharmacology of tamoxifen; an antiestrogen with antitumor properties in animals and man V. C R A I G J O R D A N

603

Studies on glucocorticoid receptors in normal and neoplastic rodent and human leukocytes: Structure, degradation, kinetics of formation and activation NIKKI J. HOLBROOK, JACK E. BODWELL, DIRK B. MENDEL and A L L A N M U N C K

637

Progestin treatment, progesterone receptors, and breast cancer KATHRYN B. HORWITZ, SCOT M. SEDLACEK, CAROYLN D'ARVILLE a n d LISA L. WEI

659

XII

Neurotransmission Steroid hormones, receptors and neurotransmitters GARY DOHANICH, BRUCE NOCK and BRUCE S. McEWEN

. . . . ο ....

.

701

New Systems, Techniques The steroid receptor of Achlya ambisexualis ROBERT M. RIEHL and DAVID 0 . TOFT . . . . . . . . . . . . . . . . .

733

The receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: Similarities and dissimilarities with steroid hormone receptors LORENZ POELLINGER, JOHAN LUND, MIKAEL GILLNER and JAN-AKE GUSTAFSSON . . . . . . . . . . . . . . . . . . . . . . .

755

Separation and characterization of isoforms of steroid hormone receptors using high performance liquid chromatography JAMES L. WITTLIFF . . . . . . . . . . . . . . . . . . . . . . . . . . .

791

Author Index

815

Subject Index

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

817

M O L E C U L A R ORGANIZATION OF THE ESTROGEN RECEPTOR SYSTEM

Akira M u r a y a m a Department of Physiological Chemistry, The Tokyo M e t r o p o l i t a n Institute of Medical Science, Honkomagome, Bunkyoku, Tokyo 113, Japan

Introduction Background Method Results A. Estrogen receptor system of the cytosol of porcine uterus. a) Basic estrogen receptor molecule estrogen receptor molecule

(vero-ER) and the proteolyzed

(secto-ER).

b) "8S" estrogen receptor-forming

factor.

c) Subunit structure of "8S" estrogen receptor-forming d) Purification of basic estrogen receptor molecule estrogen receptor-binding

factor.

(vero-ER) and

factors.

B.

Estrogen receptor system of the nuclei of porcine uterus.

C.

Molecular m e c h a n i s m of the translocation of estrogen receptor

from

the cytoplasm into the nucleus. a) Molecular m e c h a n i s m of "5S" estrogen receptor

formation.

b) M e c h a n i s m of the nuclear translocation of estrogen receptor. D.

Regulation of the reactivity of basic estrogen receptor molecule (vero-ER) w i t h estrogen receptor-binding

factors.

a) Strongly hydrophobic domain of basic estrogen receptor molecule (vero-ER). b) R e g u l a t i o n of association and dissociation of basic receptor molecule

estrogen

(vero-ER) w i t h estrogen receptor-binding

Discussion References

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

factors.

2 Introduction

Steroid hormone receptors are intracellular receptors, which recognize the hormones not on the cellular membranes, but in the inside of the target cells.

Since steroid hormones labeled with tritium of high specific

activity became available in the early 1960s (1,2), intensive studies have been made on steroid hormone receptors leading to many interesting proposals on the mode of action of steroid hormones (see reviews 3-13). However, there are as yet many discrepancies between the proposed molecular models of steroid hormone receptors, and little is known on the reactions of the receptors in the target cells to induce the hormonal responces. Precise information of the molecular organization of the steroid hormone receptor system is an important basis of the study of the action mechanism of the hormones.

We presented recently a new molecular model of the structural constitution of estrogen receptors (14-18) and progesterone receptors (19), which are

Θ

Estradiol

strongly hydrophobic domain 2.9S ER

Kd a 10 "M Kd = 10 Μ IMg 1 mM)

Bs

'5iER-FF (component A)

"5S'ER

^ Kd ^10*8M (Mg2* 1 mMI "6S"ER-FF (component Bl6

* "6S" ER

Sec/o-ER Kd => 10 Μ Kd = 10 Μ (M, 1 mMI

3.6SER

Fig. 1.

(.2SER

'8SER-FF Icomponent B)6 •(component Al

Molecular constitution of the estrogen receptor system

"8S" ER

(171

3 very similar to each other.

We showed that there is one basic estrogen

receptor (ER) molecule (vero-ER) (sedimentation coefficient, 4.5S; Stokes radius, 44 A) in the estrogen target tissues (14,18).

Vero-ER interacted

specifically with the endogenous components designated as ER-binding factors (ERBFs) ["5S" ER-forming factor ("5S" ER-FF), (component A);

"6S" ER-FF,

(component B).; "7S" ER-FF, (component B) ·(component A); "8S" ER-FF, b ζ (component B),•(component A)] to form estrogen receptors ["5S" ER, (verob ER)·("5S" ER-FF);

"6S"ER, (vero-ER)·("6S" ER-FF);

(component B)2·(component A);

"7S" ER, (vero-ER)·

"8S" ER, (vero-ER)•(component B)^·(component

A)] with various molecular constitutions (15-18).

Vero-ER is proteolyzed

by the endogenous proteases (20) to form various receptor fragments [sectoER (sedimentation coefficient, 4.5S; Stokes radius, 35 A); "3.8S" ER;

"4.2S" ER;

"2.9S" ER, etc.], which still bind with estradiol, but no

longer bind with ERBFs (14,18) (Fig. 1).

This chapter reviews the recent studies of our laboratory, and presents new conceptions of the molecular organization of the estrogen receptor system.

Background

Steroid hormone receptors are extracted in the cytosol fractions with the conventional buffers without utilizing detergents, in contrast to the membrane-bound receptors.

In spite of the facile extractability, study of

steroid hormone receptors on the molecular level is extremely troublesome. Two majour obstacles seem to have been disregarded until now in the study of steroid hormone receptors.

First, steroid hormone receptors undergo

complicated association and dissociation with endogenous protein factors, appropriate understanding of which is an important basis of the study of the receptor phenomena.

Second, steroid hormone receptors are extremely

susceptible to the modifications by the proteases coextracted with them. Through the proteolytic modifications, the binding site of the receptors to the hormones are slightly affected, while the capability of the receptors to undergo specific reactions with the endogenous protein factors is lost. The binding site to the hormone is only a part of the whole receptor molecule.

It is important to protect the steroid hormone receptors from the

4 endogenous proteases, and to separate them from the protein factors which associate with them with high affinities.

The following studies on the

molecular organization of the estrogen receptor system were undertaken by taking special care on these respects.

Methods A.

Buffer.

The following buffers were utilized.

TEMA [10 mM Tris, 1.5 mM EDTA, 1.5 iM

2-mercaptoethanol, 0.25 mM antipain (21), pH 8.0] buffer. of antipain) buffer.

TMA (TEMA devoid of EDTA) buffer.

TEM (TEMA devoid

P^EMA (n mM potas-

sium phosphate, 1.5 mM EDTA, 1.5 mM 2-mercaptoethanol, 0.25 mM antipain, pH 6.5) buffer.

Β.

Ρ EM (Ρ EMA devoid of antipain) buffer, η η

Porcine (gilt) uteri (18).

Gilt (female pig before her first conception) uteri weighing less than 80 g/uterus were collected at the local slaughter house immediately after the animals were killed.

The uteri were removed of connective and adipose

tissues, and immediately used for the experiments.

C.

Assay of estrogen receptor-binding factor (ERBF) activity (16). -13

The sample (0.1 ml in TEMA buffer) to be assayed was mixed with 2 χ 10 mol of basic estrogen receptor molecule (vero-ER) labeled with [ H]estradiol (assuming one molecule of vero-ER binds with one molecule of estradiol) in 0.1 ml of TEMA buffer, and then kept for 5 h at 1°C.

The mixture was then

subjected to sucrose gradient (5-20%) centrifugation in TEMA buffer, and ER activity appearing at 8S ("8S" ER), 6.5S ("6S" ER), 5.5S ("5S" ER), or 4.5S (vero-ER) was estimated.

One unit of "8S" ER-forming factor ("8S"

ER-FF), "6S" ER-FF, or "5S" ER-FF activity -13was defined as the amount of the respective factor to complex with 1 χ 10

mol of vero-ER to form "8S" ER,

"6S" ER or "5S" ER under the assay condition. D.

Estimation of the equilibrium dissociation constants (Kd) of the basic

estrogen receptor molecule (vero-ER) for estrogen receptor-binding factors (ERBFs) (22). Aliquots of respective ERBF-preparations (0.5 units in 0.1 ml TEMA buffer)

5 were incubated with preparations of labeled vero-ER ranging from 1 χ 10 Μ to 1 χ 10~ 9 Μ at 1°C for 5 h.

^

The complex, (vero-ER)· (ERBF), was sepa-

rated from vero-ER by sucrose gradient (5-20%) centrifugation.

It was

assumed that the dissociation of the complex, (vero-ER)·(ERBF), into veroER and ERBF during the process of the centrifugation was negligible, since the obtained sedimentation patterns of the estrogen receptors were symmetrical.

The equilibrium binding data obtained were plotted according to

Scatchard (23) assuming that one molecule of estradiol binds to one molecule of vero-ER.

Estimation of the apparent equilibrium dissociation con2+ stants in the presence of Mg was carried out similarly in TMA buffer (24)

Results

A.

Estrogen receptor system of the cytosol of porcine uterus.

a) Basic estrogen receptor molecule (vero-ER) and proteolyzed estrogen receptor molecule (secto-ER) (14,18).

The estrogen receptor (ER) of the

fresh cytosol sedimented at 8S ("8S" ER) in the low salt conditions, and at 4.5S in the presence of 0.4 Μ KCl or 0.4 Μ NaSCN (Fig. 2).

Similar remark-

able variation of the sedimentation pattern of steroid hormone receptors depending on the salt conditions have been widely reported.

The molecular

model of steroid hormone receptors is required to explain consistently the salt-dependent variation of the sedimentation pattern of the receptors. To solve this problem, it is necessary to separate the basic constituents of the receptor system from each other, and to reconstitute the association and dissociation.

When the cytosol was incubated in the presence of NaSCN for 12 h at 4°C and then analyzed, ER sedimented at 2.9S in the presence of 0.4 Μ NaSCN, and at 4.5S in the low salt conditions or in the presence of 0.4 Μ KCl (Fig. 2).

However, when the incubation of the cytosol with NaSCN was

carried out in the presence of antipain (0.25 mM), a protease inhibitor of microbial origin (21), the modification of the sedimentation pattern of ER through the incubation could be prevented (Fig. 2).

These results indicated

that the proteolytic modification of the cytosolic ER took place during the

6

Fig. 2. Sedimentation analysis of native and proteolyzed estrogen receptors (18). Sucrose gradient centrifugation was carried out either in TEMA buffer (A), in TEMA buffer containing 0.4 Μ KCl (D), or in TEMA buffer containing 0.4 Μ NaSCN (C). Labeled cytosol ( — Ο — ) . Labeled cytosol incubated in the presence of 0.4 Μ NaSCN for 12 h at 4°C ( — O " — ) . Labeled cytosol incubated in the presence of 0.4 Μ NaSCN and 0.25 mM antipain for 12 h at 4°C ( — # — ) . Fraction III (Fig. 3B) ( - & — ) . Fraction III + unlabeled cytosol ( — A — ) . Fraction II (Fig. 3A) ( — Q - — ) . Fraction II + unlabeled cytosol ( — • — ) . Fraction IV (Fig. 3B) (—57.—).

incubation with NaSCN in the absence of an appropriate protease inhibitor. We utilized antipain in the following study to prevent the eventual proteolytic modification of ER by the endogenous protease.

When the labeled cytosol prepared in TEMA (see method) buffer was subjected to a two-step gel filtration on a Sephadex G-150 column in TEMA buffer in the presence of 0.4 Μ KCl and in the presence of 0.4 Μ NaSCN, ER with a Stokes radius of 44 A (fraction III, Fig. 3B) and a sedimentation coefficient of 4.5S (Fig. 2) was obtained as a sole ER activity in the eluate. This indicated that there is one basic estrogen receptor molecule in the uterine cytosol.

The basic receptor molecule with a Stokes radius of 44 A

and a sedimentation coefficient of 4.5S was designated as vero-ER (vero, taken from the Latin verus).

Vero-ER by itself did not undergo self-associ-

60

50

AO

STOKES

30

RADIUS (A)

Fig. 3. T w o - s t e p gel f i l t r a t i o n of the labeled cytosol (16). (A) L a b e l e d cytosol w a s s u b j e c t e d to gel f i l t r a t i o n on Sephadex C - 1 5 0 in T E M A b u f f e r c o n t a i n i n g 0.4 Μ KCl ( Ο ), or in TEM b u f f e r c o n t a i n i n g 0.4 Μ KCl ( φ ). (Β) F r a c t i o n I ( Δ ) or f r a c t i o n II ( A ) w a s s u b j e c t e d to gel f i l t r a t i o n 3 on Sephadex G - 1 5 0 in T E M A buffer c o n t a i n i n g 0.4 Μ N a S C N . , [ H]estradio1 content; , a b s o r b a n c e at 280 nm. The a r r o w s i n d i c a t e the p e a k s of e l u t i o n for Blue D e x t r a n (BD), c a t a l a s e (Cat), a l d o l a s e (Aid), BSA, egg a l b u m i n (EA), and m y o g l o b i n (Myo).

ation and - d i s s o c i a t i o n depending o n the salt conditions

(Fig. 2).

When

the fraction of v e r o - E R w a s m i x e d w i t h unlabeled cytosol, ER sediraented a 8S under the low salt conditions suggesting the presence of a component ("8S" ER-forming factor, "8S" ER-FF) which binds w i t h vero-ER to form "8S ER in the cytosol

(Fig. 2).

When gel filtration of the labeled cytosol w a s carried out in TEM

(TEMA

devoid of antipain) buffer in the presence of 0.4 Μ KCl, ER eluted in the fractions

(fraction II, Fig. 3A) w i t h a smaller Stokes radius

compared to vero-ER.

(35 A) as

This indicated that vero-ER was proteolyzed by a

protease during the gel filtration in the absence of antipain. olyzed receptor with a Stokes radius of 35 A and a sedimentation cient of 4.5S w a s designated as secto-ER

The prote coeffi-

(secto, taken from the Latin

8 secare).

Secto-ER no longer possessed the capability to form "8S" ER when

mixed with unlabeled cytosol (Fig. 2). presence of 0.4 Μ NaSCN (Fig. 2).

Secto-ER sedimented at 2.9S in the

When secto-ER was subjected to gel fil-

tration in the presence of 0.4 Μ NaSCN, ER was eluted in the fractions with a Stokes radius of 24 A (fraction IV, Fig. 3B).

The ER of fraction IV

sedimented at 2.9S in the presence of 0.4 Μ NaSCN, and at 4.5S in the absence of NaSCN (Fig. 2).

These results indicated that in the presence of

0.4 Μ NaSCN, secto-ER is dissociated into "2.9S" ER fragment (Stokes radius, 24 A) and a counterpart with a similar molecular size.

b) "8S" estrogen receptor-forming factor ("8S" ER-FF) (15,18).

To analyze

the cytosolic factor ("8S" ER-forming factor, "8S" ER-FF) which binds with vero-ER to form "8S" ER, the unlabeled cytosol was subjected to gel filtration on a Sephadex G-150 column under the low salt (TEMA) conditions, and the eluate was divided into fractions as shown in Fig. 4.

1 BD 3000 -

Τ

I

1

I

Cat Aid Μ

Each fraction

1

BSA

EA

Myo

J

J

J

(A)

-

ο

ΪΪΑ

in

BCDE

F

G

Η

I

1500

radius(A)

(>5B)

50

t58_52)

40 STOKES

(52_50)

30 RADIUS (A)

0.5

i a 9

20

(50-48) (48-46) (46-38) (38-28) (28-19)

u

CE

/ 60

B

UJ

\

ου

1.0

(19>)

Fig. 4. Gel filtration of "BS" ER and "8S" ER-forming factor (15). Gel filtration of the labeled and unlabeled cytosol was carried out on a Sephadex G-150 column (1.6 χ 96 cm) In TEMA buffer. — Ο — , [ H]estradiol content; , absorbance at 280 nm. The arrows Indicate the elution peaks of the standards (see the legend to Fig. 3).

g

was assayed for the activity [ER-binding factor (ERBF) activity] to bind with vero-ER (sedimentation coefficient, 4.5S) to form ER with a higher sedimentation coefficient.

The main peak of "8S" ER-FF was eluted in the

fractions with a Stokes radius of approximately 51 A (fraction C in fig. 4B).

Other ERBF activity was not detected in the eluate.

"8S" ER-FF was

assumed to be a protein, since it was destroyed by trypsin, but not by DNase and RNase.

"8S" ER-FF did not bind with estradiol.

When a mixture

of vero-ER and "8S" ER-FF was subjected to gel filtration under the low salt (TEMA) conditions, ER eluted in the fractions with a Stokes radius of 68 A similar to the cytosolic "8S" ER (Fig. 4A) .

The levels of vero-ER and "8S" ER-FF of the cytosol preparations of various specimens of gilt uteri are shown in Fig. 5. ER was relatively small.

Variation of the level of

The ER-rich tissues contained approximately 2-

fold ER as compared to ER-poor tissues.

In contrast, the content of "8S"

ER-FF varied over 50 times among the tissues.

Positive correlation was

1

S 60

>

(Λ

• ··

5> 50 _ J2

1 §

Ο ι 11 I tr LLI

00

30

-

•

20

•

1 ·· •V* * τ» ·

-

• ·· ·

10

-

_

·

·

• •

•

J

«_

• •

· ·

·

·

·

•

• ·

k

.

1

1

l

Fig. 11. A n a l y s i s of the m o l e c u l a r forms of the c y t o s o l i c e s t r o g e n r e c e p t o r b i n d i n g factors In the p r e s e n c e of 0.4 Μ KCl at lower and h i g h e r t e m p e r a t u r e s (17). Gel f i l t r a t i o n of the u n l a b e l e d cytosol w a s c a r r i e d out on a S e p h a d e x G-150 c o l u m n in TEM buffer c o n t a i n i n g 0.4 Μ KCl at 4 ° C (A), a n d at 25°C (B) .

of the cytosolic estrogen receptor-binding factors (0.4 Μ KCl) conditions at lower

(4°C) and higher

(ERBFs) in the h i g h salt

(25°C)

temperatures.

The unlabeled cytosol w a s subjected to gel filtration in TEM buffer

contain-

ing 0.4 Μ KCl at 4°C and at 25°C respectively, and the eluates were assayed for ERBF activity. ER-forming factor

When the gel filtration w a s carried out at 4°C,

"7S"

("7S" ER-FF) which binds w i t h vero-ER to form ER sediment-

ing at 7S ("7S" ER) under the low salt conditions w a s eluted in the fractions with a Stokes radius of around 49 A (fraction Β in Fig. IIA).

Com-

ponent Β (basic unit of "6S" ER-FF) was further detected in the eluate (fraction H, in Fig. IIA).

"7S" ER-FF was shown to be a 1:2 complex of

component A and component Β (17).

These results showed that "8S" ER-FF

is dissociated into "7S" ER-FF and component Β in the high salt (0.4 Μ KCl) conditions at 4°C.

As expected, w h e n the gel filtration of the cytosol

was carried out at 25°C (condition of activation of the cytosolic

receptor),

"7S" ER-FF disappeared, and component A and component Β were eluted in the

20 respective fractions (fractions E' and H', Fig. IIB). ty was observed in the eluate.

No other ERBF activi-

These results supported strongly the assump-

tion that under the activation conditions, component A is liberated from the binding with component B, and then forms stable complex with vero-ER to form "5S" ER during the subsequent process of cooling the cytosol (Fig. 12).

The sedimentation patterns of of the reconstructed estrogen receptors and the cytosolic estrogen receptors are shown in Fig. 13.

"6S" ER, "7S" ER

and "8S" ER are present only under the low salt conditions, and dissociated into vero-ER and ERBFs in the high salt (0.4 Μ KCl) conditions. stable in the presence of 0.4 Μ KCl.

"5S" ER is

Under the low salt conditions, re-

constructed "5S" ER sedimented at 5.5S, however, the cytosolic "5S" ER migrated to the bottom fractions (Fig. 13A).

When the reconstructed "5S"

ER was mixed with the unlabeled cytosol and then analyzed by sucrose gradient centrifugation, ER migrated to the bottom fractions under the low salt conditions similar to the cytosolic "5S" ER (Fig. 13A).

These results indi-

ο

"8S" ER low salt, Λ "C

Vero-ER

"7S'ER-FF

O.iMKCI,

°

0

"5S*ER-FF

C U M KCl, 25"C

"5S"ER

Fig. 12.

Molecular mechanism of "5S" ER formation.

21

cated the presence of a cytosolic component (component C) which binds with "5S" ER under the low salt conditions.

b) Mechanism of the nuclear translocation of estrogen receptor (33).

It

was expected that difference in the capabilities to translocate into the isolated nuclei might be observed among the various forms of estrogen receptor (ER) reflecting the regulating mechanism of the translocation of ER from the cytoplasm into the nucleus.

Accordingly we compared the capabil-

ities of various forms of ERs to translocate into the uterine nuclei in an in vitro system.

The results are summarized in Table II.

The amount of

ER translocated into the nuclei was estimated by subtracting the amount of ER adsorbed on the nuclear envelopes from that of ER bound to the whole nuclei.

Vero-ER·Ε (vero-ER bound with estradiol) possessed an outstandingly

high capability to translocate into the nuclei as compared to the associated forms with ERBFs.

This suggested strongly that ER is translocated from the

Fig. 13. Sedimentation analysis of cytosolic and reconstructed estrogen receptors (18). Sucrose gradient centrifugation was carried out in TEMA buffer (A), and in TEMA buffer containing 0.4 Μ KCl (Β). Vero-ER ( — # — ) . Reconstructed "8S" ER ( _ Q _ ) . Reconstructed "7S" ER ( — Ο — ) . Reconstructed "6S" ER ( — Q — ) . Reconstructed "5S" ER ( — Δ — ) . Cytosolic "5S" ER ( — ^ ). Reconstructed "5S" ER + unlabeled cytosol ( — ) .

22 cytoplasm into the nucleus as vero-ER-E, and the translocation is suppressed through binding with ERBFs.

Astonishingly, in contrast to the previous

proposal (3,26,27,31), translocation of "5S" ER-Ε into the nuclei was nearly nil.

We mentioned above that "5S" ER in the cytosol is complexed with com-

ponent C under the low salt conditions.

The complex, ("5S" ER·E)·(component

C), possessed relatively high affinity for the nuclear envelopes, but did not translocate into the nuclei.

The adsorption of the complex to the

nuclear envelopes might have been taken as the translocation into the nuclei previously.

The slight nuclear translocation of ER observed when "6S" ER·Ε

and "8S" ER"Ε were incubated with the isolated nuclei is due to the liber2+ ation of vero-ER-E under the incubation condition caused by Mg added to the incubation medium to protect the nuclei (34). 2+ of Mg

The remarkable effect

on the dissociation of vero-ER from ERBFs is mentioned in the fol-

lowing section.

Table II.

The partially proteolyzed ERs (secto-ER-E and "3.8S" ER-E),

Capabilities of various forms of estrogen receptors to translocate into the porcine nuclei in an iri vitro system (33) .

ER· [''HIE

ER-[ H]E bound to the nuclei finol

ER- [ 3 H]E adsorbed to the nuclear envelopes

ER- [•'HIE translocated into the nuclei

flTDl

fmol

Translocation

%

226

35

191

75

65

45

20

8

110

37

73

29

100

40

60

24

133

110

23

9

Secto-ER- [•'hIE

85

68

17

7

"3.8S" ER-(3H]E

74

55

19

Β

Vero-ER- [-^HlE

"5S" ER-[3H]E

"6S" ER*[^H]E

"8S" ER· I^IE ("5S" ER-[3H]E) • (oonponent c)

^ R . [ 3 H]E (250 final) was incubated with the nuclei or nuclear envelopes in 0.32 Μ sucrose - 1 rrM MgCl 2 - 10 πΜ Tris-HCl - 0.25 irM antipain, pH 8.0, for 3 h at 4°C.

^obtained by subtracting the amount of ER adsorbed to the nuclear envelopes from that of ER bound to the nuclei.

23

which no longer interact with ERBFs, did not translocate into the nuclei. These results indicated that the binding site of vero-ER to ERBFs play important roles in the regulation of the translocation of ER from the cytoplasm into the nucleus.

It was assumed that the cytosolic protease might

reduce the hormone-sensitivity of the target cell through diminishing the amount of ER capable to translocate into the nucleus.

D.

Regulation of the reactivity of basic estrogen receptor molecule

(vero-ER) with estrogen receptor-binding factors.

a) Strongly hydrophobic domain of basic estrogen receptor molecule (vero-ER) (35)•

Vero-ER was shown to possess an extremely strong hydrophobic domain,

which is concealed through binding with estrogen receptor-binding factors (ERBFs).

Vero-ER freed from ERBFs was bound to a phenyl-Sepharose column

under the low salt (TEMA) conditions, and could not be eluted even with TEMA buffer containing 50% ethylene glycol (Fig, 14A).

Most of the strongly

hydrophobic proteins of the cytosol are eluted from the column with the low salt buffer in the presence of 50% ethylene glycol.

To elute vero-ER

from the column, it was necessary to utilize ethanol as a polarity-reducing agent.

Vero-ER could be eluted quantitatively from the column with TEMA

buffer containing 30% ethanol (Fig. 14A).

Dissociation of [ H]estradiol

from the receptor was negligible during the process of the elution from phenyl-Sepharose.

In contrast, reconstructed "5S" ER, "6S" ER and "8S" ER passed straight through the phenyl-Sepharose column in TEMA buffer (Fig. 14B).

This showed

that the strongly hydrophobic domain of vero-ER is concealed in the complexes of vero-ER with ERBFs.

The association and dissociation of vero-ER

with ERBFs is regulated mainly by ionic forces.

As mentioned above,

vero-ER is dissociated from ERBFs under the high salt (0.4 Μ KCl) conditions favorable for the hydrophobic interactions.

Accordingly, it is assumed

that the strong hydrophobic domain and the binding sites with ERBFs occupy different positions on vero-ER molecule.

Both secto-ER and "3.8S" ER passed straight through the column under the

24

FRACTION

NUMBER

Fig. 14. A n a l y s i s of h y d r o p h o b i g i t y of e s t r o g e n r e c e p t o r s w i t h a p h e n y l Sepharose c o l u m n (35). 1.6 χ 10 c p m of e s t r o g e n r e c e p t o r s in 1 m l of T E M A b u f f e r w e r e a p p l i e d (beginning a t the f i r s t a r r o w ) to 0.2 m l c o l u m n s equilibrated with TEMA buffer. The c o l u m n s w e r e w a s h e d 5 - t i m e s w i t h 1 m l T E M A b u f f e r c o n t a i n i n g 50% e t h y l e n e g l y c o l (the s e c o n d a r r o w ) . The c o l u m n s w e r e finally w a s h e d 5 - t i m e s w i t h 1 m l of T E M A b u f f e r c o n t a i n i n g 30% e t h a n o l (the third a r r o w ) . (A) V e r o - E R ( — O — ) . (B) R e c o n s t r u c t e d " 5 S " E R ( — β — ) ; r e c o n s t r u c t e d " 6 S " ER ( — * — ) ; r e c o n s t r u c t e d " 8 S " ER ( — # — ) . (C) S e c t o - E R (—Δ—); " 3 . 8 S " ER ( - - Ο - ) .

low salt conditions

(Flg. 14C), indicating that the strong hydrophobic

m a i n of vero-ER w a s totally destroyed by the endogenous protease.

do-

The c o n -

comitant removal of the strong hydrophobic domain and the binding sites w i t h ERBFs suggested that they occupy each other close positions o n vero-ER molecule.

b) Regulation of association and dissociation of basic estrogen receptor molecule

(vero-ER) w i t h estrogen receptor-binding factors

(22,24).

The

affinity of vero-ER w i t h ERBFs as estimated from the apparent dissociation constant 2.6 χ Ι Ο

(Kd) v a l u e s [2.5 χ 1 0 ~ 9 Μ ("8S" ER-FF); 3.0 χ 1 0 ~ 9 Μ ("6S" ER-FF); -10

Μ ("5S" ER-FF)] is very h i g h in TEMA buffer

(Fig. 15).

The a f -

finity of "5S" ER-FF for v e r o - E R is one order higher than the affinity of

25 "6S" ER-FF or "8S" ER-FF.

The stronger affinity of "5S" ER-FF to vero-ER

is an important basis of "5S" ER formation from "8S" ER as mentioned in the previous section. 2+ Astonishingly, Mg

at very low concentrations influenced significantly the

interaction of vero-ER with ERBFs.

The apparent Kd values of vero-ER for

9 ERBFs [3.5 χ 10~ 8 Μ ("8S" ER-FF); 6.0 χ ΙΟ" 8 Μ ("6S" 2+ ER-FF); 2.7 χ 10~ Μ ("5S" ER-FF)] observed in the presence of 1 mM Mg were all increased over 2+ 10 times as compared with in the absence of Mg (Fig. 15).

In Fig. 16 is shown the effect of Mg into the isolated nuclei.

2+

on the translocation of vero-ER

In the absence of ERBFs, vero-ER was translocated

into the nuclei almost quantitatively independent of the concentration of 2+ Mg . This indicated that the translocation of vero-ER dissociated from 2+ ERBFs is not influenced by Mg . As mentioned above, ERBFs inhibited the nuclear translocation of vero-ER under the conventional assay conditions 2+ (containing 1 mM Mg

for the protection of the nuclei).

The inhibitory

effects of ERBFs on the nuclear translocation of vero-ER were reduced dras-

Fig. 15. Scatchard analysis of the affinity of vero-ER with estrogen receptor-binding factors in the absence or presence of 1 mM MgCl (24).

26

•ο Ol

σ

.Ω 3

υ c

Ο NO "Ui *

MgCI2 (mM) 2+ Fig. 16. Effect of Mg on the nuclear translocation of vero-ER-E in the absence and presence of estrogen receptor-binding factors (24). Vero-ER-E (200 fmol) ( — O — ) ; vero-ER-E (200 fmol) + "5S" ER-forming factor (3 units) (—·—); vero-ER-E (200 fmol) + "6S" ER-forming factor (3 units) ( — * — ) ; vero-ER-E (200 fmol) + "8S" ER-forming factor (3 units) ( — · — ) ; labeled cytosol ( — α — ) .

tically by increasing the concentration of Mg In the presence of 5 mM Mg

in the incubation medium.

, the inhibitory effects of "8S" ER-FF and

"6S" ER-FF on the translocation of vero-ER into the nuclei were nearly nil. The inhibitory effect of "5S" ER-FF on the nuclear translocation of vero-ER 2+ disappeared completely in the presence of 10 mM Mg . These results showed 2+ that Mg

promoted the dissociation of YSXA-ER from ERBFs and restore the

capability to translocate into the nuclei.

As mentioned above, vero-ER

in the uterine cytosol is complexed with "8S" ER-FF in the low salt condi2+ 2+ tions when Mg is absent. In the presence of Mg (10 mM), however, the cytosolic receptor sedimented not at 8S, but migrated to the bottom frac2+ tions (24). Purified vero-ER sedimented at 4.5S in the presence of Mg (10 mM) (24). Accordingly it was assumed that, in 2+ the cytosol, vero-ER dissociated from "8S" ER-FF in the presence of Mg is bound to other macromolecule under the low salt conditions. In accord with the assumption, 2+ the promoting effect of Mg on the translocation of the cytosolic receptor 2+ into the nuclei was not evident (Fig. 16).

Anyhow, Mg

play important roles in the action mechanism of estrogen.

is expected to

27 Discussion

In this chapter, we presented new aspects on the molecular organization of the estrogen receptor system.

In the early 1970s, Mueller et^ a^l. proposed

presciently that "8S" ER is a complex of basic "4S" ER, which is common to different aggregates, and a subunit which does not bind estradiol

(4,36-38).

By incubating the labeled cytosol at higher temperatures they observed not "5S" ER formation, but formation of modified "4S" ER which no longer gives "8S" ER (4, 36-38).

They considered that the basic "4S" ER interacts with

a cytosolic factor which is dialyzable and ether soluble to form modified "4S" ER (4,37,38).

The possibility of the interaction of lipophilic lower

molecular components with the receptor remains, but the more frequently encountered modification of the basic "4S" ER into modified "4S" ER is caused by the endogenous protease.

Puca et al. reported for the first time the proteolytic modification of

2+ estrogen receptor by the endogenous Ca

-requiring protease (39).

They

further carried out detailed analysis of the estrogen receptor system by utilizing NaSCN to inhibit aggregation and dissociation of receptor

(40).

They proposed that "2.8S" ER is the basic subunit of estrogen receptor, and that "4S" ER (dimer), "5S" ER (tetramer), and "8S" ER (octamer) are formed through the self-association of the basic subunit (40). Modification

2+ of receptor by Ca

-not requiring protease takes place, however, in the

presence of NaSCN.

The disappearance of the property of the cytosolic

receptor to aggregate and dissociate through the NaSCN-treatment (40) is attributed to the proteolytic destruction of the binding site of the native receptor with receptor-binding factors.

The "2.8S" ER which they

observed in the presence of NaSCN, and proposed to be the basic subunit of estrogen receptor (40) is obtained from the proteolyzed receptor through dissociating at the nick by the chaotropic salt. Sherman e_t al. utilized antipain (21) for the first time to protect steroid hormone receptors from the endogenous proteases (41-43).

They reported

detailed analysis of the proteolyzed receptor (mero-receptor, "2-3S" receptor) and the relation to the native receptor (41-43).

The concentration

(50 mM) of the protease inhibitor utilized in their studies, however, might

28 be too high to be applied widely for the analysis of the receptor system.

Molecular organization of steroid hormone receptor systems might be similar to each other (44).

We showed that the oviduct progesterone receptor system

is similar to the uterine estrogen receptor system (19).

Colvard & Wilson

reported "8S" androgen receptor-promoting factor which binds with "4S" receptor to form "8S" receptor (45).

Joab et_ al^. reported that "8S" proges-

terone receptor contains a non hormone binding protein component which is common to "8S" receptors for other steroid hormones (46).

Receptor-binding

factors might be common for the receptors of different steroid hormones. It was proposed in various reports from O'Malley's laboratory that "6S" progesterone receptor is a complex of two different basic "4S" receptors (receptor A and receptor B) (7, 47, 48). It might be possible that receptor A and receptor Β suffered proteolytic modifications during the process of the purification.

Reconstruction of "6S" progesterone receptor from recep-

tors A and Β has not yet been accomplished.

The results presented in this chapter suggested strongly that vero-ER with the free binding sites to ER-binding factors and open hydrophobic domain is the activated receptor which translocates from the cytoplasm into the nucleus.

The temperature-dependent process for the activation of the recep-

tor (3,26,27,31) is assumed to be necessary to dissociate vero-ER from ER-binding factors.

King & Greene reported the exclusive localization of

estrogen receptor by utilizing monoclonal antibodies generated against estrogen receptor (49). for

It appeares that they utilized proteolyzed receptor

the production of the antibodies.

The possibility remains that

their antibodies recognized proteolyzed receptors, but failed to recognize native receptor. Proteolyzed receptor is easily detected in nuclei (25), but not in the fresh cytosol (14,18,41).

the uterine

Further detailed

study would be needed to solve the problem.

References

1.

Glascock, R.F., Hoekstra, W.G.: Biochem. J. 72^, 673-682 (1959).

2.

Jensen, E.V., Jacobson, Η.I.: In: Biological Activities of Steroids in

29 Relation to Cancer. 161-178 Press, New York 1960.

(G. Pincus & E.P. Vollmer Eds.), Academic

3.

Jensen, E.V., DeSombre, E.R.:

4.

Mueller, G.C., Vonderhaar, Β., Kim, U.H., Mahieu, L.M.: Ree. Prog. Horm. Res. 28, 1-49 (1972).

Ann. Rev. Biochem. 41, 203-230

(1972).

5.

Gorski, J., Gannon, F.: Annu. Rev. Physiol. 38, 425-450

6.

Yamamoto, K.R., Alberts, Β.Μ. : Annu. Rev. Biochem. 4_5, 721-746

(1976).

7.

Schräder, W.T., O'Malley, B.W.: In: Receptors and Hormone Action. 1 8 9 224 (B.W. O'Malley & L. Birnbaumer Eds.), Academic Press, New York 1978.

8.

Bualieu, E.E.: Klin. Wschr. 56, 683-695

9.

Clark, J.H., Peck, Jr.E.J.: Female Sex Steroids, Receptors and Function. Springer Verlag, Berlin 1979.

(1976).

(1978).

10. Baxter, J.D., Rousseau, G.G.: In: Glucocorticoid Hormone Action. 1-24 (J.D. Baxter & G.G. Rousseau Eds.), Springer Verlag, Berlin 1979. 11. Katzenellenbogen, B.S.: Ann. Rev. Physiol. 42, 17-35

(1980).

12. Kanazir, D.T.: In: Hormonally Active Brain Peptides. 181-214 (McKerns & Pantic Eds.), Plenum Publishing Corporation, New York 1982. 13. Moudgil, V.K.: In: Principles in Recepterology. Ed.), Walter de Gruyter, Berlin 1983.

273-379

(M.K. Agarwal

14. Murayama, Α., Fukai, F., Hazato, T., Yamamoto, T.: J. Biochem. 88, 955-961 (1980). 15. Murayama, Α., Fukai, F., Hazato, T., Yamamoto, T.: J. Biochem. 88^, 963-968 (1980). 16. Murayama, Α., Fukai, F., Yamamoto, T.: J. Biochem. 88^, 969-976

(1980).

17. Murayama, Α., Fukai, F., Yamamoto, T.: J. Biochem. 88, 1457-1466 18. Murayama, Α., Fukai, F.: J. Biochem. 89, 1829-1837

(1980).

(1981).

19. Murayama, Α., Fukai, F., Yamamoto, T.: J. Biochem.

1305-1315

20. Murayama, Α., Fukai, F., Murachi, T. : J. Biochem. 95_, 1697-1704

(1980). (1984).

21. Suda, H., Aoyagi, T., Hamada, M., Takeuchi, T., Umezawa, H.: J. Antibiotics 25, 263-266 (1972). 22. Murayama, Α., Fukai, F.: J. Biochem. 92, 2039-2042 23. Scatchard, G.: Ann. N.Y. Acad. Sei. 5^, 660-672

(1949).

24. Fukai, F., Murayama, Α.: J. Biochem. 95, 1227-1230 25. Murayama, Α., Fukai, F.: J. Biochem. 90, 823-832

(1982).

(1984).

(1981).

26. Jensen, E.V., Suzuki, T., Kawashima, T. Stumpf, W.E., Jungblut, P.W., DeSombre, E.R.: Proc. Natl. Acad. Sei. U.S. 59_, 632-638 (1968). 27. Gorski, J., Toft, D., Shamala, G., Smith, D., Notides, Α.: Ree. Prog. Horm. Res. 7A_, 45-80 (1968). 28. Giannopoulos, G., Gorski, J.: J. Biol. Chem. 246, 2530-2536

(1971).

29. Stancel, G.M., Leung, K.M.T., Gorski, J.: Biochemistry 12^, 2137-2141 (1973).

30 30. Rochefort, Η., Baulieu, Ε.Ε: Endocrinology 84, 108-116 (1969). 31. Jensen, E.V., DeSombre, E.R.: Science 182, 126-134 (1973). 32. Puca, G.A., Bresciani, F.: Nature 218, 967-969 (1968). 33. Murayama, Α., Fukai, F.: J. Biochem. 94, 511-519 (1983). 34. Tata, J.R.: In: Methods in Enzymology Vol. 31, 253-262 (S. Fleisher & L. Packer Eds.), Academic Press, New York 1974. 35. Murayama, Α., Fukai, F.: FEBS Letters 158, 255-258 (1983). 36. Vonderhaar, B.K., Kim, U.H., Mueller, G.C.: Biochim. Biophys. Acta 208, 517-527 (1970). 37. Vonderhaar, B.K., Kim, U.H., Mueller, G.C.: Biochim. Biophys. Acta 215, 125-133 (1970). 38. Mueller, G.C., Cowan, R.A.: Adv. Biosciences 1^, 55-76 (1974). 39. Puca, G.A., Nola, E., Sica, V., Bresciani, F.: J. Biol. Chem. 252, 1358-1366 (1977). 40. Sica, V., Nola, E., Puca, G.A., Bresciani, F.: Biochemistry 15_, 19151923 (1976). 41. Sherman, M.R., Pickerling, L.A., Rollwagen, F.Μ., Miller, L.K.: Fed. Proc. Fed. Am. Soc. Exp. Biol. 37_, 167-173 (1978). 42. Sherman, M.R., Berzilai, D., Pine, P.R., Tuazon, Fe.Β.: In: Steroid Hormone Receptor Systems. 357-375 (W.W. Leavitt & J.Η. Clark Eds.), Plenum Publishing Corporation, New York 1979. 43. Sherman, M.R., Tuazon, F.B., Miller, L.K.: Endocrinology 1Ό6, 17151727 (1980). 44. Hazato, T., Murayama, Α.: Biochem. Biophys. Res. Commun. 98, 488-493 (1981). 45. Colvard, D.S., Wilson, E.M.: Endocrinology 109, 496-504 (1981). 46. Joab, I., Radanyi, C., Renoir, M., Buchou, T., Catelli, M.G., Binart, N., Mester, J., Baulieu, E.E.: Nature 308, 850-853 (1984). 47. Schräder, W.T., Kuhn, R.W., O'Malley, B.W.: J. Biol. Chem. 252, 299307 (1977). 48. Coty, W.A., Schräder, W.T., O'Malley, B.W.: J. Steroid Biochem. 1Ό, 1-12 (1979). 49. King, W.J., Greene, G.L.: Nature 307, 745-747 (1984).

Acknowledgements This review is dedicated to the memory of the late Prof. Tadashi Yamamoto (1917-1981) who inspired me with his profound insights. I thank Dr. F. Fukai for his help in the preparation of this manusctipt.

STRUCTURE,

PROPERTIES

AND

SUBCELLULAR LOCALIZATION

OF T H E CHICK

OVIDUCT

P R O G E S T E R O N E RECEPTOR.

J a n Mester,

Jean-Marie

Gasc, Thierry

Buchou,

Jack-Michel

Renoir,

Joab, Christine Radanyi, N a d i n e Binart, M a r i a - G r a z i a Catelli,

Irene

Etienne-Emile

Baulieu INSERM U 33, Lab. Hormones, 94270 Bicetre, France

I.

Introduction

Although a considerable effort has been invested hormone now

receptors since their discovery

that

data

concerning

their

primary

in the study of

in the early sixties, structure

as w e l l

steroid

it is only

as

subunit

composition are becoming available. This breakthrough is to be thanked for mainly

to

the

progress

that

has

been

achieved

in

the

techniques

of

purification of these proteins w h i c h are not only relatively unstable, but also present at low concentrations in target tissues and h a v i n g a tendency to

aggregate

under

a

non-activated

receptor

problem

by

posed

variety forms

tendency

of

by

to

conditions.

molybdate

form

has

aggregates,

Stabilisation helped and

to

design

of

affinity derivatives makes it possible to obtain high purity of

several

purified

kinds receptor

immunohistological

of

steroid hormone preparations

studies.

receptors.

have

In this

been

Antibodies

raised

chapter, we

results concerning the chick oviduct progesterone

and

of

the

surmount

the

suitable preparations

against used

such

in

shall review our

recent

receptor.

II. Background

1)

Chick oviduct as a target tissue for progesterone. The term "progesterone" is in fact a m i s n o m e r insofar as the chicken

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

is

concerned

as

there

is

no

gestation

to

maintain.

However,

progesterone is secreted during the egg-laying cycle of the h e n and is apparently

important

in its regulation. Their effects in the oviduct

fall roughly into two categories. First progesterone is an inducer of synthesis

of

(immature)

chick has

several

leads to development

of

the

egg

white

proteins.

to be first exposed of the tubular

the oviduct. After w i t h d r a w a l

For

this,

the

to estrogen priming

which

glands in the m a g n u m p o r t i o n of

of estrogen,

the

tubular

gland

cells

respond to progesterone by synthesis of large quantities of egg white proteins,

mainly

ovalbumin,

conalbumin,

ovomucoid

and

lysozyme.

A

secondary treatment of estrogen-primed, w i t h d r a w n chick w i t h e s t r o g e n has a similar effect on the synthesis of these proteins in the tubular glands,

and

induces,

progesterone least

partly

antagonizes

in

addition,

tissue

growth.

The

effects

and estrogen on the egg white p r o t e i n synthesis additive certain

of

(see

reviews

the

estrogen

1,

2).

Second,

effects,

for

growth and d i f f e r e n t i a t i o n of the immature oviduct

of

are at

progesterone

instance

tissue

(3, 4), as w e l l as

growth of the "withdrawn" oviduct tissue induced by estrogen

treatment

(5-7). In terms of specific p r o t e i n synthesis, progesterone has often a transient "antiestrogen" effect instance,

it retards

conalbumin synthesis estrogen + (6).

the

: during a short period of time, for

estrogen-induced

increase

in the rate

(6, 8). Similar observations hold also

progesterone-induced

Induction of ornithine

changes

in DNA-polymerase

decarboxylase,

an enzyme

for

of the

activity

implicated

in

polyamine synthesis and known to be active in growing tissues

(9) is

another

These

estrogen effect

observations

(10)

inhibited

by progesterone

(11).

are correlated with, and perhaps a consequence of,

decrease in the nuclear (and total cellular) estrogen receptor

the

content

of the oviduct cells after treatment w i t h estrogen and progesterone as compared

to estrogen alone. For more

progesterone

information on the effects

in the chicken oviduct, see the reviews cited above

2) and references

therein.

of (1,

33 Early studies on the chick oviduct progesterone receptor. Since the first report of Sherman et al. (12) it is known that the high

affinity progesterone-binding

recovered

in the high-speed

protein of the chick oviduct

supernatant

of

the tissue

is

homogenate.

Consequently, it has been assumed that, as the other steroid hormone receptors, also this one is a cytoplasmic protein. (This assumption is at present questioned Already

: see section V).

in the immature chick oviduct, progesterone 3

levels of

the order

hormone binding molecule.

of

10

molecules

per

cell

receptor is at

as determined

by

(13), assuming one progesterone binding site/protein

Estrogen priming

leads to tissue differentiation

so that

eventually some 90 % of the cells become 4organized in tubular glands ; at

this

stage,

there

are

some

5-7.10

progesterone

binding

sites/cell. Estrogen withdrawal is followed by a progressive but not rapid loss of the progesterone receptor : while the tissue regresses from 1-2 g to about 100 mg after 2 weeks and remains without further change,

the

progesterone

receptor

concentration

is

approximately

halved at 2 weeks of estrogen withdrawal and again after the following 2-3 weeks

(13). Secondary

estrogen administration

induces again an'·

augmentation of the progesterone receptor level, probably by the novo synthesis, after an

6 h lag (14).

Treatment in vivo with progesterone "translocates" only a relatively small fraction of the total cellular

progesterone

receptor

to the

nucleus. While this "translocation" may not imply a shift from the cytoplasm to the nuclear compartment in the anatomical sense, the data reflect the fact that a portion of the total receptor population has become

associated

with

the nucleus

sufficiently

tightly

to

resist

solubilisation by the (low-salt) homogenization buffer. The proportion of

the

"nuclear"

receptor

decreases

during

the

hours

following

progesterone administration and by 6 h it differs little from that in untreated controls) . Gel filtration studies suggested heterogeneitey of the chick oviduct progesterone receptor : as many as five species differing by their

34 Stokes radii order

were identified and numbered I - V, in decreasing

(15). The largest

(non-activated The

two

(form I) is probably a mixture of a "native"

; see further) receptor and heterogeneous

following

progestin-binding

species, proteins

on

the

other

in monomeric

hand,

are

aggregates.

authentic

state. They can be

easier

resolved by ion-exchange chromatography and are now currently referred to a "subunit A" identification

(= form

was

III) and

subsequently

"subunit

made

B"

possible

(= form II). Their by

the

use

of

a

photolabile progestin R 5020, which can be covalently attached to the binding proteins by exposure to UV light (16). Their molecular weights were found analysis

to be

108K (B) and

79K

(A), respectively, by

SDS-PAGE

(see 17, 18 for review). Finally, the forms IV and V turned

out to be fragments formed from the A and/or Β subunits by action of an endogenous, Ca

2+

-activated protease (19, 20).

II. Definition of "native" and "activated" ("transformed") receptors. 3 The

H-progesterone-receptor

complexes formed

in the soluble fraction of

the homogenate of chick oviduct tissue, at low ionic strength and at low temperature, display the characteristics of what has been termed "native", i.e. non-activated

form. This means

nuclei or to their components

that they do not bind

to

isolated

(chromatin, DNA), nor to certain synthetic

polyanions (phosphocellulose ; ATP-agarose). Within < 1 h at incubation at > 20°C or at high ionic strength acquire

the capacity

to bind

(> 0.3) the receptor-hormone

to the above-mentioned

structures, although the binding is frequently change of properties has been initially

solid

incomplete

complexes

supports

or

(21, 22). This

termed "activation" since it is

presumed that binding to the nuclear components implies that the receptor is active in mediating hormonal effects on gene expression. Until present, no

in vitro

test

of

receptor

"activity"

is available

and

therefore

"activation" remains a concept based on hypothesis rather than facts. It is also to be printed out that the binding of receptors to the cell nucleus does

not

always

lead

to hormonal

effects

(for

ex.

in

the

case

of

antiestrogen-estrogen receptor complexes of the chick oviduct, 23, 24).

35 As

a

result

of

"activation",

physico-chemical

progesterone receptor are altered

properties

of

the

: the hormone receptor complexes become

smaller as reflected by a change of their sedimentation coefficient 8S to 4S) and R is tightened, Acquisition

s

leading

of

(from

(from 7.7 nm to 5.2 nm) and the binding of the hormone to a longer half-time

affinity

for nuclei,

of dissociation

chromatin, DNA and

(25, 27).

the change of

physico-chemical properties of the chick oviduct progesterone receptor have always

been

observed

to

occur

simultaneously.

"transformation", is preferred by some authors

An

alternative

term,

(19) to cover the ensemble

of the changes on receptor characteristics (see also section IV.4).

III. Methods

All methods are described in detail in the articles cited. The procedure we have developed for purification of the different forms of the progesterone receptor are schematically represented by a flowchart

(Fig.

1). Affinity

chromatography was performed by loading the high-speed supernatant of the homogenate of estrogen-stimulated of NADAC-Sepharose

(immature) chick oviducts onto a column

(28) ; the gel was washed

containing KCl and urea and eluted eluates

were

then

subjected

elution was carried

to

with 2 μΜ

fractionation

out by applying

sequentially with 3 H-progestin

(29, 31). The

on DEAE-cellulose

a continuous

buffers

where

or discontinuous

KCl

gradient. The choice of buffers throughout purification was dictated by the forms of receptor desired to obtain. The non-activated

form had to be protected by

including 20 mM Na2MoO^ ; in addition, the concentration of urea had

to

be kept < 2.5 Μ during the affinity gel washing procedure since above this limit, a significant proportion of the receptor becomes transformed (32). To purify

the "activated"

receptor, KCl

(0.3 Μ) was added

to 4S

to the

cytosol > 1 h prior to loading onto the affinity native (26, 30). When the receptor was to be crosslinked with glutaraldehyde, phosphate buffers were used instead of Tris. Antibodies

raised

against

the progesterone

receptor

IgG-RB and monoclonal BF4) are characterized 33, 35.

(polyclonal

in Table I and

IgG-G,

references

36 Fig. 1 : Progesterone receptor purification flow-chart (refs. 28, 31). Chick oviduct tissue

Homogenization in 3 vols, of buffer Ultracentrifugation

High speed supernatant

Alternative procedures

+ Na.MoO, (20 mM) 2 4

+ KCl (0.3 Μ ) > 1 h at 0°C

Chromatography on NADAC-Sepharose / \ (ref. 28)

Eluate

Eluate

\

/

Chromatography on DEAE-Celluose (elution with KCl gradient)

\ Purified non-transformed (8S) receptor

Two forms of the purified transformed receptor eluted at 0.08 Μ (subunit A) and > 0.2 Μ KCl (subunit Β)

Iimnunohistological experiments were performed with paraffin sections of the tissue which had been fixed by one of the following procedures (36) : -

Bouin's

fluid

for 60 to 80 min, followed

by several washes

in 70 %

ethanol. - Carnoy's fluid for 60 to 80 min, followed by several washed in absolute ethanol.

37 - Glutaraldehyde

(0,5 %) in 0,1 Μ Soerensen buffer pH 7.4 for 90 min,

followed by several washed in either Soerensen buffer or PBS. - Absolute ethanol + 17, acetic acid for 60 to 80 min, followed by several washed in absolute ethanol. Dehydration was made in graded ethanol when needed, and terminated in 1-butanol before embedding

in paraffin.

For antibody

reactions,

the

sections were deparaffinized, rehydrated and rinsed with PBS. They were then incubated first in 3 % non-immune serum of the animal species in which the second antibody was raised. The excess of serum was removed and the sections were incubated with the first antibody (90 to 120 min). After washing in PBS, sections received the second biotinylated antibody (dilution 1:400 ; 30 min). The second antibody was removed by washing with PBS and complexed with avidin-biotin-peroxidase the

peroxidase

activity

tetrahydrochloride

was

revealed

by

(30 min). After washing, 3-3'-diaminobenzidin

(DAB) (0.5 mg/ml) in presence of 0.01 %

·*"η

^

7.6 Tris buffer. Sections were then rinsed, dehydrated and mounted.

TABLE I : Antibodies against the chick oviduct progesterone receptor (PR)

Antibody

Raised Non-den 90% labeling efficiency of the receptors by removing low molecular weight thiols and by adjusting the time 3 and temperature of the reaction, the concentration of receptor and [ H]Dex-Mes, and most importantly the pH of the reaction solution (31) . We had previously determined that a-keto mesylates preferentially reacted with thiols and that virtually no reaction occurred unless the thiol group was ionized (14).

Accordingly,

when the pH of 3 the reaction solution at 0°C was varied from 6.7 to 8.6, the yield of [ H]Dex-Mes—labeled

98K receptors increased from M 8 %

to

•^92% of the available receptors in ( N H ^ ^ S O ^ precipitated rat liver cytosol (as determined by [^H]dexamethasone binding at the same pH) (31; see also Section IIB).

It could be calculated from these data that, if a

single amino acid was being labeled by Dex-Mes, that amino acid had a pK^ of "W.4. While this appears low for the pK a of a cysteine, the pKa of the 3 -SH group in bovine serum albumin is 7, 3 8 1 - 3 8 8 ( 1982) .

B.C.,

Litwack,

G.:

Endocrinology

83. G r i p p o , J.F. , T i e n r u n g r o j . W., D a h m e r , M . K . , H o u s l e y , P.R., Pratt, W . Β . : J . iol. Chem. 2J58, 1 3658-1 3664 ( 1983) . R . Η . S. :

84. D u b o s , R.J., T h o m p s o n 5 0 1 - 5 1 0 (1938). Liao,

S.:

85.

Liang, Τ. ( 1974).

86 .

B a r r a c k , E.R., Coffey 7 2 6 5 - 7 2 7 5 (1980).

J. Biol. Chem.

J. Biol. Chem.

87 . F e l d m a n , Μ., K a l l o s , J. 256, 1 1 4 5 - 1 1 4 8 (1981).

D.S. :

2_49,

J. B i o l .

Hollander,

4671-4678

Chem.

V.P.:

124,

255,

J. Biol.

Chem

349 88. L i a ο , S., Sraythe, S., Tyraoczko, J . L . , R o s s i n i , G . P . , C h e n , C., H i i p a k k a , R.A.: J. B i o l . C h e m . 255 , 5 5 4 5 5551 (1980). 89. T y m o c z k o , J . L . , S h a p i r o , J., S i m e n s t a d , D . J . , Nish, J. S t e r o i d B i o c h e m . J_6, 5 9 5 - 5 9 8 (1982). 90. C h o n g , M . T . , L i p p m a n , M . E . : 3002 (1982).

J. B i o l . C h e m . 2_57,

91. R o s s i n i , G . P . , B a r b i r o l i , B.: C o m m u n . U 3 , 8 7 6 - 8 8 2 ( 1983).

Biochem.

Biophys.

A.D.:

2996Res.

92. H u t c h e n s , T . W . , M a r k l a n d , F . S . , H a w k i n s , Ε . F . : Proc e e d i n g s of the 64th Annual M e e t i n g of the E n d o c r i n e S o c i e t y , a b s t . 568, (1982). 93. H u t c h e n s , T . W . , M a r k l a n d , F . S . , H a w k i n s , Ε.F.: B i o p h y s . Res. C o m m u n . 105, 20-27 ( 1 9 8 2 ) . 94. T y m o c z k o , J . L . , P h i l l i p s , M . M . : 142-149 (1983).

Endocrinology

Biochem. 112,

95. S i n g h , V., E e s s a l u , T., G h a g , S., M o u d g i l , V.: Excerpta M e d i c a , A b s t r a c t s of the 7th I n t e r n a t i o n a l C o n g r e s s of E n d o c r i n o l o g y , a b s t . 2095, E l s e v i e r S c i e n c e P u b l i s h e r s Co . , Inc . , New York ( 1984) . 96. H o u s l e y ,

P.R.,

P r a t t , W . B . : Fed. Proc . 43, a b s t . 908( 1984)

97. M i l l e r , A . S . , S c h m i d t , T . J . , L i t w a c k , G.: Excerpta Medica, A b s t r a c t s of the 7th I n t e r n a t i o n a l C o n g r e s s of E n d o c r i n o l o g y , a b s t . 1056, E l s e v i e r S c i e n c e P u b l i s h e r s Co., Inc . New York ( 1984) . 98. Van B e l l e , H.:

Cell C a l c i u m

99. A r any i , P., N a r a y , Α.: 272 (1980). 100. Van B o h e m e n , C . G . , P h y s i o l . 4. 9 5 - 1 1 0

2, 4 8 3 - 4 9 4

J. S t e r o i d

E l i a r d , P.H., (1983).

102. Z o l l e r , M . J . , T a y l o r , 8368 ( 1 9 7 9 ) .

S.S.:

(1981).

Biochem.

VZ,

267-

Rousseau, G.G.:

Mol.

J. B i o l . C h e m . 254,

8363-

103. H a t h a w a y , G . M . , Z o l l e r , M . J . , T r a u g h , J . Α . : C h e m . 2_5_6, 1 1442-1 1446 ( 1981 ).

J.

104. H o p p e , J., F r i e s t , W.: ( 1979) .

141-146

Eur. J. B i o c h e m .

105. S c h m i d t , T . J . , M i l l e r - D i e n e r , C h e m . 219, 9 5 3 6 - 9 5 4 3 (1984).

93,

Α., L i t w a c k , G.:

Biol.

J.

Biol.

INTERACTION

OF

V i r i η der

Mo u d g i 1

Κ.

NUCLEOTIDES

WITH

STEROID

HORMONE

RECEPTORS

Biochemical E n d o c r i n o l o g y L a b . , Department of Biological Sciences, Oakland U n i v e r s i t y , Rochester, Michigan 48063

Introduction

Under to

physiological

enter

tor

their

proteins

receptor

its

to

sites

chromatin sponse

is

called

interaction

atin

form

complex

alteration

conditions,

target

(2,3).

regulation

to

The

be

of

gene

expression

receptors

seem

to

and

in

triggering

have

chemical data

techniques

which

aided

in

the

ported plasm.

resulted

the

teraction in

of

to

be

Upon are

physiologic

Recently, and

from

by

a

with

predominantly

to

that

rat

the

in

into

and

the

recepbio-

ligands. such

The

approaches

for

(4).

the

binding

hormone

mechanism

the

re-

hormonal

established,

hormonal

hormones,

nuclear

hormone

employed

receptors

localized

relocate

in

Steroid

uterus

steroid

of

in

to chrom-

hormonal

histochemical

two-step

the

prior

a

been

both

response.

steroid-

nuclear

with

events

yet

radiolabeled

of

availability

not role

studies

hormones,

believed

has key

recep-

The

eliciting of

believed

temperature-dependent

receptors

for

studied

using

estradiol of

a

hormonal been

formulation

absence

complexes

trogen

a

generally

play

a

undefined,

sequence

but

tors

of

essential

complete

are

specific

'transformation' yet

interaction

to

The

as

with

complexes.

undergo or

certain,

hormones

interact

hormone-receptor

'activation'

appears

steroid

and

thought

with

(1).

cells

the

in-

Accordingly,

have

been

target-cell

recyto-

receptor-hormone nucleus

under

conditions.

nuclear

localization

progesterone

has

of

been

unoccupied demonstrated

receptors in

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

the

for target

es-

352 cells The

by

use

receptor

of

may,

therefore,

with

the

a

the a

sites.

process

In

be

complexed not

bind

capacity at

can

elevated

tion

of

(see

review

by

the

such

clei,

with to

been

temperature

in gel

by

1).

The

and

now

nonactivated

an

agent

which

have

A

been

number

lieved no

to

factor

of

recently

appeared

factors the

to

be

target

nuclear

high of and

on

conditions,

hormone,

by

by

treatments

other

complexes for

and

have

are

on

dilu-

activated

isolated

resins

unstable

can

of

receptors

the

be

against and

results

This

maintained

sodium

capacity)

pro-

activation

preparations.

presence

(2,

in

procedures

receptor

nu-

altered

ion-exchange

receptors

and

binding

ionic

affinity

preliminary

progesterone-

cellular

influence

The

receptor

the

the

that

binding

Thus,

from

purification

steroid

steroid

(19).

purified

reported

of

of

in

as

complexes

studies

and

of

nonactivated

complexes

crude

form

stabilizes

(loss

activation

tivation

in

correct,

thought

can

increased

overcome,

be

to the

receptors

presence

lengthy

to

be

lead with

steroid

phosphocellulose,

the

only

the

under

mobility

associated may

event(s).

filtration

previous

been

prove

(5-8).

homogenate

complex

could

steroid-receptor

and

and

(1),

their

their

vitro

steroid-receptor

form

hormone

(9,10).

the

develop

kinetics

loosely

extraction

form

a

is

receptors

to

of

the

postulations

nuclei in

approaches

steroid-receptor

their

cytosol, ref.

that

temperature,

hormones

in

inactivation

upon

other

fraction

with

intranuclear

low

performed has

binding the

acquired

activation

problem

receptor

these

isolated

be

Since

nonactivated

had

at

cytosol

steroid

systems,

DMA-cellulose

dissociation

mote

the

treatments

10-18).

of

Should of

cytosols

do

the

its

representing

cell-free

tissue

and

association

activation

in

represent

nucleus,

tighter

nuclear

i m m u n o h i stochern i c a 1 a n d

recovered

molybdate, thermal blocks

on

heat

ac-

glucocorticoid

receptors

reported

are

(20,21).

have

process

of

necessary

been

receptor for

which

activation.

heat-activation

beWhile

of

353 purified

chicken

cellulose purified

have

been

which

reported

forms

produce

the

(24).

tentiates' (25-27).

5S

activated 8S

to An

4.5S form

influence

with

of

trix

i_n_ vi t r ο .

have

been

form

of

under

and

be

to

mutant

the

an

CEM-7

(31).

process

of

also

has

been role

step

activation

of

A

in

in

the

rat

in

the

the

nonactinuclear

ma-

activation

activation intact

of

cells

of

normal

resistant

activation

factors,

receptors

'po-

vitro

response

be

to

uterine

Zn++

glucocorticoid to

re-

converts

receptor

cellular

steroid

in

towards

shown

reported of

to

a

receptor

that

that

conditions,

been

(28-30).

exact

on

steroid

nuclei

receptor

in

factors

protein,

reported

affinity

cell-free

of

reported to

0Ν A the

cellular

estrogen

factor

studies

obligatory

cells The

a

androgen

has

glucocorticoids

of

fective

to

been

receptor

reduced most

of

a

on

complexes

many

activator

have

identified

Although

receptors

appears

has

(20),

conferred

activation

form

Wilson

the

glucocorticoid

cells

form,

shows

performed

4S

androgen

also

the

receptor

the

and

have

which

be

Furthermore,

estrogen

activated

binding

receptor

only

glucocorticoid-receptor

Colvard

They

could

(21).

a complex

cytosol

vated

system

(22,23).

progesterone

capacity

nonactivated

reconstituted

ceptors

oviduct

binding

if

de-

any,

remains

to

in be

established.

Mechanism

A

number

chanism

of of

tivation the

4S

is

Nielsen been the

activation

hypotheses activation

has

to

monomers This

of

5S or

been

of

steroid

have of

been

steroid

explained

by

transformation

of

the

addition

of

consistent

with

(36)

showed

which

suggested

that

sedimentation

the a

a

proposed

to

receptors

explain

rat

uterine

second and

the

steroid

model

estrogen binding

studies order

the

me-

(1,13,32-34).

dimerization

kinetic

of

receptors

the

non-steroid

activation

properties

hormone

of

Not ides

reaction.

receptors

on

receptor

subunit

It

accompanying

Ac-

based

(35).

and has

changes

are

two

in

dif-

354 ferent shown

processes that

(37,38).

estrogen

monomeric

receptor

and

quent

and

separate

event.

units

(7-9S

receptor

to

3.4S)

that

and

(43,44)

postulated

the

are

receptor,

receptors

may

difficult

to

thesis

be

been that

that

and

requires

only

transformation

of

steroid

be

described

steric ation

mechanism of

Measurement The

extent

measuring

of

nuclei

Toft

(46)

be

binding

Weichman

activation

and

or

John

comparable, can

be

have

H]estradiol

from

exponential

process.

the

dissociation the

netics

[

of

slow

ATP

is

the

by

steroid It

one

is

hypo-

steroid

(34).

reported

The

from

favour in

a 1..

with

of

studied

result

et

steroid

process.

an

the

of

generally

this allo-

dissoci-

[

determined

complexes

to

phosphocellulose.

(47) of

have All

three

using

H]estradiol from

the

dissociation,

and

another determining

methods

appear

portion

of

ATP-Sepharose.

receptor fast

by

a greater

demonstrated

The

by

target

Miller

introduced

activation

although

estrogen

component

Hjestradiol

Sherman

form

to

of

progesterone-

hormone

appears

detected

(15)

[

receptor;

of

sub-

associated

the

steroid-receptor

measurement

Notides

for

proteolysis

of

subse-

a consequence

activation

by

a

(1,41,43-45).

component from

of

receptor a3 n d

later)

ATP-Sepharose.

qualitatively

activated

tion

to

of some

presence

DNA-cellulose

involves

and

as

the

activation

of

Moudgil

RNA

recently

in

receptor

(42).

native

may c o n s e q u e n t l y

receptor binding

as

activation

receptors

subunits

receptor

and

and

that

receptor to

of

the

cell

method

which

receptor

their

the

the

or

of

accepted

contain

process of

laboratory

(to

in

have

change

follows

receptors

may

in

the

activation

systems

with,

widely

dissociation

involved

explain

since

receptor

and

(39)

dissociation

glucocorticoid

oligomers

Gorski

dimerization The

has

(5,20,40,41) have

and

a conformational

concurrently

activation,

receptors

Sakai

induces

that

the

follows

component

from

the

activated therefore,

dissociaa

two-

results

nonactivated one.

The

provide

ki a

355 sensitive

criterion

ceptor

general

The

in

monitoring

of

has

receptor

complexes

glycerol

or

upon

earlier. more

widely

used.

with

criterion

two

states

it

one

molecular

is

by

of

re-

molecules

are

transforming

form

to

observe receptor

salt-free to

agents

receptor

of

steroid-

in

converted

measuring

possible

sedimentation

nonactivated

7-9S and

the of

and

The

as

gradients

treatment

from

the

transformation

sediment

sucrose

direct

change

been

This

distinguishing

receptor

analysis

eties

for

(48,49).

a

3-5S

described

activation

gradual, to

moi-

is

complete

another

following

activation.

Influence

of

nucleotides

Munck

and

Brink-Johnsen

cific

binding

of

Subsequently,

in

forms,

steroid was

binding

receptor version In

and

suggested was to

other

loss

of

that

form

that

incompetent

during ATP

it

was

an

(52).

partially

restored

phosphatase

steroid

to

established

is

be

its

release

from

at

mouse

by

an

of

from

and

the

the

be

that

slowed

and

its

ATP-Mg

Moudgil

and

that

the

(L-cells) by

the

the

John

cytosol

nucleotides

in

generating

receptor

binding

systems.

can

presence

(44)

in

of

ad-

of

binding

(capable

con-

thymocytes in

the

process.

capacity

rat

It

nucleus,

ATP-dependent

binding

with

exists

percursor.

the

fibroblast

can

L-cells

(52,53).

other

generate

spepro-

receptor

demonstrated

mouse

25 °C

glucocorticoid

for

to

that

dependent

that

steroid, was

incubation

A role

energy

(51)

subsequently

receptors

reported

required

rebind

binding

by

an

receptors

non-steroid-binding

to

from

ATP.

of

be

a

inhibitors

increased with

initially

Additionally,

be

forms

may

incubation

of

incubated

had

hormone

proposed

binding

cytosol

an

ATP

from

heat-treated

ported

was

following

steroid

studies,

of

(50)

it

glucocorticoid

cytosol dition

a

steroid

glucocorticoids

cess. two

on

re-

aliquots active

hormone)

remains

Although

it

is

356 apparent latory roid

that

either

component(s)

binding,

steroid ceptor

occurs

Preliminary

observations

Toft,

ATP

with

steroid

made

unpublished)

cytosol

from

cow

uterus

observations

led

to

enhancement ATP

could

or

to

Sepharose

yield from

an hen

be

its 4B

affinity oviduct an

were

adsorbed

buffers

This

interaction have

phosphates, tion

are

is

a

regu-

direct

ste-

evidence

that

of

re-

cAMP

and

reported

systems general

the

to

AMP.

for

phenomenon

binding.

whether

effect

this

of

the

linked

ribose

via

a

spacer

6-carbon

progesterone-receptor

salt be ATP

with

a majority resin

or

the

and

free

reversible, over

main

other

complexes

(NH^J^SO^ of

the

could

of

this

eluted

(54,55).

in

nature,

nucleoside

features

and

com-

be

ligand ionic

to

triinterac-

I.

avian

interaction

indicating

to

to

covalently

The

Table

as

(Moud-

ATP

was

column,

for

of

progesterone

fractionated

to

laboratory

addition

a direct

affinity

high

found

in

Toft's

that

ATP

When were

preference

cleotide-receptor other

was

summarized

Originally

by

containing

a

its

ATP-Sep harose

with

Dr.

enhanced

attributed

resin.

over

in

metabolism.

cytosol

plexes

to

some

phosphorylation

investigations

through

passed

and

of

or

facilitate

receptors

showed

These

with

provides

a result

crude

added

itself to

lacking.

of

and

protein

phosphorylated that

as

Interaction

gil

receptor be

information

binding is

the may

that (Table

progesterone

receptor,

has

now

observed

ATP

binding

II).

been

by

steroid

the in

ηu-

many

receptors

357 T a b l e I. Properties ftΤP-Seρharοse and 1.

Ionic

2.

Ligand

3.

Binding

4.

Selective

5.

Nature Specificity

Steroid-Receptor Rema ins Intact

Reproduced

II.

with

Interaction

Receptor Progesterone

Estrogen

Glucocorticοi d

Androgen Vi tami η- DJ

Steroid

permission

Receptor

studies were and

discovered

Chick, Rat Chick

(54,55),

the

chick the

1

with

ftTP-Sepharose

Reference Moudgil & Toft (54,55) L e a v i tt e t a l . ( 5 6 ) Moudgil et al. (47) M i l l e r & T o f t (46) Moudgil & Eessalu (58) K a t z e n e l l e n b o g e n et al. (59) Moudgil & Weekes (60) Moudgil & John (47) M c B l a i n e t al . ( 6 1 )

hen

M o u d g i l et al. (62) M u l d e r et al. (63) H a u s s l e r & Pike (64)

reference

Activation

utilized

that

from

reference

Receptors

Hen Rat Mouse

In

ATP-Sepharose

of

from

Oviduct Liver Mammary Gland Oviduct Prostate Intestine

of

previous

permission

Animal C h i c k , hen Hamster Rat Ra t

with

preparations

Complex

Tissue Oviduct Uterus Brain Uterus

Requirement

soon

Specificity

Reversible

7.

Reproduced

H i g h s a l t d i s r u p t s the interaction A T P is a n e c e s s a r y constituent ftTP is p r e f e r r e d o v e r other nucleotides Y i e l d s p u r i f i c a t i o n of 100 X - f o l d or h i g h e r

Divalent Cations Not Required

6.

Table

of the I n t e r a c t i o n Between Steroid-Hormone Receptors

for

1

ATP-Sepharose

(NH^^SO^-precipitated

to

study

the

progesterone

receptor

in

the

interaction receptor. freshly

Binding

receptor between It

was

prepared

cy-

358 tosol

had

1 i111e

activation could

occur

hormone shown that 0°C

step

had

the been

no

The

rat

retention

at

2 3°C

binding

the

for

eluted tion,

with the

1Μ

maximum

FRACTION

on

of

ATP

(Fig.

to

receptor

for

method

Cytosol

acetonide

binding

at

subsequent

enhanced

of

an

receptor-

(47).

However,

The

that

subsequently

receptor

ATP-Sepharose this

5

was

periods 1).

and

ATP-Sepharose

H]triamcinolone

time

resin

to

activation

ATP-Sepharose.

Employing

extent

NUMBER

[

various

adsorbed

KCl.

of

immobilized

with

affinity

quantitatively

ATP-Sepharose binding

glucocorticoid 3

incubated

no

to

to

liver

showed

for

before

requirement

binding

incubation

was

affinity

required

(46).

complex

for

or is

receptor

activated

receptor

and

be

could

receptor to

activa-

ATP-Sepharose

10

F I G . 1. E f f e c t of h e a t - a c t i ν a t i ο η on the b i n d i n g of glucocort i c o i d - r e c e ρ t o r , c o m ρ 1 e χ to A T P - S e p h a r o s e . Rat liver cytosol was incubated [ H ] t r i a m c i n o l o n e acetonide for 4 h at 0 ° C . Al i quots (0.5ml) containing [3H]triamcinolone acetonide-receptor c o m p l e x w e r e i n c u b a t e d a t 2 30 C f o r t i m e s s h o w n , c o o l e d o n ice and c h r o m a t o g r a p h e d on i d e n t i c a l 2- m 1 c o l u m n s of ATP-Sepharose. The c o l u m n s were w a s h e d with lOmM Tris-HCl b u f f e r , 2 0% g l y c e r o l , l O m M K C l , pH 8 a n d the a d s o r b e d c o m p l e x e s w e r e rec o v e r e d w i t h the s a m e b u f f e r c o n t a i n i n g IM K C l . Taken with permission from ref. 47.

359 ranged

between

binding heat the

of

50-70?

of

the

receptor-hormone

treatment)

ATP-Sepharose

uptake

cytosol

has

of

the

receptor

therefore,

has

application

activation

of

steroid

conveniently

used

hormone in

shown

(46).

assays

by

The salt

to

or

parallel

ATP-Sepharose

in

the

receptors.

batch

receptor.

(activated been

atography,

be

nuclear

to

total

complexes

The

for

chrom-

measurement resin

multiple

of

can

also

sample

mea-

surements .

Inhibitors

The

of

ATP

functional

tablished

with

chapter.

The

step to

in

the

provide

tion,

the

pounds

mechanism

have

and

sodium

of

famycin to

to

latter

known

binding

later an

the

have

with

action.

been

do

nuclei,

an

effort

interac-

These

with com-

AF/013

5'-phosphate

not the

this

Several

rifamycin

block

in

interfere

pyridoxal

agents but

isolated

to

(1,65,66).

(ATA),

These

In

sought.

appear

es-

important

receptor-ATP

(o-phe.),

process,

action

of

is

with

have

an the

also

o-phenanthroline,

receptors

antibiotic

inhibitory that

may

been

of

DNA

demonstrated acid

effects

also

be

interfere cell-free

in-

DNA-cellulose

and

blocks

a metal

on

is the

a

that

and to

be

has

RNA

been

of

Rishown

polymerases;

meta1 - contaiηiηg

triphenylmethane

activities

binding

chelator,

meta11 ο proteiηs .

derivative

activities

Aurintricarboxylic

transferases,

discussed

ATP-Sepharose

molybdate.

been

represent

which

acid

not

be

to

process

identified

has

may

hormone

definition

steroids

AF/013

enzymes. with

steroid

binding

(19,65-70).

that

interfere

the

of

receptor

inhibitory

suggests

will

o-phenanthroline

steroid

teraction

ATP

binding

aurintricarboxylic

the

of

and

this

binding

ATP-Sepharose

The

of

been

include

(Rif.), (PLP)

certainty nucleotide

additional

receptor

with

significance

inhibitors

compounds

binding

of

steroid

dye,

nucleotidyl receptors

to

360 ATP-Sepharose to

block

tween cule

ATP

the

inhibitor

which

receptor these

(58,60,71,72). binding

may

be

due and

to

for

Although

are

not

characterization

and

identification

the

steroid

of

these

inhibitors

roid-receptor liver

receptors are

systems

glucocorticoid

should are

the

of

a Schiff

appears base

the

receptor

DNA

binding

modes

present,

prove

applicable

and

and exact

inhibitors

on

at

of

l y s i n e of

ATP the

clear

5'-phosphate

formation

a critical

necessary

(65,69,70).

Pyridoxal the

of

their

in

various

acceptor

valuable.

The

generally

summarized

in

to

moleof

action use

III

the

of the sites

effects

different

Table

be-

for

sterat

receptor.

Table I I I . E f f e c t s o f V a r i o u s I n h i b i t o r s on t h e B i n d i n g o f [ ^H ] d e x a m e t h a s o n e - R e c e p t o r C o m p l e x t o D N A - C e l 1 u l o s e a n d ATP-Sepharose C o n c e n t r a t i o n Ί S p e c i f i c LJH J de x a m e t h a s o n e b i n d i n g Compo und Used DNA-Cel1ulose ATP - S e p h a r o s e Control (no inhibitor)

—

100

100

NaV03

lOmM

75

74

Na2W04

lOmM

3

0

Na2Mo04

1 OmM

0

0

5mM

17

60

14

34

0

0

0

7

0

0

PL Ρ ATA Heparin o-Phe . Ri f .

O.OlmM 30 0u g/m1 3m Μ 1 75ug/ml

F r e s h l y e x c i s e d l i v e r s from b i l a t e r a l l y a d r e n a l e c t o m i z e d adult m a l e r a t s w e r e h o m o g e n i z e d a t 0°C i n 2 v o l u m e s ( V / w ) o f T r i s b u f f e r ( 2 0 m Μ T r i s - H C l , 12mM t h i o g l y c e r o l , 10 - g l y c e r o l and 0.3mM ρ h e n y 1 m e t h y 1 s u 1 f o n y 1 f l u o r i d e , pH 7 . 5 ) . The h o m o g e n a t e was c l e a r e d by c e n t r i f u g a t i o n a t 1 5 0 , 0 0 0 x g f o r 60 m i n . The r e s u l t i n g c y t o s o l was c o m p l e x e d w i t h 2 0 η Μ [ J H ] d e x a m e t h a s o n e f o r 2 h at 0°C. The e x c e s s s t e r o i d was r e m o v e d by t r e a t m e n t w i t h a c h a r c o a l s u s p e n s i o n and a l i q u o t s o f c y t o s o l w e r e i n c u b a t e d w i t h t h e c o m p o u n d s l i s t e d i n t h e t a b l e and w e r e h e a t activated (23°C, 1 h). P o r t i o n s ( 0 . 5 m l ) were u s e d to m e a s u r e D N A - c e l l u l o s e and A T P - S e p h a r o s e b i n d i n g . Taken w i t h p e r m i s s i o n from r e f e r e n c e 66.

361 Effects

While

of

the

obscure, of

ATP

on

the

functional it

is

activated

clear

steroid

activation

significance that

receptors.

of

steroid

hormone

when

is

free

solution

mobilized

form

and

to

the

effect

solic

gain

in

of

insight

the

It

than

into

nucleotide

on

tissue

cytosols

containing complexes

for

the

quired

the

at

0-4°C,

properties

of

Sepharose

( 41 , 4 3 - 4 5 , 7 3 ) ( F i g .

ATP-dependent nuclear

tions

in

and

in

the

its

of

effects ATP

of

steroid

modifier sive

is

shown

method

vitro.

ventional activation and

is

imize

the

receptor

is

Figure

used

out

for

at

structural

components.

5.

studying

4).

cyto-

with

prepared

as

shown

and

lOmM

complexes

ATP

ac-

by

their

and

receptor

ATP-

found of

more

may

reported

conditions

for

be

additive

a

biological

A

comprehen-

ATP

com-

offers activation

over

the

activation. less

liver

of

conΑΤ Ρ-

cumbersome

(0-4°C)

metabolism rat

3) the

receptor

by

advantages

receptor

and

be

receptor

quantitative,

assay

(Fig.

to

to

altera-

heat-transforma-

steroid

of

forms

by

Furthermore,

activation. of

several

alterations

Initially

were

process

achieving

mild

ATP,

progesterone-

Transformation

the

has

the

of

freshly

DEAE-Sephacel

nucleotide

activation

convenient,

carried the

in

procedure

modes

on

(Fig.

receptors,

of

The

receptor

of

of

When

im-

assumption

accompanied

ATP

studying

above

function(s)

cytosol was

process

process

another

of

the

for

the

DNA-cellulose

and

the

an

receptor

heat

of

in

present

incubated

accelerates

scheme

plexes

in

of

ATP

is

2).

sedimentation

(43).

Since

of

different by

estrogen-,

counterparts

mobility

rate

property

that

activation

nuclei,

transformation

binding

transforming

tion

isolated

remained

a

influenced

on

were

activated

binding

their

it

has is

possible

are

steroid-receptor

increased

The

to

binding

investigated.

glucocorticoid-receptor

receptors

binding

presumed

the

was

min

is

Based

the

receptor

40-60

ATP

when

steroid

target

of

receptors

(ATP-Sepharose).

more

steroid

ATP-Sepharose

aspects it

of

which the

min-

cytosol

glucocorticoid

362

ATP

(mM)at

0°C

FIG. 2 . A c t i v a t i o n of s t e r o i d - r e c e p t o r c o m p l e x e s by A T P . A s e r i e s of a l i q u o t s c o n t a i n i n g steroid-receptor complexes were i n c u b a t e d at 0°C for 4 0 - 6 0 min w i t h lOmM T r i s - H C l buffer containing different concentrations of ATP (pH 8 ) . The samples were subsequently charcoal-treated and i n c u b a t e d w i t h isolated nuclei from rat liver (GR), chick o v i d u c t (PR), and rat uterus (ER). The n u c l e a r s u s p e n s i o n s were w a s h e d with 1ow-saltbuffer and the n u c l e a r - b o u n d c o m p l e x e s were e x t r a c t e d w i t h high-salt Tris-HCl b u f f e r ( 1 Μ K C l ) , pH 7 . 4 . G R , g l u c o c o r t i c o i d receptor; PR, progesterone receptor; ER, estradiol receptor.

F I G . 3. E f f e c t of A T P - t r e a t m e η t on the r e s o l u t i o n of c y t o s o l glucocortic o i d - re c e ρ to r c o m p l e x e s on OEAE-Sephacel. Rat liver cytosol containing [3H]triamcinolone acetonide-receptor complexes was incubated f o r 1 h a t 0° C w i t h 1 O m K A T P (pH 8) or w i t h 5mM Na2W04. Samples (0.5ml) were chromatographed over identical 5m 1 D Ε A Ε Sephacel columns pre-equilibrated with buffer containing lOmM Tris-HCl, 12mM t h i o g l y c e r o l , 20':· g l y c e r o l , l O m M KCl, pH 7 . The r e s i n - b o u n d r e c e p t o r was e l u t e d by a s t e p - w i s e increase in ionic strength. T w e n t y 1-ml fractions were collected each with above buffer containing 0 . 1 5 M or 0 . 5 K KCl. Small aliquots (0.5ml) were used for the m e a s u r e m e n t of radioactivity.

363

5 - 2 0 Ά sucrose y.control

gradients »—t'ATP

υ ι ο δ >ϊ 0:

20 FRACTION

40 NUMBER

FIG. 4. E f f e c t o f A T P - t r e a t m e η t on t h e r a t e o f s e d i m e n t a t i o n of s t e r o i d - r e c e p t o r c o m p l e x e s from t a r g e t tissue cytosols. The s t e r o i d - r e c e p t o r c o m p l e x e s p r e p a r e d from hen o v i d u c t (PR), rat u t e r u s (ER) and r a t l i v e r (GR) c y t o s o l s w e r e i n c u b a t e d at 0 ° C f o r 4 0 - 60 m i n w i t h l O m M T r i s - H C l b u f f e r , pH 8 (0 0) or T r i s - b u f f e r c o n t a i n i n g l O m H A T P (I 1). P o r t i o n s ( 0 . 2 m l ) of the a b o v e p r e p a r a t i o n s were l a y e r e d over 5-20' l i n e a r sucrose gradients. The g r a d i e n t s were c e n t r i f u g e d at 1 5 0 , 0 0 0 x g (SW r o t o r ) f o r 16 h ( P R , E R ) o r a t 2 7 0 , 0 0 0 x g ( v e r t i c a l r o t o r ) f o r 2 h (GR). F r a c t i o n s w e r e c o l l e c t e d by p i e r c i n g the b o t t o m o f the g r a d i e n t tubes and a l i q u o t s w e r e used for radioactivity mea s u r e m e n ts.

receptor,

activation

demonstrated

in

many

of

steroid

receptor

receptors

systems

by

(Table

ATP IV).

has

now

been

364

Increased

binding

to:

ATP-Sepharose DNA-Cellulose Phosphocellulose

Η

- R — * H R — » H R *

•

Isolated

nuclei

Slow •

Temperature

4 pH • Ionic

strength

Cytosol

Change

in sedimentation

Change

in

Altered

dilution

Gel

dissociation

mobility

ion-exchange

rate

size on resms

filtration

Phosphatase Phosphorylated ATP.

compounds

4°C

FIG. 5. A c t i v a t i o n o f s t e r o i d - r e c e p t o r c o m p l e x e s i_n v i t r o . A c o m p r e h e n s i v e scheme f o r s t u d y i n g a c t i v a t i o n o f steroid-recept o r c o m p l e x e s in c e l l - f r e e s y s t e m s . H, s t e r o i d hormone; R, r e c e p t o r ; HR, h o r m o n e - r e c e p t o r complex (no η a c t i ν a ted ) ; H R * , s t e r ο i d - h o r m ο η e - r e c e ρ t ο r complex (activated).

Table

Progesterone

A c t i v a t i o n o f S t e r o i d R e c e p t o r s by A T P C o n d i t i o n of S y s tem Activation References Rat liver 5 - 1 0 mM ATP » John & M o u d g i l (43) , 4 ° C , 4 0 min M o u d g i l & John ( 4 4 ) 2 - 4 mM A T P , Rat liver A n d r e a s e n (74 ) 0 . 1 Μ KCl , 0° C 1 0 mM A T P , Rat liver B a r n e t t e t al . ( 7 5 ) 1 5 ° C , 3 0 miη M C F - 7 e e l 1 s 10 mM A T P , 1 h C h o n g & L i ppman ( 7 6 ) 10 mM A T P , 0 ° C H o l b r o o k et al . ( 7 7 ) Rat thymus cells Rat u t e r u s 10 mM A T P , 4 ° c , M o u d g i 1 & E e s s a l u 4 0 min (45) M C F - 7 e e l 1 s 10 mM A T P , Nawata et a l . ( 7 8 , 0°C , 1 h 79) Moudgil et a l . (41) Hen o v i d u c t 10 mM ATP , 4 ° C , 40 min

Reproduced

with

Receptor Glucocort icοid

Estrogen

IV.

permission

from

reference

1

365 A number which sol

of

receptor

Sepharose) are

compounds

interfere

to an

pounds,

which

Table

have the

been

state.

in T a b l e is n o t

identified

ability

acceptor

binding

summarized

discussed

with

of A T P

(nuclei, Effects

V.

fully

The

to

over

the

DNA-cel1ulose

of

mode

some of

understood

of

the

and

these

action

at

years

transform

of

ATP-

inhibitors

these

present,

cytο-

has

com-

been

earlier.

V.

C h e m i c a l i n h i b i t o r s of the p r o c e s s r e c e p t o r a c t i v a t i o n by A T P .

Control PLP o - P h e n a n t h r o l i ne Sodium levamisole Sodium tungstate Sodium molybdate Rifamycin AF/013 Heparin ATA

progesterone-

[ 3 H ] R 5 0 2 0 - receptor complex b o u n d to A T P - S e p h a r o s e

Concentration mM (ug/ml )

Inhibitor

of

c!

100 84 83 84 59 1 9 11 0 0

- -

5 3 10 10 10 (175) ( 300 ) 0.02

F r e s h l y p r e p a r e d hen o v i d u c t c y t o s o l w a s c o m p l e x e d w i t h TD n~M [ 3 H ] R 5 0 2 0 for 2 h at 4 ° C . A s e r i e s of t u b e s w a s s e t up (in d u p l i c a t e ) in a final v o l u m e of 0.5 ml a n d c o n t a i n e d 0 . 1 5 ml of r e c e p t o r c o m p l e x , 20% g l y c e r o l , 10 m Μ A T P and d i f f e r e n t inh i b i t o r s at c o n c e n t r a t i o n s s h o w n in the t a b l e . The c o n t e n t s of t u b e s w e r e m i x e d a n d i n c u b a t e d for 1 h at 4°C . Following t h i s , 0.5ml D e x t r a n - c o a t e d c h a r c o a l s u s p e n s i o n w a s a d d e d to e a c h t u b e to r e m o v e f r e e n u c l e o t i d e s and i n h i b i t o r s . After c e n t r i f u g a t i o n at l O O O x g f o r 5 m i n , p o r t i o n s ( 0 . 7 m l ) of s u p e r n a t a n t w e r e u s e d to m e a s u r e the e x t e n t of r e c e p t o r a c t i v a t i o n by u s i n g A T P - S e p h a r o s e b a t c h a s s a y s . Taken with permission from r e f e r e n c e 41.

Significance receptors

Several receptor creases tor

of

lines

the

of

evidence

function. the

(44,80).

extent The

interaction

suggest

Addition of

between

steroid

of

a role

ATP

to

binding

sterοid-receρtοr

ATP

and

for

target

ATP

steroid

in

cell

steroid cytosol

by g l u c o c o r t i c o i d

complexes

are

in-

recep-

activated

366 upon can of

incubation also

ATP,

effects

be

tween

on

involve

the

receptors

have

demonstrated

dialdehyde, receptor.

Based

been

are

mediated

ceptors

via

is

not

by

which

activated

receptor

is

in

activated ATP

forms.

of

out

in

the

the

receptor

influence

on

the

latter

if

ATP

(Table

supbe-

II).

Addi-

site

on

McBlain

Toft

(81)

and

to

all,

of

of

site(s)

stewho

21 - 31 -

ATP,

avian

available

binding

is

ATP-binding

linked

not

view

progesterone

information, the on

may

process

interaction

derivative

other

Although

of

The

from of

8S

presence

both

favour

of

oviduct other

of

receptor.

the

knowledge

for

of

ATP the

it

effects steroid

re-

an

activated

receptor

'native'

metastable

structure,

easily

ceptor

disrupt

8S

is

steps

disrupted.

oligomeric

a

and

form

that (and

(5,82).

the

then

activa-

perhaps

of

consequence with-

The

nonac-

vitro

may

structure

the

readily.

molecule,

behaves ATP

which

more

direct

native

molecule

mean

receptor

hypothesis

hormones)

an

shift

receptor

oligomeric

into would

nonactivated

the

the

cytosol

receptor

favours

enzyme-catalyzed

the

This

ATP-Sepharose

progesterone

the

transforms ATP.

com-

ATP-Sep harose

and

oligomeric

and

and

nonactivated

to

resins

steroid of

s t e r o i d - r e c e ρ tor

However,

and

free

activated a

the

Binding

nonactivated

to

dissociation

transforms

understood.

bind

involvement

tivated

ATP

form

polyanion

chick

receptor of

may

present

tion

in

influences ATP

to

the

state

equilbrium

The

and

some,

completely

an

binds

of

aldehyde

nucleotide

requires

that

studies

(41).

analog

capacity

immobilized an

activation

(1,41,43-47,54,55,73).

mechanism

plex

this

that

This

demonstrating

irreversibly

on

proposed

its

of

an

8).

n o n - h y d r o 1iζ a b l e

triphosphate

direct.

and

from

that

be

be

reports

existence

come

can

has

to

receptors to

(pH a

binding

metabolism,

numerous

steroid clues

its

hormone

appears

the

ATP

1

5 -[@ , γ - i m i d o ]

ATP

roid

The

lOmM

substituting

the

by

tional

with

by

adenosine

activation

ported

0°C

of

possibly of

at

achieved

bind

as

with

(perhaps

by

a re-

367 competing ganized

In

in

recent

have of

for the

shown

steroid

forms

to

receptors,

as

well by

Auricchio

of

enzyme

effects

lated,

is

the

hormone

is

role

as

is

becoming

(86)

and

sults that

of

Fig. ATP.

of

of

7).

mouse

of

To

with

prepare

cytosol.

[

uterine

or

the A in

particular

receptor

steroid The

for

studies

40

has

from

to

be

the

are model for

the phos-

unrerepresteroid on

the

cytosol

been

this

and

good

(84,85),

laboratory

a

the

sub-

significance

is of

a

estrogen

tissue

[ ^H ] d e χ a m e t h a s o n e

have

Reshown

phosphoprotein

glucocorticoid

liver

treated

of

Phos-

demonstrated.

receptor

tissue,

was

are

established.

phosphorylation

hepatic

nM

receptors

progesterone

(87,88)

Pjorthophosphate

with

to

emphasis

physiologic

remains

glucocorticoid

The

es-

receptors

that

perform

cell

the

Whether

processes

are (75,

purification

related

target

steroid

receptors

restore

hypothetical a

the

processes

cytosol.

are

two

to

re-

binding

partial

ATP

molybdate

cytosol

that

steroid

reported

chapter

6 with

steroid

the i n t a c t 32

complexed

of

suggested

utilizes

this

receptors

liver

or-

inactivation

ATP-Sepharose

been

events

in

evident

cubated

0°C .

or

present.

of

preliminary

in

transformation

have

phosphorylation.

rat

(88)(Fig.

and

at

glucocorticoid

the

receptor

of in

for

of

phosphorylation phorylation

sodium

transformation

receptor

clear

shown

for

like

apparently

sequence

Phosphorylation

It

inhibitors

also

a 1 . (84)

the

subunits

temperature-dependent block

has

the

et

speculated

strates

It

ability

of

not

senting

and

discussed

phorylation

the

DNA-cellulose

which

trogen-binding ATP

keep

ρ hosρhory1 atiοn-deρ hosρhory1 ation

83). an

to

structure)(82).

inhibit

nuclear,

influenced

required

phosphatase to

(19,65,75).

binding

bonds

native

years,

been

ceptor

certain

with

mince was 40:'

mesylate

was

iη-

used

to

( ΝΗ^ ) ^S04 for

3

h

at

368

FIG. 6. A hypothetical model o f g l u c o c o r t i c o i d a c t i o n in a t a r g e t cell showing possible i n v o l v e m e n t of ATP. G , glucoc o r t i c o i d ; R, r e c e p t o r ; G R c , g l u c o c o r t i c o i d - r e c e p t o r complex. T a k e n w i t h p e r m i s s i o n f r o m r e f . 7 5.

The

glucocorticoid-receptor

graphy mi de tor

over

gel

revealed

which

DEAE-Sephacel

{7%)

the on

presence the

Many

questions

about

roid

receptors

remain

tors,

metabolism

under

and

other

of

of

the

of

specific

may

circumstances

added of

Whereas,

result the

by

in

chromato-

SDS-po1yacryla purified

P-containing

of

function

unanswered. ATP

purified

partially 32

several

availability a

was

DNA-cel1ulose.

electrophoresis

depended

ditions,

complex

ΜgC1^ ATP

binding

effects

nucleotide

bands

(Fig.

under

recep-

7 ). by

certain on

may

the be

stecon-

recep-

meta-

369 1 0 % SOS-PAGE of Photphorylottd

GRc

FIG. 7. P h o s p h o r y l a t i o n of rat l i v e r g l u c o c o r t i c o i d receptor by i n c u b a t i o n o f [ 3 2 p ] _ 0 r t h o p h o s p h a t e w i t h l i v e r t i s s u e mince. The l i v e r m i n c e was s u s p e n d e d in a p h o s p h a t e - f r e e J o k l i k medium p l u s ΜgC12 and i n c u b a t e d f o r 3 - 4 h a t 3 7 ° C . The m i n c e was w a s h e d w i t h c o l d h o m o g e n i z a t i o n b u f f e r c o n t a i n i n g 2 5mM N a F a n d homogenized. The c y t o s o l was f r a c t i o n a t e d w i t h s a t u r a t e d ammonium s u l f a t e , d e s a l t e d on S e p h a d e x G - 7 5 c o l u m n and was incub a t e d w i t h 5 0 n M u n l a b e l e d d e χ a m e t h a s ο ηe m e s y l a t e f o r 3 h . Under i d e n t i c a l c o n d i t i o n s , l i v e r mince was i n c u b a t e d w i t h unl a b e l e d p h o s p h a t e and the r e c e p t o r was f r a c t i o n a t e d and d e s a l t e d as a b o v e b u t i n c u b a t e d w i t h 50nM [ ^ H j d e x a m e t h a s o n e mesy l a t e for 3 h at 0°C. The p r e p a r a t i o n s were t h e n s u b j e c t e d to SDS-electrophoresis a n a l y s i s on 10'. a c r y l a m i d e g e l s . Gels w e r e s l i c e d i n t o 2 - 3mm p i e c e s , d i g e s t e d w i t h p r o t o s o l and were mixed with s c i n t i l l a t i o n c o c k t a i l (67:3:30; toluene:protosol: T r i t o n X - 10 0 ) c o n t a i n i n g 0 . 5 g O m n i f l u o r . Phosphorylation performed i n p r e s e n c e (0 0) and a b s e n c e ( I ·) of MgCl?.

bolized formed tial

of

by or

the

receptor.

reported

steroid

Although

suggesting

receptors

has

these been

no

studied

views, explored

have

the

been

enzymic

recently.

perpoten-

370

116 Κ

97 Κ

67 Κ

43 Κ

29 Κ

2

3

Ια

2α

3α

FIG. 8. P h o s p h o r y l a t i o n o f c a l f thymus hi s t o n e s u s i n g purif i e d GR a s a p r o t e i n k i n a s e . P u r i f i e d GR w a s d i a l y z e d a n d i n c u b a t e d w i t h h i s t o n e s , Μ g 2 + a n d / o r C a 2 + and [ γ - 3 2 Ρ ] Α Τ Ρ . After c o m p l e t i o n o f t h e r e a c t i o n , t h e TCA p r e c i p i t a t e d s a m p l e s w e r e a p p l i e d t o 10:« P o l y a c r y l a m i d e s l a b g e l s a n d electrophoresis was p e r f o r m e d as d e t a i l e d i n r e f . 9 0 . Far l e f t , m o l . wt. mark e r s ; l a n e 1 , h i s t o n e s w i t h 5mM Μ g C 1 2 ; l a n e 2 , h i s t o n e s with 5mM C a C 1 2 ; l a n e 3 , h i s t o n e s a n d 5m Μ Μ g C 1 2 + 5 0 n M u n l a b e l e d T A . L a n e s l a - 3 a a r e an a u t o r a d i o g r a p h o f l a n e s 1 - 3 . Arrows indic a t e the p o s i t i o n s of r e c e p t o r and p h o s p h o r y l a t e d histones. Taken w i t h p e r m i s s i o n from r e f . 90.

371 Protei η kinase The

postulation

tein

kinase

chick

nase

oviduct

near

rat

in

ions.

The

porated

c h a i n 2 k+ i n a s e the

and

reaction

tein (H3,

H4)

by

Our

of

Miller

of

histones

The

mixture purified

results et

by

steroid an

tance gene

receptors

expression

phorylation

of

teration

the

in

processes

which

ceptor

of

and be

or

who

by

by

be

20893.

the

of

acknowledged.

peptides kinon-

purified

also

the

to

is

may

proteins

other

work

all

be

of

of

thymus

is

to

pro-

hi s t o n e s

shown

in

Figure

observations

phosphorylation

kinase of

activity

at

this

critical

the

well be

by

presence ligand

extent

assessed

in

which

of

(89).

steroid

have

been

phos-

the

al-

Other

phosphorylation

include

time,

recognized,

genes

of

impor-

regulation

involved

specific

influenced

the

light

receptor.

Since

hormones of

in

calf

reported

may

myosin

steroid

the

of

protein

action. proteins

of

preliminary

accurately

receptor, and

proThese

protein

gizzard

receptor

the

receptors

related

This

Reading

be

of

Acknowledgements : fully

increase

of

transcription

ATP

to

with

steroid

might

was

kinase

addition

glucocorticoid

chromatin

of

muscle

The

have

hormone

other

activation affected

well

cannot

steroid

dalton

exhibited

turkey

glucocorticoid

role

(89).

molybdate-stabilized

Phosphorylation

significance

enzymatic for

skeletal

purified

possess

al.

90,000

receptor

hi s t o n e s ,

appeared

(91)

itself et

with d i f f e r e n t protein sub32 [γP]ATP and d i v a l e n t c a t 32 from [ γ P ] A T P was i n c o r -

50 μ Η

(90).

agree

al.

and

receptor

phosphate

thymus

ions

may

Garcia

incubated of

and 2+ rabbit

biological

but

was

phosphorylation.

8.

110,000

laboratory,

presence

Ca

by

glucocorticoid

and

calf

receptors

receptor

provided

that

radioactive

into

Mg

a steroid

this

liver

the

steroid

progesterone In

homogeneity

strates

of

was

reported

activity.

activated

of

that

activity

investigators of

activity

of

re-

binding shown

to

nucleotides.

was

manuscript

supported by

Dr.

Ν.

by

N.I.H.

Eliezer

Grant is

AM-

thank-

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Internatl.

STEROID HORMONE RECEPTOR DYNAMICS:

THE KEY TO TISSUE RESPONSIVENESS

Thomas G. Muldoon Department of Endocrinology Medical College of Georgia Augusta, Georgia 30912

Introduction By d e f i n i t i o n , s t e r o i d hormone receptors are the s u b c e l l u l a r components which, to the best of our current knowledge, single-handedly dictate q u a l i t a t i v e responsiveness of the cell to the respective hormone.

Tissue s p e c i -

f i c i t y of receptor l o c a l i z a t i o n to responsive c e l l s was e s t a b l i s h e d in the pioneering studies from Jensen's laboratory (1,2) and confirmed by an extensive s e r i e s of autoradiographic analyses (3).

Impetus into

investiga-

tions of receptor nature and function was afforded by c r i t i c a l

demonstra-

tions of the biochemistry of these molecules by a number of groups

(4-7),

defining t h e i r proteinaceous nature and various physicochemical means of detecting and quantifying them.

This early work set the stage for extra-

polation from the q u a l i t a t i v e role of receptors as effectors of i n t r a c e l l u l a r retention to q u a n t i t a t i v e assessment of t h e i r function as determinants of s p e c i f i c hormonal responsiveness.

A number of the chapters in t h i s

book are devoted to various aspects of such s t u d i e s ; the aim of t h i s presentation i s to focus on several areas of receptor regulation and l o c a l i z a tion which have represented a central thrust of the research in our laboratory within recent y e a r s .

In terms of i n t r a c e l l u l a r receptor d i s t r i b u t i o n ,

our studies of the s i g n i f i c a n c e of microsomal components w i l l be presented. Receptor level and f u n c t i o n a l i t y in r e l a t i o n to d i f f e r e n t i a l

gene expres-

sion w i l l be addressed by summarizing our studies on androgen receptors of the rat ventral prostate.

Regulation of a s p e c i f i c sub-population of s t e -

roid hormone receptors within a responsive t i s s u e w i l l be exemplified by addressing our observations in the estrogen-LHRH-LH response system.

Fi-

n a l l y , the complex regulation of the estrogen and progesterone receptors

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

378 by both steroidal

and proteinaceous hormones in mammary tissue will be pre-

sented as representative of the diverse and divers ways in which

respon-

siveness is subservient to regulators of steroid hormone receptor

Intracellular Receptor

activity.

Distribution

The steroid receptor literature is replete with measurements of specific binding activity in cytoplasmic and nuclear compartments of hormonallyresponsive cells.

This approach has been useful

toward understanding

tions and interactions of assorted natural and synthetic hormonal and for elucidating the gross features of the steroid-receptor

complex

association with chromatinic components of the nucleus, generally dered to be a critical

element of the mechanism whereby genetic

is altered by the hormone. several

laboratories

consi-

expression

Of recent vintage are a series of studies

(8-11) which present a strong case for

from

intranuclear

localization of most, if not all, steroid hormone receptors within cells.

ac-

agents,

intact

These findings indicate that receptors designated as cytoplasmic

are actually those which are not strongly bound to chromatin and are leached out into the cytosolic fraction under homogenization and ultracentrifugational

conditions.

manner of visualizing

It is important to note that this change in our

intracellular receptor distribution does not compro-

mise the validity of the multitude of studies performed to determine tionships between receptor binding and functional

rela-

response.

In an analysis of subcellular distribution of estradiol

in castrate rat

uterine cells at intervals after administration of radiolabeled estradiol (12), we observed that nuclear estrogen receptor content diminished

sharply

between 3-5 hours without a concomitant rise in cytoplasmic receptor content (Figure 1); this was the first demonstration in a normal

tissue of a

phenomenon subsequently to be referred to as receptor "processing"

(13).

Strikingly, we were able to show that the complexes lost from the nucleus at this time became associated with the microsomal

fraction of the cell.

Between 5-10 hours after estradiol

microsome-associated

complexes re-entered the nucleus

exposure, these

(Figure 1), leading to a second nuclear

379 peak which was also observed later in mouse uterus (14,15).

The

transient

residence of the nuclear receptors on the microsomes was unexpected and suggested a role for the vacuolar membrane system as a conduit, shuttling steroid-receptor complexes to and from nuclear sites; this phenomenon is clearly distinct from the microsomal-associated receptor forms described by Jungblut and his co-workers

(16-18), which appear to be biosynthetically

developing species of the mature receptor molecule.

FIGURE 1. Subcellular distribution of estradiol in uteri of 2 - w e e k s 1 - o v a riectomized rats following estradiol injection. Animals were administered 0.25 uCi (1.25 pg) of estradiol at time zero and killed in groups at the specified intervals. The designated fractions were separated by differential centrifugation of the uterine homogenate. The data represent compartmental binding as a function of total specifically-bound estradiol recovered at each time point.

In a separate series of experiments, we obtained further evidence for a functional

role of the microsomes in receptor-mediated events.

of 4-mercuriestradiol

Assessment

as an affinity-labeling agent for the estrogen re-

ceptor revealed that this compound was possessed of robust inherent estrogenicity, but no detectable interaction with or effects upon the nuclear genetic material

of the cell

(19,20).

This seemed to defy classical

con-

cepts of the mechanism of steroid hormone action, but in fact only strengthened a growing body of evidence that extranuclear sites of action, particularly those areas associated with translational

processes

involved in the response of the cell to Steroid hormones. tigated the subcellular distribution of radiolabeled

(21-26), are

When we

inves-

4-mercuriestradiol

following administration in vivo (20), we observed a rapid, elevated and prolonged association of this compound with the microsomes, relative to

380

the i n t e r a c t i o n of e s t r a d i o l with t h i s f r a c t i o n (Figure 2; compare with the estradiol data of Figure 1).

The slow and gradual accumulation of small

amounts of the mercury d e r i v a t i v e within nuclei i s a l s o in contrast to the pattern of e s t r a d i o l uptake by nuclei and r e f l e c t s the lack of s p e c i f i c teractions in t h i s

in-

organelle.

FIGURE 2. S u b c e l l u l a r d i s t r i b u t i o n of 4-mercuriestradiol in uteri of 2weeks' ovariectomized rats following 4-mercuriestradiol i n j e c t i o n . Animals received 1.0 uCi of [ 3 H ] 4 - m e r c u r i e s t r a d i o l i n t r a l u m i n a l l y at time zero. Groups were k i l l e d at the i n t e r v a l s shown and the s u b c e l l u l a r d i s t r i b u t i o n of s p e c i f i c a l l y - b o u n d s t e r o i d in each f r a c t i o n was determined. Results are expressed as the percentage of total recovery in each f r a c t i o n . At longer i n t e r v a l s after introduction of the mercury d e r i v a t i v e , we have found that nuclear l e v e l s decline concomitantly with a second increase in microsomal content.

Schematically, the i n t e r a c t i o n s are described in

Figure 3. In recent i n v e s t i g a t i o n s , we have quantified a population of microsomal estrogen receptors which are d i s t i n c t from the cytosol receptors and can be s o l u b i l i z e d in hypotonic buffer.

This l a t t e r property d i s t i n g u i s h e s

these binders from c a l f uterine microsomal receptors described by Parikh et al_. (27), which are not r e a d i l y s o l u b i l i z e d .

The concentration of these

binding s i t e s fluctuates in response to estrogen treatment and at various i n t e r v a l s of the estrous cycle in much the same fashion as do cytosol ceptors.

re-

A s a l i e n t difference from the cytosol form of the receptor i s the

i n a b i l i t y of the microsomal receptor complexes to transform upon heating to a moiety which i s capable of t r a n s l o c a t i n g into i s o l a t e d n u c l e i .

As

shown in Figure 4, conditions which are conducive to transformation of ÖS

381 cytosol receptors to the 5S nuclear form simply lead to d i s s o c i a t i o n of the more l a b i l e microsomal e s t r a d i o l - r e c e p t o r complexes.

FIGURE 3. A mechanism of action c o n s i s t e n t with the a c t i v i t y of 4-mercurie s t r a d i o l (HgE). I n i t i a l i n t e r a c t i o n with the estrogen receptor causes transformation to a 5S complex (HgER 1 ) which accumulates r a p i d l y and extens i v e l y in the microsomes. Small amounts slowly enter the nucleus, but do not interact with chromatin in a s p e c i f i c manner. These complexes are subsequently found in a s s o c i a t i o n with the microsomes.

FIGURE 4. Failure of microsomal estrogen receptors to undergo transformation to a nuclear form. Extracted microsomal receptors were incubated with [ 3 H ] e s t r a d i o l for 2 hrs at 4°C in the presence ( — ) or absence ( · - · ; o-o) of excess unlabeled e s t r a d i o l . One sample (o-o) was further incubated for 30 min at 24°C, and a sample of uterine cytosol (Δ-Δ) was treated i d e n t i c a l l y . Sucrose gradient c e n t r i f u g a t i o n a l a n a l y s i s (15.5 h r s , 350,000 χ g) was performed with l l x C-labeled BSA as an external marker (4.4S, arrow). Note difference in scale between c y t o s o l i c and microsomal samples. An i n t r i g u i n g feature of the microsomal receptor system i s that detachment of the receptors from the membranes by s o l u b i l i z a t i o n imparts to the receptor-exhausted microsomes a newly-acquired acceptor c a p a b i l i t y for cytosol

382 estradiol-receptor complexes.

As shown in Table 1, the capacity of this

acceptor activity parallels rather closely the level of depleted microsomal receptors.

TABLE 1. Acceptor capability of microsomes for cytosol removal of microsomal estrophiles

Group

receptors

following

Concentration of specific binding sites (fmoles/mg protein)

KA (lO^ir1)

Experiment I Control Exhausted Reconstituted

1.31 4.12 1.01

126.7 16.9 156.8

2.04 2.01 2.04

111.5 37.3 87.4

3.10 2.16 3.14

114.7 63.8 132.9

Experiment II Control Exhausted Reconstituted Experiment III Control Exhausted Reconsti tuted

Uterine microsomes were prepared and suspended in buffer. One-third was taken to measure the Control binding level by saturation analysis. The remainder was extracted into steroid-free buffer and the pellet, representing Exhausted microsomes, was re-isolated by centrifugation. Half of these resuspended pellets were submitted to saturation analyses, and the other half was mixed with either an equivalent volume (Experiment I), 3 volumes (Experiment II) or 4 volumes (Experiment III) of original cytosol; these samples represented the Reconstituted microsomal preparations. Following mixing, the cytosol-microsome samples were submitted to saturation binding analysis, using the pelleted, resuspended material as source.

The perinuclear situation of microsomal

components suggests that they may

be included in the newly-described intact cell the vast majority of cellular receptors.

"nuclear" localization of

Our findings indicate a function-

al role for these very active protein-synthesizing organelles as both primary sites of steroid hormone action (through receptor interactions) and as regulators of access of hormone-receptor complexes to functional sites (through their role as acceptors for these complexes).

nuclear

383 Receptors and Differential

Gene

Expression

Repercussions of the eruption of molecular genetics in recent years been felt in the area of steroid hormone action. transcriptional

have

Regulation of specific

events in the chick oviduct by estrogen and

progesterone

(28-30) was exploited as a working system for investigation of hormonal control of gene expression, and progress in this area was swift and revealing.

Work has advanced to the stage where hormonal

loci of action are be-

ginning to be mapped to specific DNA sequences in the vicinity of genes (for recent work, see 31), but the mechanism whereby the

inducible

interaction

between steroid-receptor complexes and chromatin components alters transcription is still not

specific

understood.

In recent studies performed in collaboration with Professor W.I.P. Mainwaring, we have undertaken an analysis of changes in androgen receptors of the castrate male rat ventral

prostate during the course of constant expo-

sure to testosterone by silastic implant.

Our observations have been com-

pared w i t h patterns of androgen-induced protein synthesis during this period.

We have presented our data in preliminary form (32).

tion in this model

Enzyme

system occurs in a distinct biphasic manner.

zymes involved in cell growth and normal

functioning

(including

glucose-6-phosphate dehydrogenase, acid phosphatase and alkaline

induc-

Those enaldolase, phospha-

tase) are maximally stimulated within 1.5 days of androgen exposure, and their levels are dramatically reduced by 2 days. replication

Enzymes involved in DNA

(thymidine kinase, thymidylate synthetase, DNA polymerase a

and DNA unwinding protein), on the other hand, are present in very small amounts at 2 days, but the levels rise synchronously to a peak at 3 days and then slowly recede. criptional

level.

Our studies indicate that control

The mechanism whereby nuclear acceptor activity

over the course of this constant hormonal differential

is at the trans-

stimulation, allowing

changes

temporally

expression of specific genes, will be the focus of our con-

tinuing investigations

into this

phenomenon.

In attempting to define a molecular basis for the observed effects on selective genetic triggering, we have studied ventral tor dynamics during an identical

prostate androgen recep-

testosterone-replacement regimen.

The

384

r e s u l t s (Figure 5) revealed two d i s t i n c t peaks of nuclear androgen receptor binding a c t i v i t y , mirroring the respective patterns of the two sets of enzymes induced. the t r a d i t i o n a l

The i n i t i a l

nuclear retention phase appeared to involve

s t e r o i d - s t i m u l a t e d a s s o c i a t i o n with s p e c i f i c nuclear chro-

matinic components, since i t was o p e r a t i o n a l l y concomitant with cytosol ceptor depletion.

re-

Extensive processing of these receptors followed t h e i r

nuclear accumulation.

At 2.5-3 days into testosterone treatment, the nu-

clear l e v e l s again began to r i s e , but, in t h i s case, the increase was not at the expense of cytosol receptors, which showed only moderate and gradual decreases in l e v e l s during t h i s period.

FIGURE 5. Fluctuations in ventral prostate androgen receptor l e v e l s lowing testosterone implantation into 7-day castrate r a t s . C y t o s o l i c and nuclear ( — ) receptor concentrations were measured as a function days (%-6) after implantation of a testosterone p e l l e t . The castrate vels (-T group) are presented for comparison.

fol( —) of le-

In a previous study (33), we found differences in the androgen-binding properties of the two i n t e r c o n v e r t i b l e forms of cytosol ventral prostate androgen receptors observed by Wilson and French (34,35) and not a t t r i b u t a b l e to endogenous protease a c t i v i t y .

The larger (8S on glycerol gradients) of

the two forms has a 10-fold higher a s s o c i a t i o n rate constant for 5a-dihydrotestosterone than does the smaller 4S form.

Thus, the r e c e p t i v i t y and

the responsiveness of the c e l l to androgen can be expected to be high when the 8S:4S r a t i o i s high.

Glycerol gradient analyses of the receptor forms

at selected i n t e r v a l s of androgen exposure in our model system are shown in Figure 6.

In the absence of androgen replacement, the cytosol

androgen

receptors of the castrate rat t i s s u e are predominantly 8S in nature,

indi-

cating that the c e l l s are h i g h l y responsive to androgen, as judged by t h e i r

385 receptiveness.

The distribution shifts during the first phase of nuclear

binding, such that the 4S form of cytosol processing wave of this event.

receptor is prevalent during the

Between days 2 and 3 of androgen

stimula-

tion (the interval during which the second nuclear surge is occurring), the form of the cytosol

receptor reverts back to the 8S and remains thus for

the duration of this second stage of response.

These findings have been

substantiated by measurements of association rate kinetics for the interaction between androgen and the cytosol

receptors from tissue at the selec-

ted intervals of stimulation.

AxUU Μ FIGURE 6. Glycerol gradient centrifugation of rat prostate cytosol from castrate animals subjected to testosterone replacement for various intervals (in days). Samples containing 6 mg protein/ml were incubated with [3H]mibolerone. Sedimentation patterns were developed in 10-35;, glycerol gradients at 365,000 χ g for 75 min in a vertical rotor. The top of the gradient is to the right of each figure. Bovine γ-globulin (6.6S) sedimented at fraction #14 and BSA (4.4S) at fraction #21 under these conditions .

The second wave of androgen-induced transcription of DNA-replicating

en-

zymes occurs as the .cells pass into the replicative phase of their cycles; this does not, however, begin to explain the mechanism whereby these genes are selectively activated at this time, as opposed to earlier intervals of identical

androgen stimulation

(particularly those times during which the

first wave of protein synthesis is occurring). between the two stages of this overall

The harmonious

synchrony

response and the changes in nature

and amount of androgen receptor activity suggest a functional

correlation

which should become clearer with our projected refinements of the experimental

protocol.

386 Direct Regulation of Nuclear

Receptors

In most instances where steroid hormone receptor regulation has been studied the regulator has been shown to initially alter the subcellular

distribu-

tion of the receptors, which is probably largely a reflection of a change in the affinity or extent of interaction of the receptor with genetic material

in the nucleus (for review, see 36).

Occasionally, however, a curious

situation is encountered wherein levels of nuclear receptor are altered in the absence of any accompanying effect on cytosol

receptor content.

An in-

teresting example is the action of progesterone on estrogen receptors of the hamster uterus.

In this system, progesterone selectively

depresses

nuclear estrogen receptor binding (37) by a mechanism which requires transcriptional

and translational

ted by acid phosphatase (39).

intact

circuitry (38), and appears to be media-

We have reported a similar effect in the rat

anterior pituitary, and have shown tissue specificity, since

progesterone

has no effect on the hypothalamic estrogen receptors of the same animals (40).

The mechanism appears to be different from that of the hamster

uter-

us since nuclear localization of the progesterone is not required even though cytosol

progesterone receptor does seem to be involved.

Along these lines, our curiosity was piqued by our observation that LHRH administration in vivo caused nuclear estrogen receptors of the rat anterior pituitary to increase w i t h o u t any concomitant decrease in cytosol receptor level.

estrogen

In contrast to the progesterone studies mentioned above,

this study involved the action of a peptide hormone known to interact with cell membrane receptors on a species of protein localized within the nucleus.

Conditions were established for investigating this phenomenon in

vitro, using incubation of dispersed anterior pituitary cells in the presence of LHRH (41), and we were able to show the effect on whole cells (Figure 7).

The highly active LHRH analog, (D-Ala 6 ,

des-Gly10)LHRH-N-

ethylamide (designated LHRH-A), had a greater intrinsic activity in elevating nuclear estrogen receptor than did LHRH, and was maximally at a 10-fold lower concentration

(Figure 8).

effective

TRH, on the other hand, was

totally devoid of such activity over a wide concentration range (Figure 9) (42).

387

FIGURE 7. Estrogen receptor l e v e l s following incubation of i n t a c t c e l l s with LHRH for 30 min. Whole a n t e r i o r p i t u i t a r y c e l l s were incubated with 1,5 or 10 ng of LHRH per p i t u i t a r y f o r 30 min at 37°C, and estrogen receptor binding was estimated in the nuclear and extranuclear compartments of the c e l l s . The receptor level observed in control groups of c e l l s was a r b i t r a r i l y assigned a value of zero, and the p o s i t i v e or negative differences in the l e v e l s observed in treated c e l l s was plotted at each dose l e v e l . Values represent t r i p l i c a t e determinations of the number of experiments shown in parentheses. A s t e r i s k : Ρ < 0.05. [Reprinted, with permission, from Singh and Muldoon ( 4 1 ) ] .

0 1

1 0

10

100

1000

Peplicle Hormone Level (p m o l e s ' p i l u i l a r y t

FIGURE 8. Response of e s t r a d i o l receptor to LHRH and LHRH-A in i n t a c t p i t u i t a r y c e l l s . Enzymatically dispersed p i t u i t a r y c e l l s from castrated, lowdose estrogen primed rats were incubated with increasing concentrations of either LHRH or LHRH-A for 30 min at 37°C, followed by estimation of cytosol (A), nuclear (B) and the sum total (C) receptors. Values represent 10-24 observations in 6-18 separate experiments. A s t e r i s k : Ρ < 0.05, compared with c o n t r o l . [Reprinted, with permission, from Singh and Muldoon ( 4 2 ) ] .

388

T R H (ρ moles/pituitary)

FIGURE 9. E f f e c t s of TRH on estrogen receptor populations in intact p i t u itary cells. Increase (+) or decrease ( - ) in femtomoles of c y t o s o l , nuc l e a r and total estrogen receptors per 10 p i t u i t a r y equivalents i s compared to control values (assigned value of z e r o ) , r e s u l t i n g from incubation of p i t u i t a r y c e l l s from castrated, low-dose estrogen primed rats with TRH f o r 30 min at 37°C. Each value i s a mean ± SEM of 3 separate experiments. A s t e r i s k : Ρ < 0.05, as compared with c o n t r o l . [Reprinted, with permission, from Singh and Muldoon ( 4 2 ) ] . Of s i g n i f i c a n t mechanistic importance was the observation that LHRH was capable of a l t e r i n g nuclear estrogen receptor when incubated with i s o l a t e d nuclei (Figure 10).

In attempting to explain t h i s unexpected f i n d i n g , we

are forced to entertain the p o s s i b i l i t y that i n t e r n a l i z a t i o n of LHRH precedes an i n t r a c e l l u l a r mechanism of d i r e c t action of t h i s hormone.

FIGURE 10. E f f e c t of LHRH on estrogen receptors of i s o l a t e d anterior p i t u i t a r y cytosol or n u c l e i . Cytosol and nuclei were prepared and incubated separately with 1-1000 ng of LHRH per p i t u i t a r y for 30 min at 37°C. E s t r o gen receptor a c t i v i t y was then determined in each f r a c t i o n . Values are means + SEM of (n) experiments. A s t e r i s k : Ρ < 0.05, compared with c o n t r o l s incubated in the absence of LHRH. [Reprinted, with permission, from Singh and Muldoon ( 4 1 ) ] .

389 The concept that i n t e r n a l i z a t i o n of peptide hormones represents e x c l u s i v e l y the c e l l u l a r mechanism for degradative elimination of the hormone (43,44) has been questioned (45) on the b a s i s of a number of studies in which i n tranuclear l o c a l i z a t i o n of protein hormones has been observed at e x t r a lysosomal l o c i .

A p o s s i b l e d i r e c t action of LHRH on cell nuclei i s brought

into the realm of f e a s i b i l i t y by the report of s p e c i f i c r e c e p t o r - l i k e binding s i t e s for LHRH on nuclear membranes (46). In considering the mechanism whereby LHRH enhances nuclear estrogen receptor a c t i v i t y , i t therefore becomes important to determine whether cAMP functions as a mediator of t h i s a c t i o n , under the presumption that such mediation should not be required i f the action of the hormone i s indeed directly intracellular.

Of great s i g n i f i c a n c e to such studies i s the ob-

servation that cAMP i s not involved in the mechanism by which LHRH induces LH release from the a n t e r i o r p i t u i t a r y (47-50), in s p i t e of the fact that LHRH does cause increased i n t r a c e l l u l a r cAMP accumulation (51).

In Figure

11 i s presented evidence that LHRH i s capable, at 100 pmoles/pituitary, of s i g n i f i c a n t l y elevating cAMP l e v e l s in a n t e r i o r p i t u i t a r y c e l l s .

The level

of LHRH required was an order of magnitude higher than that which causes maximal nuclear estrogen receptor s t i m u l a t i o n .

The action of the above-

mentioned hyperactive analog of LHRH was l e s s pronounced than that of LHRH, in contrast to the opposite pattern in stimulating nuclear estrogen receptor (see Figure 8).

RELEASING HORMONE CONCENTRATION (PMOLE5 PITUITARY;

FIGURE 11. The influence of LHRH and LHRH-A on c y c l i c AMP production in anterior p i t u i t a r y c e l l s in suspension. C e l l s were incubated for 30 min at 37°C with the indicated l e v e l s of LHRH or LHRH-A. Concentrations of cAMP in the c e l l s and released into the medium were measured by radioimmunoassay. Numbers in parentheses are separate experiments at each dosage.

390 When we attempted to reproduce with dibutyryl cAMP the effects of LHRH on nuclear estrogen receptors, we were unable to do so, using either intact cells (Table 2) or isolated nuclei (Table 3), and amounts of cAMP orders of magnitude greater than the maximally-LHRH-stimulated level.

TABLE 2. Effects of LHRH or DBcAMP on cytosol and nuclear estrogen receptor levels in whole cells under suspension or culture conditions % of control Agent

nuclear ER suspension

cytosol ER culture

suspension

culture

LHRH (pmoles per pituitary) 0 .3

112 ± 1 .6

76

+

3..4

70 ± 1.8

10

166 ± 5 .3

256 ± 8..5

62

+

2..1

50 ± 6.5

100

192 ± 6 .1

256

5,.9

56

+

3,,2

82 ± 0.7

104 ± 0..5

66

+

2.,6

80 ± 1.5

-

34

+

2..0

1.0

102

+•

+

DBcAMP (nM) 0.1 10

95

+

1 .1 .

98 ± 4.,0

-

100

116

+

1 .7 ,

168

+

3,.3

20

+

2.,8

9 ± 4.1

1000

97

+

6.,4

95

+

7..7

30

+

5..4

20 ± 10.1

112

+

2.,2

88

+

3..0

26

+

2.,6

23 ± 8.8

10,000

Fresh anterior pituitary cells in suspension or from 3-day culture were incubated at 37°C for 30 min in the absence (control) or presence of the indicated levels of LHRH or DBcAMP. Cells were collected and subfractionated into cytosol and nuclear fractions for determination of estrogen receptor content. Each value is the mean ± SEM of triplicate determinations, using groups of 20 animals per experiment.

We conclude from these studies that LHRH has a direct specific stimulatory effect on nuclear anterior pituitary estrogen receptors, independent of involvement of cAMP.

This action of LHRH enhances the sensitivity of the

cells to estrogen, precipitating the documented estrogen enhancement of responsiveness to LHRH (52,53) and providing an explanation for the phenomenon of LHRH self-priming, whereby one exposure to LHRH sensitizes cells to a second LHRH stimulus (54,55).

391 TABLE 3. Effect of varying l e v e l s of DBcAMP or LHRH on estrogen receptor binding in i s o l a t e d cytosol and nuclear f r a c t i o n s from anterior p i t u i t a r y homogenates % of control cytosol

nuclei

LHRH (pmoles per pituitary) 112,.0 ± 1.5

(4)

10

121..1 ± 4.0

(4)*

221

± 18

(4)*

100

110,.5 ± 7.2

(4)

385

± 36

(4)

1000

95,.2 ± 3.1

(4)

590

ζ 70

(4)*

1.0

92..2 i 3. 5

(4)

DBcAMP (nM) 0.1

92,,4

± 5.9

(4)

6 .0

(4)

1.0

95,,6

±11 .8

(8)

136,.1 ± 23 .0

89,.0 ±

(8)

10

77,,5

± 4.2

(8)*

83,.6 ± 15 .3

(8)

100

73.,9

± 2.5 ( 1 2 ) *

79,.7 ± 10 .7

(8)

1000

62.,5

± 5.2 (12)*

139.,4 ± 21 .8

(8)

5000

67,.7

i l l .0

(6)*

83.,6 ± 20 .9

(4)

10,000

33.,0

ι

(2)*

87..3 ±

(2)

3.7

8 .6

Cytosol and nuclei were prepared and incubated for 30 min at 37°C with the level of effector indicated. The samples were then cooled to 4°C and the nuclei were washed with b u f f e r . Estrogen receptor content was assessed in each f r a c t i o n and expressed as the percentage of that present in samples incubated without added agent. A s t e r i s k : Ρ < 0.05, as compared with cont r o l s . Values are mean ± SEM for the number of experiments shown in parentheses . D i v e r s i t y of Receptor Regulation Numerous examples appear in the l i t e r a t u r e for regulation of s t e r o i d hormone receptor nature, level and f u n c t i o n a l i t y as a function of a wide array of e f f e c t o r s , hormonal and otherwise.

As a representative of the complex-

i t y which can be involved, we w i l l o u t l i n e some of our observations on normal mouse mammary gland estrogen receptor c o n t r o l .

392 Estrogen receptors of the mouse uterus are maintained and regulated by estrogen in much the same manner as in the rat uterus (12,56). no effect on these receptors of the uterus.

Prolactin has

In marked contrast,

prolactin

is the most potent factor y e t detected for controlling the level of estrogen receptors in mouse mammary tissue (57,58).

Administration of prolactin

to virgin mice results in dramatic stimulation of cytosol estrogen activity (Figure 12).

The receptor in the stimulated state exists as a

more highly-aggregated form than in the virgin animal greater physiological estradiol.

receptor

and, of probably

importance, has a diminished rate of interaction with

We have recently described an analogous situation for androgen

receptors of the rat ventral

prostate (33); in this system, we also find

two molecular forms of the receptor which differ with respect to their avidity for androgens.

In Figure 12 is also presented a pattern of respon-

siveness of the mouse mammary estrogen receptors to prolactin in the thyroidectomized animal.

We have previously observed an involvement of thy-

roid hormones in regulation of estrogen receptors in another tissue, the rat anterior pituitary (59).

Comparison of the two panels of Figure 12

demonstrates that thyroidectomy enhances the sensitivity of the mammary estrogen receptor response to prolactin, since it has been shown that removal of the thyroid does not result in increased prolactin secretion Moreover, it has been reported that endogenous thyroid hormones

(60).

inhibit

the mammotropic actions of prolactin.

FIGURE 12. Effect of prolactin (PRL) on mouse mammary gland estrogen receptor level and nature. Intact (left panel) or thyroidectomized (right panel) virgin mice were injected with prolactin and killed 18 hours later (o), or injected daily with prolactin for 3 days and killed 1 hour after the final injection ( A ) . Equivalent amounts of cytosol protein were incubated with [ 3 H]estradiol and applied to sucrose gradients prepared in low ionic strength buffer. The arrow represents bovine serum albumin at a value of 4.4S.

393 The effect of p r o l a c t i n on mammary t i s s u e estrogen receptors i s seen when the animals are treated with estradiol

(Table 4).

I t was o r i g i n a l l y

that the estradiol d i r e c t l y regulated i t s receptor l e v e l s in t h i s

thought

tissue

as i t does in the uterus; however, the data of Table 4, in which bromocryptine suppression of p r o l a c t i n eradicates the p o s s i b i l i t y of estradiol hancing estrogen receptor a c t i v i t y , demonstrate that the estradiol

en-

action

i s i n d i r e c t and mediated through stimulation of p r o l a c t i n secretion. TABLE 4.

Influence of p r o l a c t i n on mouse mammary gland estrogen receptors Estrogen receptor l e v e l s as % of control

Treatment

Cytosolic

Nuclear

100 ± 9

100 ± 7

Estradiol, 0.5 yg/day χ 30, sc in o i l

61 ± 5

500 ± 43

CB-154*, 100 yg/day χ 30, ip in sal i ne

87 - 8

111 i 17

E s t r a d i o l + CB-154

59 ί 5

133 ± 8

386 ± 38

273 ± 26

Control a

Rat p r o l a c t i n , 50 yg/day, twice d a i l y χ 5, sc in o i l a

Untreated, 58-day old v i r g i n mice *CB-154 = bromocryptine I t can also be seen from Table 4 that prolactin-induced estrogen receptor i s heavily concentrated in the c y t o s o l .

When estradiol

i s subsequently

administered, nuclear t r a n s l o c a t i o n occurs and progesterone receptor i s induced (58). sue i s bimodal.

Thus, the primary control of estrogen receptors in t h i s

tis-

P r o l a c t i n regulates the level of estrogen receptors and

e s t r a d i o l regulates t h e i r f u n c t i o n a l i t y .

Neither hormone, in the absence

of the other, i s capable of e l i c i t i n g maximal estrogenic response.

This

i s o l a t e d instance of multifaceted regulation of a s i n g l e receptor system in a t i s s u e - s p e c i f i c manner exemplifies the complexity with which multiple

394 homeostatic hormonal environments are regulated and maintained. Concluding Remarks In t h i s presentation, work from my laboratory has been d i s c u s s e d , dealing with several s p e c i f i c studies chosen to exemplify d i f f e r e n t aspects of the ways in which regulation of s t e r o i d hormone receptor turnover occurs and i s related to hormonal responsiveness.

To adequately portray the paradox-

ical features of both uniqueness and universal commonness of s t e r o i d a l receptor systems, I have d e l i b e r a t e l y selected four d i f f e r e n t t i s s u e s y s tems, i . e . , uterus, prostate, hypothalamus-adenohypophysis, and mammary gland.

While the q u a n t i t a t i v e features of control and function are v a r i -

able, the basic denominator remains constant.

For instance, the estrogen

receptors of the uterus and mammary gland are p r i n c i p a l l y regulated by estradiol

and p r o l a c t i n , r e s p e c t i v e l y , whereas the fundamental

processes

of control of receptor dynamics and concentration appear to be common to both t i s s u e s .

The overall process of receptor r e g u l a t i o n , j u s t as the

mechanism of s t e r o i d hormone a c t i o n , appears to be a highly-conserved evolutionary t r a i t .

However, the development of t i s s u e and c e l l u l a r

dif-

f e r e n t i a t i o n which has evolved with time has given r i s e to both subtle and profound changes in the control of responsiveness to a given hormone or group of hormones.

This chapter i s designed as a mere introduction to t h i s

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61.

Mittra, I.: Nature 248, 525-526 (1974).

AN ENDOGENOUS LIGAND FOR TYPE II BINDING SITES IN NORMAL AND NEOPLASTIC TISSUES Barry M. Markaverich and James H. Clark

Department of Cell Biology Baylor College of Medicine Houston, Texas 77030

Abstract

The rat uterus contains two classes of specific nuclear estrogen binding sites which may be involved in estrogen action. the classical estrogen receptor are stimulated uterine gen

in the nucleus by estrogen

hyperplasia.

treated

rats

3

[ H]-estradiol measurable

to

exchange

quantities

nuclear

result

dilution

from

estradiol

the

under

represent

(Kd

10-20nM)

conditions which

cause

Dilution of uterine nuclear fractions from estro-

prior

quantitation

results

of

the

occur

dilution

in

type

are not affected by dilution. ing

Type I sites

(Kd InM) and type II sites

of

an

estrogen

increase

II site.

binding

(3-4

Estimates

sites

fold)

in

independently a

specific

binding to these sites.

of

protein

the

of type I sites

These increases in type II sites

of

follow-

concentration

endogenous

by

inhibitor

The inhibitor activity

of

and [3 Η ] —

is present in

cytosol preparations from rat uterus, spleen, diaphragm, skeletal muscle and

serum.

Sephadex

Preliminary

characterization

G-25 chromatography

in molecular weight ( 300). on

LH-20

chromatography

retained on this

These components

since

inhibitor

component

gas-liquid

chromatography-mass

tive

inhibitor

very

similar

304).

activity

high

is not

phenanthrene-like

Analysis

the

lipophilic resin. by

of

cytosol

of

the

inhibitor

activity

shows two distinct peaks which are

ß-peak

by

similar

(a and β) can be separated

component

is

preferentially

Partial purification of the LH-20-8

performance

liquid

chromatography

spectrometric analysis steroidal molecules

preparations

in nature

and consists of two

(molecular on LH-20

and

suggest the p u t a -

weights

302

chromatography

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

and shows

400 that

non-neoplastic

contain

both

mammary

tumors

peak

α

tissues

and

contain

inhibitor

(uterus,

β inhibitor very

liver,

components

low

to

lactating

whereas

mammary

gland)

estrogen-induced

non-measurable

quantities

of

rat

the

6-

activity.

INTRODUCTION

Estrogen

administration

in m o t i o n a n u m b e r of

true

uterine

estrogen

nuclear sites

growth.

receptor

complexes

to

I

nucleus

II

immature

sites

in e s t r o g e n

sites),

The

antagonism

of

uterotropic

responses

inhibition

of

the

type

antagonists ethylene In

this

we

II

cytoplasmic purified liquid

We will

sites

and

or

our

does

not

procedures,

Detection

and M e a s u r e m e n t

presence

II s i t e s w a s II

sites

and

ref

specific

of

an

first

increased 6.

These

and

of

of a p p r o x i m a t e l y

Inhibitory for

the

uterine

[3H]-estradiol

show binding

nuclear that

the

steroid

(5). demonstrates

the

to

nuclear

for

nuclear

binding

material

has

to been

analyzed

by

gas-

the

basis

of

two

is p h e n a n t h r e n e - l i k e

in

300.

Activity

binding

considered w h e n we observed

data

with

and

On

activity

II

that

triphenyl-

binding

This

of

type

for

[^H ] - e s t r a d i o l

chromatography

the

or

and

is s p e c i f i c

receptors.

feel

case

(4)

[^Hj-estradiol

with

I

associated the

(6) w h i c h

inhibitor

the

activation

demonstrated

clomiphene

spectrometry. we

inhibitor

as

is

to

receptor-estrogen

type

is

This

sets

stimulation

binding

or

of

estrogen

work

of

liquid

nature with a molecular weight

The

role

progesterone

this

estrogen

chromatography-mass

derivatization

to

interfere

performance

of

rats

the

hormone

H o w e v e r , we h a v e

recent

that

with

stimulation

precise

and

inhibitor

show

nuclear

by h i g h

the

s u c h as n a f o x i d i n e

summarize

include

sites.

dexamethasone

of an e n d o g e n o u s

II s i t e s .

type

as

derivatives paper

presence type

such

II

ovariectomized

associated

translocation

and

is u n k n o w n .

nuclear

mature

events

(1,2),

(3-5).

action

or

events

These

(type

the

type

to

of b i o c h e m i c a l

of

fractions

with

[^H]-estradiol

that

were

dilution,

to n u c l e a r

type

the q u a n t i t y diluted

(40

II s i t e s

to

type

of

type

(Figure

mg/ml-10

1

mg/ml)

increases

even

401

Specific Binding Ό 3 Ο

CO D

30-

• 10mg/ml ο 20mg/ml • 40mg/ml

-t-1

Ο

3

TJ (0 (0

φ

I

TO

10

20

30

40

3

[ H ] - E s t r a d i o l nM

Fig.

1.

E f f e c t of d i l u t i o n on [ ^ H J - e s t r a d i o l b i n d i n g in u t e r i n e n u c l e a r f r a c t i o n s f r o m a d u l t o v a r i e c t o m i z e d rats i m p l a n t e d w i t h a n e s t r a d i o l p e l l e t for 4 d a y s .

402 though

the quantity

to 4-fold. that

the

of nuclei

Expression quantities

nuclear

of

of

in the

nuclear

concentrations

incubation

(10

type

and

20

II

sites

to

show

increased not

the

that

type

concurrently case.

I sites

with

These

is decreased

the

3-

to

4-fold

Although these binding

curves

are

we will

increases

at

least

lower

ntn [ 3 H]-estradiol) are

(0.4-8.0

dilution,

apparent

measured

2

demonstrates at

mg/ml)

greater than levels measured at 40 mg/ml. appear

mixture

the data on a per uterine basis

show

later

in receptor

that

also

this

estimates

is

are

due

only to an increasing influence of the type II site on the assay of type I sites as we have described previously These

results

measured specific

inhibitor

sites.

To

cytosol nuclei type

examine

the

increased

nuclear

fractions

binding

of

this

of

The

possibility

results

increasing

decreases

II sites

concentration expected.

specific

The

which

decreased

concentration

suggest

uterine

binding

to

competitive

nuclear

II

or nuclear receptor

nuclear

large

cytosol

of

linearly

in

of

dilutions

of

type

related

dilution

sites.

The

of the binding of

inhibitor

[^HJ-estradiol

which

we

to

protein

would

be

identical did

These

to not

results

[ 3 H]-estradiol

of

appears

to

to type

be

a

II sites

[ 3 H]-estradiol to either the

cytosol

(6).

boiled-acid

precipitated

cytosol

from

ACTIVITY adult

tomized rat uteri on Sephadex G-25 revealed two major peaks of activity

nuclear nuclear

yg/ml)

II sites.

of the

to

as

were

(200

uterine

that

the

G E L FILTRATION CHROMATOGRAPHY OF UTERINE CYTOSOL INHIBITOR Chromatography

II

quantities

to

BSA which

cytosol

sites

type

[ 3 H]-estradiol

with

of

to nuclear

yet does not inhibit the binding of

II

demonstrate

contains a specific inhibitor

type

inhibitor

contain

of

binding

measured

[ 3 H]-estradiol binding

type

various

experiments

concentrations

inhibit

cytosol

to

inhibition was not directly

In addition,

protein

which

these

concentrations

the

(6).

further,

of

from dilution of a

of BSA were incubated with

rats

of

quantities may result

[ 3 H]-estradiol

concentrations

from estradiol-implanted

II sites.

suspension

the

that

of

or equivalent

addition

type

suggest

in more dilute

(7).

designated

a and

g

(Figure

2).

This

ovariecinhibitor

activity

was

403

A.

B. L H - 2 0

G-25

(

(p)

• ίΜ, '

Τ

J

l.J/'s»«!«»^, C. G - 2 5 ~

Ο

Fig.

2.

Λ

'

:

LiX^^V^™, , D. G - 2 5 0

'Λ ' h'

10

20

30 40 10 Fraction Number

20

30

40

Chromatography of rat uterine cytosol inhibitor preparations on Sephadex G-25 (A,C, and D) or LH-20 columns (B).

60 c o

I 40 c

20

0 Fig.

3.

10

20

30

40

Fraction Number

50

Comparison of LH-20 elution profiles from acidprecipitated-boiled cytosol preparations (100 mg fresh tissue equivalents/ml) from rat uterus (o) or an estrogeninduced rat mammary tumor (·).

404 measured in the column fractions by their ability to inhibit the binding [ 3 H]-estradiol seen

in

the

to

nuclear

void

volume

type of

II.

the

A minor

column, which

with protein since there was a significant tions.

In

addition,

peak

incubation

of

the

of activity was

is apparently rea

OD28O cytosol

ding

also

associated these

inhibitor

frac-

preparation

with 0.4 Μ KCL for 60 minutes at 4° prior to chromatography on Sephadex G-25

(TE buffer containing 0.4 Μ KCL) dissociated the inhibitor

from the void volume fractions On

the

basis

of

sizing

(raw 507.2) as markers, we and

β inhibitor

chromatography

(data not

experiments

is

of an aliquot

in

of

shown).

using

estimated

components

tryptophan

that

the

(mw 204.2) and

ATP

the molecular weight of the α

range

this

activity

same

of

300-400.

cytosol

Surprisingly,

preparation

on

LH-20

(Figure 2B) shows that the two components are more clearly resolved. determine

if

the

chromatography collected

order

was

the

D).

The

correspond

elution

individual

rechromatographed and

of

analagous

to

a

of

the

their

and

α

and

behavior

β

peak

β

on

peaks

on

Sephadex

fraction

from

LH-20

G-25, LH-20

the of

demonstrate elution

cytosol

of

that

the

this

β peaks

material

preparations

on

from on

larger

LH-20

we and

these fractions on the Sephadex G-25 column (Figure

results

Chromatography

to

To

2C

columns

Sephadex

G-25.

preparative

LH-20

columns facilitates complete separation of the α and β peak fraction due to the selective retention of the β material and has greatly purification of this inhibitor activity

COMPARISON UTERUS, MAMMARY

Since

OF

LH-20

NORMAL

ELUTION

LACTATING

PROFILES

RAT

MAMMARY

facilitated

(see below).

OF

CYTOSOL

GLAND

AND

FROM

RAT

ESTROGEN-INDUCED

OBTAINED

RAT

TUMORS

nuclear

estrogens

type

cause

II sites may

cell

play

growth

a role

be involved

(3,

4)

we

inhibitor

may

this were

the case, then rapidly proliferating

respond

to estrogen might

examine

this

possibility

in modulating

in the mechanisms by which

reasoned

show we

these

deficiencies

prepared

that

cellular

perhaps responses.

neoplastic tissues

in inhibitor

boiled-acid

this

activity.

precipitated

If

which To

cytosol

405 from rat uterus, normal lactating rat mammary gland and estrogen-induced rat

mammary

LH-20.

tumors,

and

chromatographed

aliquots

of

these

cytosols

Individual fractions were assayed for inhibitor activity.

data show that

the

larger LH-20 column (1.0 χ 50 cm: 75 ml bed

on

These volume)

used for these experiments clearly separated the a and β inhibitor peaks (Figure tumor peak

3) in rat

cytosol

uterine

contained

material,

the

cytosol.

Furthermore,

approximately

tumors

contained

although

equivalent

little

of

rat

quantities

mammary

of

the

the 3 component

and

α in

some cases the ß-inhibitor component was nonmeasurable. This

observation

has

also

human breast cancer (15).

been

extended

to

mouse

mammary

tumors

20 chromatography showed that mouse mammary gland contained inhibitor

activity

than

mammary

tumors

in the

This difference in activity appears to result in the ß-inhibitor peak material (Fig. 4, ref completed

a

series

preparations

from

chromatography. ß-inhibitor following

of

uterus

component

molecule

experiments

and

tumor

same

inhibitor

in

in uterine

15).

In addition, we have

where

were

cytosol

cytosol

mixed

was

(as compared

(since

they

readily

are

of

very

inhibitor

the

peak

28-38

(Figure

in tumors which (fractions

ß-peak

However,

biological preparations

prior

to

material

in

3)

quantitatively

recovered

to uterus) is not

in

crude

rat

due

to

At present we do not

relationship similar

between ο and β

molecular weight) and

Certainly the inhibitor activity

suggests

there

is not observed

tumors

experiments

activity. from

LH-20

that the

is

an

altered

in the uterus.

form

Whether

28-38) represents an altered or metabolized

preliminary

material

inhibitor

form the ß-material, or if perhaps the tumors

metabolize this ß-inhibitor component.

activity

cytosol

These results suggest that the tumor cytosol

there is a precursor-product

peaks

fractions

animals.

deficiency

in the ß-inhibitor component, and the low levels of

tumor

the tumor cannot

in

of

from a primary

some intrinsic degradation during the preparation. know whether

20-fold more

strain

The results of these experiments demonstrated

is indeed deficient this

mixing

chromatography.

and

Detailed studies by dilution analysis and LH-

(Figure

3)

indicate

inhibitor

remains

that

preparations

Acid-Precipitated-Boiled uterus

of

liver

to

be

the presence

(containing

is

a

and

this

form of

resolved. of

associated

cytosol

of

the

ß-

with

inhibitor β

inhibitor

406

Fig. 4.

LH-20 chromatography of mouse mammary gland (A) and mouse mammary tumor (B) cytosol. Aliquots (1 ml) of the cytosol preparations (80 mg fresh tissue equivalents/ml) were loaded on an LH-20 column and the columns eluted with TE (10 mM Tris; 1.5 mM EDTA) buffer. Fractions (0.5 ml) were collected and assayed for inhibitor activity as described in methods. Results are plotted as [ 3 H ] - e s t r a d i o l binding to nuclear type II sites (% inhibition) were buffer controls (0% inhibition) contained approximately 45,000 cpm bound. The results were obtained from a single experiment. However, we have analyzed 15-20 separate tumor cytosol preparations and very similar results were obtained.

407 components) inhibit approximately

the growth of rat mammary tumor cells in culture by

80-90%

in

4-7 days.

Conversely,

preparations

from

rat

mammary tumors (containing a, but lacking the ß-inhibitor component) had no significant effect on cell growth in identical experiments for

3-4

weeks.

inhibitor

peak

These

results

material

in

suggest

tumors

that

is

the

absence

correlated

continued of

with

the

rapid

ß-

cell

proliferation in these populations.

PURIFICATION AND PARTIAL CHARACTERIZATION OF INHIBITOR ACTIVITY

As

stated

activity HPLC

of the

inhibitor

(LH-20 6-peak component) remains to be established.

earlier,

However,

analysis

showed

from the silica times

on

elutes very

5-6 minutes

broad

of

with to

injection. a single

procedures

conditions;

to

and

of

the activity

consistently

of uv absorbance

at

254 nM

The peak is somewhat

suggesting

multiple

inhibitor

To date we have tried a number of

these

peak

can be eluted

The samples at this point are

peak

shoulders,

separate

reinjection

activity

has been repeated a number of

the inhibitor activity.

have

components which are not separated. HPLC

inhibitor

liver preparations

observe

appears

peak

identification

This experiment

following

since we

is coincident and

structural

a major

column.

3-4 separate

clean

which

positive

components

fractions)

and

(various we

elution

cannot

further

separate these components on a straight phase column. This heterogeneity of putative inhibitor molecules was supported by GCMS analysis.

The GC-MS analysis of the pooled

minutes) positively

HPLC fractions

identified a number of fatty

nents in the sample inhibitor preparations.

(5.4-5.8

acids as major

compo-

However, these are unlikely

candidates for the inhibitor since the authentic compounds did not inhibit

[ 3 H]-estradiol binding to type II sites over a wide range of concen-

trations

(0.001 nM -

100 pM).

The putative

inhibitor activity appears

to be associated with two remaining components in the sample which have a

molecular

weight

of

302 and

304.

Comparison

of

sample

spectra

to

those of known compounds in the NIH bureau of standards library suggests the inhibitor is very similar to phenanthrene-like

molecules.

408 DISCUSSION AND CONCLUSIONS

These

experiments

demonstrate

that

the adult

ovariectomized

rat

uterus

and a variety of rat tissues contain an inhibitor which interferes with [ 3 H]-estradiol inhibitor with

binding

is specific

estrogen

binding

Consequently,

if

to

type

II

for nuclear

sites

type

to cytoplasmic

this

inhibitor

in

uterine

II sites and

nuclei.

does not

or nuclear estrogen

is

involved

in

receptor

the

(6).

modulation

estrogenic response in target tissues, its effects are expressed an interaction with nuclear type II sites.

This

interfere

of

through

We currently feel that this

molecule represents an endogenous ligand for type II sites (6). Preliminary

characterization

demonstrates

of

this

inhibitor

this molecule(s) is stable to heat

in

rat

uterine

HCL, and therefore it is unlikely to be protein in nature. trypsin and

and proteinase

the

(Figure 350.

inhibitor 2) as We

appears

two

have

major

identical

to

and

the

that

HPLC

with 2

an

peak

seen

and

phenanthrene-like

its activity

chromatographs

peaks

purified

identical

chromatography

Κ do not destroy

activity

it

on

estimated

the

appears

compounds

basis of mass spectrometry of 302 and 304. the

inhibitor

molecules

identification equivalent

and

since

the

(free

fatty

derivatives

only

also is

for

compete not

At

weight

not

thin

layer

of

two

nearly

weights

to homogeneity, "identified"

present

compounds

inhibit

of

(which

on

the

Proof that these are in fact

the the

LH-20

liver

by

consist

shown),

or

time

structural

material we

feel

are good candidates for the inhibitor

measureable

acids) did

type II sites. competes

other

that

activity.

rat

molecular

purification

demonstration

biological

phenanthrene

awaits

G-25

molecular

from

uterus)

to

with

In addition,

(data not

Sephadex

material

in

cytosol

(100° χ 60'), and 0.1 Ν

in

the

sample

has these

activity

preparation

[ 3 H]-estradiol binding

to

nuclear

Although one could argue that if the putative inhibitor

[ 3 H]-estradiol binding to nuclear for

necessarily

triphenylethylene

type

II sites

[ 3 H]-estradiol binding to the estrogen the

case.

derivatives

Nuclear

type

(anti-estrogens)

compounds bind to the estrogen receptor

(7).

II even

it should

receptor, this do

not

though

bind these

Likewise, nuclear type II

sites also appear to bind this inhibitor with amazing specifity, whereas

409 we have been unable receptor

(6).

estrogen binding type

receptor (Figure

I sites

possible

to show this inhibitor

Certainly,

the

if

direct

direct

we

observed

demonstrate

with that

have

to

of

associated

with

the

effect

on

[ 3 H]-estradiol binding

to

a dilution

experiments

obtain

(6).

milligram

It

is

amounts

also

of

the

[ 3 H]-estradiol binding to type I sites would

pharmacological at

were

observed

inhibition

able

interacts with the estrogen

inhibitor

competition

were

inhibitor, competition for be

the

in vivo we would 1) or

in

that

if

concentrations

physiological

(mM).

concentrations

this

Our

data

interaction

is

unlikely · Since

we

have

inhibitor

not

been

activity

describe

any

experiments positive

able

in vivo

direct await

role

it is very

experiments,

however, are very

should

to

normal

4).

mammary

tumors

We

uterus

have

which

liver

which

inhibition cell

(Figure

were

an

this early

time to

action.

These

inhibitor. of its

Once

a

biological

Preliminary

It appears

this

that

in vitro

there is a

and

lactating

mammary

inhibitor

preparations

in

peak

the

g

and myometrial

gland

from

rat

did

not

material

cells, or rat

mammary

In contrast, inhibitor preparations from uterus

contain

cell

or

the

of

in rat mammary tumor cytosol as

3),

that

deficient

the

80% following

of

death,

at

effects

in estrogen of

promising.

growth of uterine stromal

approximately

the

straightfoward.

observed

tumor cells in culture. or

be

in the ß-inhibitor component

(Figure

inhibit

difficult

compound

identification

in vivo

compared

assess

has been made, determination

significance

deficiency

directly

for this

chemical

identification

to

growth

6

material

reduced

4-7 days of treatment. in

inhibition

culture of

cell

results

from

division

or

cell

numbers

by

Whether or not this an

acceleration

both

remains

to

of be

resolved. Although

the physiological

significance of this inhibitor remains to be

resolved, we speculate at this time that the inhibitor may act to modify or

regulate

uterotropic

responses

to

estrogen

or

perhaps

"protective" capacity in cases of hyperestrogenization.

act

in

a

Such hypothesis

are consistent with our current knowledge concerning a possible role for nuclear

type

secondary

II sites

nuclear

in

estrogen

estrogen

action.

binding

sites

We

have

are

shown

only

that

these

activated

or

410 stimulated

in

the

nucleus

hypertrophy,

hyperplasia

dexamethasone

and

under

and

conditions

DNA

progesterone

which

synthesis

antagonism

cause

(3-5).

of uterine

uterine

Furthermore,

growth

in the

rat

is associated with an inhibition of estrogen stimulation of nuclear XI sites the

(4) and these antagonists do not affect the normal functions of

estrogen

On

the

basis

of

that

nuclear

type

II

sites

may

action.

Since

nuclear

type

II

localized

on

suggested

estrogen

receptor.

the

replication

nuclear

(9), we

induced

feel

DNA

estrogen

matrix this

DNA

the

failure

synthesis)

hypothalmus

in

inhibitor

synthesis

of

by

estrogen

estrogen

to

stimulate

mammary

tumors

levels

(~ 15-20 a

fold)

Likewise, ovarian

in

human

tissues

type

II

variety

we of

nuclear

cell

growth as

block of

(hyperplasia;

the

pituitary

and

have

are

basal do

14)

estrogen

which

appear

of

type

respond

this inhibitor may be a component

as are nuclear

type

II sites.

and

in

endocrine

II

sites

in

deficiency.

estrogen

(6).

in a via

tissues

Therefore

of all

tissues

In tissues which do not normally

respond

manner, type

consequently

Conversely,

mammary

(diaphragm, spleen, liver) and these

is that

this

permanently

II sites

to

activity

lower

correlated

the

type

our hypothesis

inhibitor

be

inhibitor

of nuclear

normally

to

quantities of inhibitor

to estrogen in a proliferative

target

with

(12) mouse

nuclear

this

is

consistent

regardless of

with

levels

not

is

independent

levels

correlated

which

activity

II sites

(13,

these

(15) contain significantly

(11) or

higher

measured

tissues

in

the

the failure of

do contain significant

this

or

be DNA

stimulation

Certainly,

component

type

cancer

hypertrophy and hyperplasia

expressed.

to in

Our findings that rat and mouse

inhibitor

3-peak

dependent

these

tissues

Likewise,

appear

may modulate

such

sites

cancer

this

the

breast

Therefore

malignant

of

in

activated and

have

estrogen

implicated

estrogen

to stimulate

and human breast

deficiency

status.

sites

been

we

in

sites.

in these tissues.

nuclear

hypothesis.

tumors

has

activity

organs makes this a tenable hypothesis.

with

involved

binding

inhibiting

target

experiments

be

(10) is related to the inability of estrogen to modulate

activity of this inhibitor estrogen

these

(8) which

these secondary nuclear estrogen binding Perhaps

type

the

tissues

II sites are complexed with

functions which

do

of

these

respond

are

not

to estrogens,

sites

the

411 association of the receptor-estrogen result

in a dissociation

Under

these

observed.

conditions

Consistent

observed

following nuclei

the

this

(Figure

dilution.

from

complex with target cell nuclei may

inhibitor

cellular

with

estrogen treated nuclei

uterine

of

from

hypertrophy

hypothesis

estrogen

Since

ovariectomized

dependent.

is

1) additional this animals

feel that this dissociation of the inhibitor is

nuclear

Obviously,

the

and

our

sites.

hyperplasia

is

that

in

type

II sites

not

observed

in

shown),

we

is

(controls;

not

from nuclear lower

II

observation

nuclear

effect

type

levels

type of

are

II sites inhibitor

activity in neoplastic tissues is consistent with the elevated levels of type

II

sites

measured

these cell populations.

in

tumors

and

the

Although only

rapid

proliferation

tentative at this point

we feel that this is a reasonable model for potential proliferation by type II binding

rate

in

in time,

regulation of cell

inhibitor.

ACKNOWLEDGEMENTS

The authors would technical

like to thank Rebbecca Roberts and M.A. Alejandro

assistance,

Georgietta

Brown

David Scarff for the illustrations.

for

typing

the

manuscript,

Supported by NIH grants

for and

HD-08436.

REFERENCES

1.

Jensen, E.V., Numata, M., Brecher, P.I., DeSombre, Biochemistry of Steroid Hormone Action (Smellie, Academic Press, London 133-159 (1971)

E.R.: In The R.M.S., ed.)

2.

Shyamala, G. Gorski, J.: J. Biol. Chem. 244,

(1967).

1097-1103

3.

Markaverich, B.M., Clark, J.H.: Endocrinology

4.

Markaverich, Β.Μ., Upchurch, 14, 125-132 (1981).

5.

Markaverich, B.M., Upchurch, S., McCormack, Clark, J.H.: Biol. Reprod. 24, 171-181 (1981).

6.

Markaverich, Β.Μ., Roberts, R.R., Finney, R.W. Clark, J.Η.: J. Biol. Chem. 258,11663-11671 (1983).

S.,

Clark,

105, 1458-1462

J.H.:

J. Steroid S.,

(1979). Biochem.

Glasser,

S.R.,

412 7. Markaverich, Β.Μ., Williams, Endocrinology 62-69 (1981).

Μ.,

Upchurch,

S.,

Clark

J.Η.:

8. Clark, J.H. Markaverich, Β.Μ.: In The Nuclear Envelope and Nuclear Matrix, A l a n R. Liss, Inc., New York 260-269 (1982). 9. Pardoll, D.M., Vogelstein, Β. Coffey: Cell 19, 527-536

(1980).

10. Keiner, Κ.L.Peck, E.J., Jr.: J. Receptor Res. 2, 47-62

(1981).

11. Watson, C.S. Clark, J.H.: J. Receptor Res. 1, 91-111

(1980).

12. Watson, (1980).

107,

C.S., Medina,

D.

Clark, J.H.: Endocrinology

13. Syne, J.S., Markaverich, Β.Μ., Research 42, 4443-4448 (1982). 14. Syne, J.S., Markaverich, Β.Μ., Research 42, 4449-4454 (1982).

Clark, Clark,

the

1432-1437

J.H.,

Panko,

W.B.:

Cancer

J.H.

Panko,

W.B.:

Cancer

15. Markaverich, Β.Μ., Roberts R.R., Alejandro, Μ.A. Clark, J.H.: Res. 44, 1575-1579 (1984).

Cancer

REGULATION OF MAMMARY RESPONSIVENESS TO ESTROGEN: AN ANALYSIS OF DIFFERENCES BETWEEN MAMMARY GLAND AND THE UTERUS

Gopalan Shyamala Lady Davis Institute for Medical Research, Sir Mortimer B. Davis - Jewish General Hospital and Department of Medicine, McGill University, Montreal, Quebec, Canada

I) Introduction The mammary gland is a compound tubuloalveolar gland composed of adipose, connective and epithelial tissue elements.

At

birth the gland is still rudimentary which remains in a quiescent state until puberty at which time there is a limited development.

In the adult female during each menstrual cycle,

cyclic proliferative changes and active growth of the ductal tissue occurs but the full development and differentiation of the gland occurs only during pregnancy and lactation.

Almost

all aspects of mammary development and differentiation are under complex hormonal control and there are some apparent differences among various species with regard to hormonal regulation of mammary development and differentiation.

Most of

the classic studies on the developmental biology of the gland have been done with rodents and the conclusions derived from these experiments have been frequently applied to many species including humans. Estrogens have been reported to have a variety of effects on various cell types of mammary glands and are believed to be important for both mammary development and differentiation

(1)

However, as yet the mechanism(s) underlying the actions of estrogens in mammary tissues is poorly understood.

In fact

there are even some doubts as to whether estrogens exert their effects on mammary glands directly

(2), despite the wide docu-

mentation of the presence of estrogen receptors in this tissue

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

414

A detailed discussion of various estrogenic responses occurring in mammary glands, their relationship to estrogen receptors, and their relative importance in the overall biology of the tissues is beyond the scope of this article.

Therefore

this chapter will focus only on certain selected aspects of estrogenic influences occurring during mammary development and differentiation and the possible mechanisms underlying these estrogenic responses in this tissue.

The data used to illus-

trate various aspects of estrogen action are from our own laboratory using mouse as the experimental animal. At present, the major body of literature pertaining to the mechanism of estrogen action has been obtained with rodent uterus.

It is conceivable that estrogens may have different

effects on different target tissues of the same animal both from a qualitative and a quantitative aspect, and these differences may bear a relationship to certain unique functions characteristic to a particular target tissue.

As shown in

this article in the same animal under identical physiological conditions there are differences in responsiveness to estrogen by mammary glands and uterus.

Therefore an attempt has also

been made to discuss certain aspects of mammary responsiveness to estrogens which appear to differ from those observed in the uterus.

It is hoped that such a discussion will prove useful

in the ultimate resolution of the mechanism of estrogen action in mammary gland.

II) Estrogenic Responses in Mammary Glands A. Characterization of early and late responses A characteristic feature of all hormones including estrogens is their ability to elicit a variety of biochemical responses in their target tissues, which may be roughly catalogued into early and late responses.

The three well known responses to

estrogens in most target tissues for estrogen are changes in

415

glucose metabolism, progesterone receptor synthesis and DNA synthesis and these responses to estrogen also manifest in the mammary glands.

Based on the relative time required to

elicit each of these responses, the first wave of increase in glucose oxidation occurring between 1-2 hours after estradiol administration

(Fig. 1) and progesterone receptor synthesis

(Fig. 2) may be termed as early responses while the increase in DNA synthesis

(Fig. 3) and the second wave of increase in

glucose oxidation occurring between 18-21 hours after estradiol administration

(Fig. 1) are classified as late responses.

It

3001-

TIME (hours) Fig. 1. Effect of estradiol on glucose oxidation in mammary glands of castrated mice. A single injection of either saline ( • ) or estradiol (S3) was given to castrated virgin mice prior to killing at indicated times. The ability of the tissues to oxidize glucose was estimated according to procedures previously described (3). The data represent mean ± SEM of three experiments with duplicate determinations in each. The 100% value corresponding to saline treated animals was 44.4 ± 4.4 1 '*COj/mg mammary tissue. is, however, to be noted that while the overall pattern and time scales for initiation of the three estrogenic responses in mammary glands are similar to that previously observed in the uterus tissues.

(6), there are some differences between the two In the case of uterus, both the early and late waves

of glucose oxidation are also associated with increases in the phosphorylation of glucose as measured by the conversion of 2-deoxyglucose to 2-deoxyglucose phosphate

(7).

However, as

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Fig. 2. Temporal relationship between estradiol administration and increase in cytoplasmic mammary progesterone receptor level. Castrated virgin mice were given a single injection of 1 pg of estradiol prior to sacrifice at times indicated. The progesterone receptor levels were assayed by measuring the specific binding of ( 3 H)R5020 in cytoplasmic extracts. Each bar represents the mean ± SEM of three to five experiments. Control values for saline injected animals were: 467 fmoles/mg DNA. [From Shyamala, 1984 (4)] shown in Table 1, in mammary glands estradiol does not cause an increase in the metabolic conversion of 2-deoxyglucose to 2deoxyglucose phosphate.

Thus the conversion of 2-deoxyglucose

to 2-deoxyglucose phosphate, an easy parameter used for detecting the estrogenicity of a compound in the uterus

(7) cannot

be used for assessing estrogenic responsiveness in mammary glands.

Another striking difference between the mammary glands

and the uterus is the temporal relationship between estrogen administration and estrogen mediated increase in DNA synthesis as shown in Fig. 3.

Although in both tissues the increase in

DNA synthesis due to estrogen becomes apparent at approximately 12 hr, in the uterus the maximal increase is observed around 24 hr which returns to control value by 48 hr; however, in the

417

MAMMARY

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Fig. 3. Effect of estradiol on the in vitro incorporation of ( 3 H)thymidine into DNA in the mammary glands and uteri of castrated mice. A single injection of saline (control) or 1 pg E2 was given to castrated virgin mice before killing at the indicated times. The data represent mean ± SEM for three to five experiments. The control values represent the data obtained at 2 4 hours after injection and were: mammary glands, 2989 ± 558 cpm/mg DNA; uterus, 3834 t 403 cpm/mg DNA. [From Shyamala and Ferenczy, 1984 (5)] case of mammary glands the maximal increase in DNA synthesis occurs at approximately 48 hr after estrogen administration returning to control values only at about 96 hr.

Thus the

duration in DNA synthesis due to a single administration of estradiol is greater in mammary gland than in the uterus. However, the magnitude of response to estradiol with respect to DNA synthesis is larger in the case of uterus when compared to the mammary glands.

These relative differences in the

degree of mammary and uterine responsiveness to estradiol is most likely related to the differences in the estrogen receptor content of these tissues

(as shown later) since it mani-

fests regardless of the dose of estradiol administered to the tissue

(Table II).

The possible importance of this difference

in the pattern of DNA synthesis due to estrogen in mammary glands and uterus is discussed later in this article.

418

TABLE I. Effect of estradiol on 2-deoxyglucose metabolism in mammary glands and uterus of castrated mice. Treatment

Dose yg

Saline

mammary glands 14 . 0

-

Estradiol

1

Estradiol

2-deoxyglucose phosphate

3

14., 9 16 . 4

+ +

+

(cpm) uterus

0 ., 45

1875

2.. 3

3233

1 .9 ,

4438

+ + +

274 311 344

Three groups of ovariectomized virgin mice were given a single injection of either saline or indicated dose of estradiol for two hours prior to sacrifice. The conversion of 2-deoxyglucose to 2-deoxyglucose phosphate by the tissues was assayed exactly according to the procedure described by Gorski and Raker (8). The data represent mean ± SEM of three experiments with duplicate determinations in each experiment. TABLE II. Effect of estradiol on the in vitro incorporation of ( 3 H)thymidine into mammary and uterine DNA in castrated mice. Dose of estradiol yg

cpm/mg DNA mammary glands

0.. 25

279

1.. 0

292

3., 0

395

+ + +

36

(% of control) uterus 912

49

921

107

656

+

120

+ +

79 61

Three groups of castrated mice were injected with indicated doses of estradiol for 24 hours prior to killing. The tissues were processed for the in vitro incorporation of ( 3 Η)thymidine into DNA according to procedures previously described (5). The data represent mean ± SEM of three to five experiments.

B. Effect of prolactin on mammary responsiveness to estrogen It is known that the influence of estrogen on mammary development may be modified by virtue of its interplay with other hormones

(1).

More importantly, similar to estradiol prolactin

is also required for mammary development and differentiation (1).

Therefore, any precise understanding of the role of es-

trogens in mammary biology have to take into consideration the

419

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ο υ S

1

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Fig. 4. Effect of prolactin on the estrogenic responses in castrated virgin mice. A single injection of either saline (C) or 1 mg of ovine prolactin (PRL) was administered prior to processing the mammary tissues at indicated times. (A) Glucose oxidation at 1 hr. (B) Progesterone receptor level at 24 hr. (C) Rate of DNA synthesis as measured by in vitro incorporation of labelled thymidine into DNA at 24 hr. [Adapted from Shyamala, 1984 (4)] influence of prolactin on estrogenic responses. In castrated virgin mice, exogenous prolactin has no effect on either mammary glucose oxidation

(Pig. 4A) or progesterone re-

ceptor level (Fig. 4B) or DNA synthesis

(Fig. 4C).

If estra-

diol and prolactin are administered together, there is an increase in mammary response to estradiol as measured by all the three criteria but this increase is not significantly greater than that seem with estradiol alone

(data not shown).

Thus these data reveal that by the three criteria chosen for mammary responsiveness to estrogen, prolactin has no influence on estrogen action in mammary glands.

However, since the

animals used in these studies had not been hypophysectomized, the possibility that prolactin might be necessary for the expression of mammary estrogenic responses could not be ruled out.

For instance it has been reported that the augmentation

of both casein synthesis and lactose synthetase activity by estradiol in explants from pregnant mice depends on the presence of both prolactin and thyroid hormone

(9).

It has also

been reported that prolactin may increase the level of mammary estrogen receptors in mice

(10).

Therefore it is possible

420 that at least some of the estrogenic responses in the mammary glands is influenced indirectly by prolactin. C. Loss of estrogenic sensitivity during lactation in mammary glands Extensive studies from several laboratories have revealed that the sensitivity of normal mammary tissue to both protein and steroid hormonal stimuli may be profoundly altered in relationship to the ontogeny of mammary epithelium, i.e. the mammary glands can modulate their sensitivity to hormones as a function of various physiological states

(1).

This also appears to be

true for estrogenic sensitivity of mammary glands since while the glands from castrated virgin mice are responsive to estradiol as shown previously, by the same criteria for responsiveness to estrogen the mammary glands of castrated lactating mice are nonresponsive to estradiol

(Fig. 5).

It is, however, to be

noted that while estradiol does not stimulate glucose oxidation in mammary glands of lactating mice, the glucose oxidation in the mammary gland of saline-treated, ovariectomized, lactating mice is, in fact, approximately 8-fold higher than that seen in the mammary gland of saline-treated, ovariectomized, virgin mice.

Thus, it appears that while estradiol can stimulate

mammary glucose oxidation, this tissue is not necessarily dependent on this hormone to accelerate its metabolism of glucose. This is not surprising since it has been well established that pituitary

(13), adrenal

(14), and thyroid

(15) hormones can

influence several enzymatic reactions associated with glucose metabolism in the lactating mammary gland.

As with the mammary

gland, estradiol also did not stimulate significantly uterine glucose oxidation in lactating mice

(Fig. 5A) although it was

capable of stimulating the rate of glucose phosphorylation in the same tissue

(Table III).

Estradiol was also effective in

increasing the level of progesterone receptor DNA synthesis in uteri of lactating mice

(11).

(12) and rate of Thus, these

data indicate that while estradiol can accelerate several biosynthetic pathways in its target tissues, the mechanisms by

421

which these individual effects are mediated may be different.

200 1h 1h

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Fig. 5. Mammary and uterine responsiveness to estradiol in castrated lactating mice. Mice were castrated on day 2 of lactation, a single injection of saline (C) or 3 ug of estradiol (E) was given on day 9-10 of lactation for the times indicated prior to processing the mammary glands ( • ) and uterus (0). (A) Glucose oxidation [From Shyamala and Ferenczy, 1982 (11)] (B) Progesterone receptor level [From Haslam and Shyamala, 1979 (12)] (C) In vitro incorporation of thymidine into DNA (11).

422

TABLE III. Effect of estradiol on 2-deoxyglucose phosphorylation in uteri of castrated lactating mice Treatment

2-deoxyglucose phosphate

Saline

6501

Estradiol

(1 hr)

9065

Estradiol

(2 hr)

12772

+ + +

(cpm)

1900 980 1289

Three groups of lactating mice between days 7 and 10 of lactation were given a single injection of either saline or 3 yg estradiol for the indicated times prior to killing. [Adapted from Shyamala and Ferenczy, 1982 (11)]

III. Mammary Estrogen Receptors A. Modulation of receptor levels during development It has been widely documented that estrogen receptors are present in normal rodent mammary glands.

Moreover, it has also

been documented that levels of cytoplasmic estrogen receptor in mammary glands are modulated in relation to development such that they are present at the highest level during lactation (16,17,18,19) as illustrated in Fig. 6.

The higher level of

estrogen receptor present during lactation also appears to correlate with the secretory state of the tissue

(Fig. 6).

In contrast to the mammary glands, the uterine estrogen receptor level did not increase during lactation; while the level of estrogen receptor in the uteri from virgin mice was 3297 ± 223 fmoles/uterus, it was 2563 ± 314 fmoles/uterus in lactating mice.

Thus the increase in estrogen receptor levels

during lactation in mammary gland appear to be tissue specific and related to the secretory state of the gland. B. Molecular properties of mammary estrogen receptor Although there is extensive documentation on the mammary estrogen receptors, only limited information is available on the molecular properties of these receptors.

In part this is due

423

Fig. 6. Modulation of mammary cytoplasmic estrogen receptor as a function of lactation.

to the fact that most of the earlier studies had been done prior to the introduction of effective receptor stabilizing agents such as sodium molybdate in the receptor assays and as such were susceptible to degradation and proteolysis during assay.

This in turn led to identification of various forms of

«native» or «non-transformed» mammary estrogen receptor and this led to the conclusion that mammary estrogen receptor exhibits molecular heterogeneity as a function of development (20).

In the presence of sodium molybdate, as shown in Fig. 7

and 8, mammary estrogen receptor from both virgin and lactating mice sediment as 9S species on sucrose gradients.

How-

ever there are differences in the susceptibility of mammary estrogen receptors from different strains of mice to undergo artifactual degradation and such phenomenon is also facilitated by types of buffers used.

As shown in Fig. 9, in con-

trast to the mammary gland from Balb/c mice (Fig. 7), the gland from C 3 H mice have a greater tendency to dissociate into smaller molecular weight components which, however, can be minimized with the use of sodium molybdate.

The molecular

properties of mammary estrogen receptor estimated in the pre-

424

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FRACTION NUMBER Fig. 7. Effect of sodium molybdate on sedimentation profiles of mammary estrogen receptor from virgin mice. The arrows represent the position of standards: G = gammaglobulin, 6.6S; S = bovine serum albumin, 4.4S. Β and Τ refer to the bottom and top of the gradient, respectively. (A) Cytosol from ovariectomized virgin mice either as is ( · , Δ ) or exposed to 10 mM molybdate ( O ) was incubated with 10 nM ( 3 H)estradiol alone ( Ο , · ) or also with a 100-fold excess of unlabelled estradiol ( Δ ) prior to layering on gradients in phosphate buffer. (B) Cytosol from ovariectomized virgin mice either as is ( · ) or exposed to 10 mM molybdate ( O ) was incubated with 10 nM of (3 Η)estradiol prior to layering on gradients in phosphate buffer containing 10 mM molybdate. [From Gaubert et al, 1982 (21)] sence of sodium molybdate as the receptor stabilizing agent are shown in Table IV and as may be seen there are essentially no differences in the properties of estrogen receptor from virgin and lactating mice. The ability of estrogen receptor to undergo dissociation due

425

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FRACTION NUMBER Cytosol from lactating mice either as is ( · , Δ ) or Fig. exposed to 10 mM molybdate ( Ο , • ) was incubated with 10 nM of (3 Η)estradiol either alone ( Ο , Π , · ) or also with a 100-fold excess of unlabelled estradiol ( Δ ) prior to layering on gradients in phosphate buffer only ( Ο , Δ ) or on gradients also containing 10 mM molybdate ( · , • ) . [From Gaubert et al, 1982 (21) ]

to high ionic strength does vary between the mammary tissues of virgin and lactating mice.

The estrogen receptor from lacta-

ting mammary glands are relatively less susceptible to dissociation and this in turn is reflected in their relative inability to undergo in vitro activation due to salt. Although these findings are consistent with the nonresponsiveness of lactating mammary glands to estradiol, it is to be noted that if estradiol is administered to lactating mice, estrogen receptor is found associated with the nuclear fraction

(22). The

precise relationship between estrogen receptor status and mammary responsiveness is therefore not clear at present.

It is

426

Ε

α Ο

Ξ
> ο ο pH ο ο tn + (Ν

α os α S3
E. c o l i rRNA > p o l y

>_ p o l y U > p o l y A : U > p o l y A >

p o l y C as d e t e r m i n e d by c o m p e t i t i o n b e t w e e n the f o r m a t i o n of complexes with synthetic polynucleotides cellulose. Hollander poly

Using

and binding

(10) s h o w e d t h a t the c o p o l y m e r s , p o l y

(dG-dC), were better competitors

h o m o p o l y m e r s or h o m o p o l y m e r d u p l e x e s . e s t r o g e n r e c e p t o r to D N A c e l l u l o s e . i n d i c a t e d that

r e c e p t o r b o u n d m o r e to p o l y d(G-C) A l t h o u g h these studies

(dA-dT)

than single

and

stranded

In this a s s a y ,

d ( A - T ) w a s the m o s t e f f e c t i v e c o m p e t i t o r androgen receptors

to D N A

the s a m e c o m p e t i t i o n a s s a y , K a l l o s &

for b i n d i n g

poly of

Similar studies

with

dihydrotestosterone t h a n p o l y d(A-T)

(38).

indicated that steroid receptors

p r e f e r e n c e s for b i n d i n g

to s y n t h e t i c p o l y m e r s , t h e i r

have

ability

to r e c o g n i z e n u c l e o t i d e b a s e s t r u c t u r e s c o u l d not be inferred.

The p o l y n u c l e o t i d e s e m p l o y e d w e r e

w i t h r e s p e c t to t h e i r s i z e and s e c o n d a r y for a n a l y z i n g

(dT) c e l l u l o s e

receptor complexes

While estradiol

receptor

(dT) c e l l u l o s e , n e i t h e r the h i g h

high capacity serum albumin

(11) d i d , i n d i c a t i n g

istic p r o p e r t y of the s t e r o i d r e c e p t o r s . tide celluloses offer several advantages

01igodeoxynucleoaffinity

in l e n g t h a n d

t h r o u g h t h e i r 5' t e r m i n i , g i v i n g

oligonucleotides a directional polarity. attachment

affinity-

a character-

for use a s

They are r e l a t i v e l y h o m o g e n e o u s

a t t a c h e d to c e l l u l o s e

inter-

could

affinity-low

c a p a c i t y e s t r o g e n b i n d i n g α - f e t o p r o t e i n nor the low

matrixes.

bound

in a m a n n e r a n a l o g o u s to t h e i r

action with DNA-cellulose. b i n d to o l i g o

steroid

(11) b a s e d o n a r e p o r t by T h r o w e r et^

(39) w h o s h o w e d t h a t e s t r a d i o l

to o l i g o

A method

the b a s e r e c o g n i t i o n c h a r a c t e r i s t i c s of

receptors was developed al.

heterogeneous

structure.

The

are

the

covalent

to c e l l u l o s e o v e r c o m e s the p r o b l e m of

dissociation

511

of ligand from the cellulose under experimental conditions. The interference of secondary structure differences is minimal because of the short length of oligonucleotides and their attachment to cellulose.

Effect of Salt Concentration on Steroid Receptor Binding to Oligonucleotide Celluloses Binding of steroid receptors to oligodeoxynucleotide celluloses showed a typical bell-shaped dependence on salt concentration.

The salt dependency of binding varied significantly

among the steroid receptors.

While estradiol receptors bound

optimally in a broad range of monovalent cation concentrations (0.05 to 0.15 Μ KCl), the activated dexamethasone receptors showed a sharp optimum at 0.1 Μ KCl and with testosterone receptors, binding was maximal at low concentrations (< 0.05 M) of KCl. (10,14).

However, the binding of

all steroid receptors to oligonucleotide celluloses decreased at salt concentrations higher than 0.2 M.

Salt-dependence is

a characteristic property of protein : DNA interactions including the cAMP receptor protein (40), lac repressor and other gene regulatory proteins (41).

The salt dependence is

indicative of large electrostatic interactions between the binding domains of DNA and protein.

The binding is presumed

to be entropically driven by the counterion diffusion potential.

The binding of estrogen receptor to the oligodeoxy-

nucleotide celluloses was sensitive to the type of monovalent anions.

Anions which promoted the order structure of water

were favorable for binding while those which disrupted this structure were unfavorable.

Significant differences were

observed in the binding of estradiol receptor to oligo (dT)and oligo (dG)- celluloses in the presence of various anions. The binding of estradiol receptor to oligo (dG) cellulose was

512 less s e n s i t i v e to I , C I O , ' cellulose. anions

-

4

-

,.

and SCN

t h a n to o l i g o

The s e n s i t i v i t y of lac r e p r e s s o r to

(dT)

chaotropic

for b i n d i n g to D N A is b e l i e v e d to be due to

conforma-

t i o n a l c h a n g e s in the DNA b i n d i n g d o m a i n of the

repressor

protein.

estradiol

The differences

in the s e n s i t i v i t y of

r e c e p t o r to c h a o t r o p i c a n i o n s for b i n d i n g oligo

(dG)

to o l i g o

(dT)

c e l l u l o s e s m a y r e p r e s e n t the v a r i a t i o n s

conformational

changes

i n d u c e d by the a n i o n s

the D N A b i n d i n g d o m a i n of the s t e r o i d

and

in the

in s u b s i t e s of

receptors.

P r e f e r e n c e s of S t e r o i d R e c e p t o r s for B i n d i n g

to

01igonucleotides A c o m p a r i s o n of e s t r a d i o l r e c e p t o r b i n d i n g oligodeoxynucleotides

to

homologous

of equal a v e r a g e l e n g t h a t t a c h e d

to

c e l l u l o s e , s h o w e d that the o r d e r of a f f i n i t y w a s o l i g o oligo

(dT)

(Fig. 1).

> oligo

(dC)

>> o l i g o

(dA) > o l i g o

Androgen and glucocorticoid

(dl)

quantitative differences

(14).

corticoid receptor binding

B i n d i n g of t e s t o s t e r o n e

significant

(dT) c e l l u l o s e

(dC), w h i l e

(dT) a n d

r e c e p t o r s to o l i g o

(dA)

lose w a s r e l a t i v e l y b e t t e r than t h a t of e s t r a d i o l o r methasone

receptor

(14).

A s s o c i a t i o n c o n s t a n t s for

receptor-oligodeoxynucleotide

cellulose complexes

d e t e r m i n e d by the t e c h n i q u e d e s c r i b e d (43).

the c y t o s o l e s t r a d i o l eluted

(1 ml)

cellu-

dexaestradiol

were oligodeoxy-

to w h i c h w a s a p p l i e d a s a m p l e

receptor complexes.

The p r o t e i n

from the c o l u m n b y p a s s a g e of a b u f f e r of

salt concentration

oligo

by d e H a s e t h et. a l .

The m e t h o d e m p l o y e d a small c o l u m n of the

nucleotide cellulose

gluco-

was

testosterone

r e c e p t o r s s h o w e d no d i f f e r e n c e s b e t w e e n o l i g o (dC).

showed

Temperature-activated

to o l i g o

m a r k e d l y g r e a t e r t h a n to o l i g o

(13,42)

receptors also

similar binding preferences although there were

(dG) >

(0.15 Μ N a C l ) .

of

was

constant

Association constants

were

Figure 1. Binding of Steroid receptors to oligonucleotide celluloses: (Top left), mouse uterine estrogen receptor (46); (Top right), mouse kidney estrogen receptor (14); (Bottom left), activated dexamethosone receptor (14); (Bottom right) mouse kidney testosterone receptor (14).

514

calculated

from the e l u t i o n d a t a by using

= Vf/j^0)- w h e r e V f

the e q u a t i o n

is the v o l u m e of the f r a c t i o n and

total m o l a r a m o u n t of n u c l e o t i d e s o n the c o l u m n . obtained

Ο

K_D bsd is the

k was

from the s l o p e of the s e m i l o g a r i t h m i c p l o t of

the

r e c e p t o r p r o t e i n r e m a i n i n g o n the c o l u m n a f t e r f r a c t i o n i (Pc^)

v e r s u s the f r a c t i o n n u m b e r .

The association

r e f l e c t the r e l a t i v e a f f i n i t i e s of e s t r a d i o l the o l i g o n u c l e o t i d e

celluloses

(Table 1).

constants

receptors

The

for

observed

Table 1 A p p a r e n t a s s o c i a t i o n c o n s t a n t s for m o u s e estradiol

receptor

. oligodeoxynucleotide

NaCl

oligo(dG)

3.7 χ

104M-1

oligo(dT)

1.75 χ

104M-1

oligo(dC)

1.49 χ

104M_1

v a l u e s are c o m p a r a b l e binding

complexes

RD A p p a r e n t K Q ^^

Oligodeoxynucleotide

and Alberts

uterine

to the c o n s t a n t d e t e r m i n e d by

(4) for e s t r a d i o l

receptor binding

Yamamoto

to D N A .

p a t t e r n of e s t r o g e n r e c e p t o r p r o t e i n l a b e l e d

s t e r o i d or n o n s t e r o i d e s t r o g e n s o r a n t i e s t r o g e n s , the s a m e s e l e c t i v i t y

with

also

showed

(42).

T h e s u b s i t e s of the p o l y n u c l e o t i d e b i n d i n g d o m a i n of receptors were further characterized. h o l o r e c e p t o r to e l e v a t e d t e m p e r a t u r e s c a u s e d a r a p i d loss of b i n d i n g (dC)-, w i t h o u t a f f e c t i n g (13).

The

E x p o s u r e of

estrogen

(37° for 10-30

to o l i g o

the b i n d i n g

(dT)- and

to o l i g o

min.)

oligo

(dG)-

D i s s o c i a t i o n of the b o u n d e s t r o g e n r e c e p t o r

estrogen

cellulose from

515 oligodeoxynucleotide

c e l l u l o s e s , by i n c r e a s i n g

concentrations

of K C l , o r by c i b a c r o n b l u e F3GA s h o w e d that o l i g o

(dG)

bound

e s t r o g e n r e c e p t o r w a s m o r e s t a b l e t h a n the r e c e p t o r b o u n d oligo

(dC)- and ( d T ) - c e l l u l o s e s .

The f o r m a t i o n of the

deoxynucleotide cellulose - estrogen receptor complexes i n h i b i t e d by c i b a c r o n b l u e F 3 G A , p y r i d o x a l p h o s p h a t e , diethyl pyrocarbonate.

T h e s e s t u d i e s s h o w e d that

was

and

higher

c o n c e n t r a t i o n s of the c o m p o u n d s w e r e n e c e s s a r y to i n h i b i t f o r m a t i o n of r e c e p t o r - o l i g o

(dG) c e l l u l o s e c o m p l e x e s ,

for c o m p l e x e s w i t h o t h e r o l i g o d e o x y n u c l e o t i d e (44,45). oligo

Competition

for r e c e p t o r b i n d i n g

insoluble was

if the s o l u b l e o l i g o m e r c o n t a i n e d 8 or m o r e

n u c l e o t i d e s and w a s m o r e p r o n o u n c e d w i t h the h o m o l o g o u s (dT) t h a n w i t h the h e t e r o l o g o u s o l i g o

(dG).

These

d o m a i n of e s t r o g e n r e c e p t o r subsites: nucleotides

binding

is c o m p o s e d of two c l a s s e s

s t a b l e s i t e s w h i c h b i n d to d e o x y g u a n y l a t e

and relatively (N sites)

labile sites which

oligo

observa-

t i o n s led to the s u g g e s t i o n that the p o l y n u c l e o t i d e

sites)

of

(G

interact w i t h

other

(19,46).

A c t i v a t i o n of S t e r o i d R e c e p t o r s to B i n d

Oligodeoxynucleotide

Celluloses It is g e n e r a l l y b e l i e v e d t h a t c y t o s o l

steroid

receptors

undergo a temperature or high salt-mediated change

in c o n -

f o r m a t i o n w h i c h f a c i l i t a t e s t h e i r m i g r a t i o n to the

nucleus

a n d b i n d i n g to the n u c l e a r a c c e p t o r s i t e s . zation

the

than

celluloses

to the

(dT) c e l l u l o s e m a t r i x by the s o l u b l e o l i g o m e r

efficient

to

oligo-

is not a p p l i c a b l e

binding properties.

to DNA and

This

W h i l e the g l u c o c o r t i c o i d

receptors

r e q u i r e a c t i v a t i o n to b i n d j_n v i t r o to D N A , o t h e r r e c e p t o r s d o not

(4).

Studies on

generali-

oligodeoxynucleotide steroid

oligodeoxynucleotide

c e l l u l o s e b i n d i n g p r o p e r t i e s of s t e r o i d r e c e p t o r s

clearly

516 showed due

that no s i g n i f i c a n t

to p r e i n c u b a t i o n

of

tors at t e m p e r a t u r e s tions.

Exposure 30 m i n

controls

receptors process

was

to n u c l e a r

such an a c t i v a t i o n

bind

salt

with

step

for o t h e r or

activation

celluloses.

UTERINE E 2 R

at 2 2 °

for

unacti-

glucocorticoid an

activation

of these

steroid

recep-

concentra-

sites or to D N A .

receptors The

receptors

absence to

oligodeoxynucleotides

by a n u m b e r of l a b o r a t o r i e s

LIVER dexR

of

following

the a c t i v a t i o n

acceptor

observed

i n c r e a s e o v e r the

The b i n d i n g

e i t h e r DNA or p o l y n u c l e o t i d e s reported

high

for 30 m i n or p r e i n c u b a t i o n

(Fig. 2).

is c o n s i s t e n t

was

testosterone

to o l i g o d e o x y n u c l e o t i d e

in a 6 to 12 fold (14)

and

receptors without

to o l i g o d e o x y n u c l e o t i d e s

for b i n d i n g of

bound

to 0.5 Μ KCl

resulted

in binding

18-37°C or w i t h

However glucocorticoid

were minimally

vated

increase

the e s t r o g e n

KIDNEY E 2 R

(4,39,42,47).

KIDNEY TESTOSTERONE

RECEPTOR 1200 -

g 1000 800-

Z) ο

600-

Ρ •c O Ο

Figure 2. Activation requirements of steroid receptors for binding to oligodeoxynucleotide celluloses (14).

517 B i n d i n g of S t e r o i d R e c e p t o r s to

Polyribonucleotides

Although steroid receptors bind preferentially nificant binding strated.

King

to D N A ,

to R N A and p o l y r i b o n u c l e o t i d e s c a n be

(48) s h o w e d that u t e r i n e e s t r o g e n

(rG) >> p o l y

(rC) > p o l y

(rA) > p o l y

demon-

receptor

b o u n d to p o l y n u c l e o t i d e s w i t h an o r d e r of p r e f e r e n c e poly

sig-

where

(rl). L i a o e t

al.

(12) d e t e r m i n e d the r e l a t i v e a f f i n i t i e s of s t e r o i d

receptors

for the p o l y r i b o n u c l e o t i d e s

in

releasing

by t h e i r e f f e c t i v e n e s s

the s t e r o i d r e c e p t o r s b o u n d to D N A - c e l l u l o s e .

(rG) w a s the m o s t e f f e c t i v e p o l y n u c l e o t i d e

in this a s s a y

androgen, estrogen, glucocorticoid and progesterone tors.

Poly

(rC) and p o l y

recently

F e l d m a n e_t a_l. (49) c o n f i r m e d t h e s e s t u d i e s

(rA) w e r e a l m o s t

d e m o n s t r a t e d that in i n h i b i t i n g (rU) >> p o l y

(rA) = p o l y

(rG),

ineffective.

the b i n d i n g of

r e c e p t o r to DNA the o r d e r of a f f i n i t y w a s , p o l y

More and

estrogen (rG) > p o l y

(rC).

It is, t h u s , e v i d e n t that s t e r o i d r e c e p t o r s r e c o g n i z e ferent nucleotide base structures. h a v e the h i g h e s t a f f i n i t y and

All steroid

for

dif-

receptors

for g u a n i n e , the l o w e s t for

intermediate affinities

R o l e of A c c e s s o r y

for

recep-

(rU) w a s a b o u t half as e f f e c t i v e as p o l y

but poly

Poly

adenine

pyrimidines.

Proteins

A l t h o u g h m a n y l i n e s of e v i d e n c e h a v e c l e a r l y

demonstrated

t h a t s t e r o i d r e c e p t o r s b i n d to s p e c i f i c s i t e s o n the D N A , a v a i l a b l e e v i d e n c e h a s not e s t a b l i s h e d solely responsible specific genes.

is

for the r e g u l a t i o n of e x p r e s s i o n of

T h e k i n e t i c s of b i n d i n g

w i t h the p r o p o s e d r e g u l a t o r y r o l e . supporting

that this b i n d i n g

the

is not

consistent

T h e r e are s e v e r a l

the c o n c e p t that f u n c t i o n a l

b i n d i n g of

reports

steroid

518 r e c e p t o r s to g e n o m i c D N A r e q u i r e s a d d i t i o n a l [see r e v i e w by S p e l s b e r g ejt £ 1 . ( 5 0 ) ] .

nuclear

proteins

Complete removal

of

h i s t o n e s and the bulk of n o n h i s t o n e p r o t e i n s from c h i c k oviduct chromatin resulted tightly bound proteins. DNA

for P - R b i n d i n g (51)

receptor

the s p e c i f i c i t y

(P-R) b i n d i n g

following

r e m o v a l of p r o t e i n s from c h r o m a t i n r e v e a l e d

yielded a fraction

(CP-3) c o n t a i n i n g

nucleoacidic

that

which

proteins

t i g h t l y b o u n d to D N A w i t h e n h a n c e d P - R b i n d i n g .

Similar

findings were also reported

prostate chromatin

for a n d r o g e n

receptor-rat

(52) and for e s t r o g e n r e c e p t o r s

calf-uterine chromatin

(53).

In n o n - t a r g e t

in the

t i s s u e s s u c h as

the s p l e e n and the e r y t h r o c y t e w h i c h show little P - R r e m o v a l of h i s t o n e s a n d the bulk of n o n h i s t o n e increased binding acceptor sites

indicating

in m a s k i n g

T h e p r o t e i n s of H o w e v e r , the

s t i t u t e d c o m p l e x of C P - 3 p r o t e i n s w i t h D N A s h o w e d b i n d i n g of P - R ( 5 0 , 5 4 , 5 5 ) .

Further

in two

Monoclonal

pro-

(13-18 k

antibodies

r a i s e d a g a i n s t these p r o t e i n s b l o c k e d p r o g e s t e r o n e to c h r o m a t i n or to the N A P - D N A c o m p l e x

guanidine

These

t e i n s w e r e c h a r a c t e r i z e d as small m o l e c u l a r w e i g h t

binding

CP-3

fractions

w h i c h p r o m o t e d e n h a n c e d b i n d i n g of P - R to D N A . (50).

recon-

f r a c t i o n a t i o n of the

h y d r o c h l o r i d e b u f f e r e d at pH 6, r e s u l t e d

in n a t u r e

CP-3

marked

p r o t e i n s by m o l e c u l a r s i e v e c h r o m a t o g r a p h y using 6M

and hydrophobic

binding,

proteins,

t h a t m a y be i n v o l v e d

in n o n - t a r g e t t i s s u e s .

f r a c t i o n did not b i n d to P - R d i r e c t l y .

Da)

of

tissues.

r e m o v a l of h i s t o n e s a n d bulk of n o n h i s t o n e p r o t e i n s , (NAP)

chroma-

binding

from t a r g e t a n d n o n t a r g e t

E x a m i n a t i o n of p r o g e s t e r o n e sequential

few

than native

in that r e c e p t o r

f o l l o w e d s a t u r a t i o n k i n e t i c s and r e f l e c t e d o r i g i n of the c h r o m a t i n

a

This structure designated NAP was a

more physiologic template tin o r d e p r o t e i n i z e d

in a f r a c t i o n c o n t a i n i n g

receptor

(56).

DNA

s e q u e n c e s w h i c h are t i g h t l y b o u n d to the C P - 3 p r o t e i n s are a c l a s s of

intermediate

communication).

repetitive DNA

(Spelsberg,

personal

The s i g n i f i c a n c e of t h e s e s i t e s in the

p r o d u c t i v e b i n d i n g of P - R r e m a i n s to be

elucidated.

519 In a d d i t i o n to the n u c l e a r p r o t e i n s , w h i c h u p o n b i n d i n g specific sites on genomic DNA enhance ing to s u c h s i t e s , t h e r e are c y t o s o l stimulate steroid binding nucleotide polymers.

steroid receptor proteins which

to i m m o b i l i z e d D N A o r

T h r o w e r et a l .

also

synthetic

(39) s h o w e d t h a t

t i o n of u t e r i n e c y t o s o l to S e p h a d e x g e l

filtered

addi-

uterine

c y t o s o l i c e s t r o g e n r e c e p t o r s s t i m u l a t e d b i n d i n g of the t o r s to o l i g o

(dT) c e l l u l o s e .

n e e d for c y t o s o l eluted oligo

from o l i g o

It a p p e a r e d t h a t d u r i n g

a n d d e s o r p t i o n of e s t r o g e n r e c e p t o r ( s ) affinity matrix.

fresh

the

from o l i g o

adsorption (dT)

cellu-

f a c t o r or f a c t o r s r e m a i n e d b o u n d to O n e f a c t o r was s h o w n to be

the

heat-stable,

n o n d i a l y z a b l e , s e n s i t i v e to p r o n a s e , p r o t e i n a s e κ, p e p s i n S t a p h . a u r e u s p r o t e a s e V , b u t r e s i s t a n t to t r y p s i n o r trpysin.

Such factors were present

a

receptor

(dT) c e l l u l o s e c o l u m n , to r e b i n d

l o s e , an a c c e s s o r y

recep-

T h a n k i et a_l. (57) d e t e c t e d

f a c t o r s for m o u s e k i d n e y e s t r o g e n

(dT) c e l l u l o s e .

to bind-

in c y t o s o l s

and

chymo-

from

kidney

u t e r u s and lung b u t n o t in b r a i n and s k e l e t a l m u s c l e

cyto-

sols.

oligo-

The h e a t - s t a b l e

nucleotide celluloses

f a c t o r w a s e f f e c t i v e w i t h all as b i n d i n g m a t r i c e s .

Prior

incubation

w i t h the m a t r i c e s a l s o s t i m u l a t e d r e c e p t o r b i n d i n g

and

there

w a s no i n d i c a t i o n t h a t the factors d i r e c t l y r e a c t e d w i t h holoreceptor. to DNA

The r o l e of s u c h f a c t o r s

is u n k n o w n a t this

in s p e c i f i c

time.

D u r i n g o u r s t u d i e s o n the a c c e s s o r y p r o t e i n s for the of e s t r o g e n r e c e p t o r to o l i g o d e o x y n u c l e o t i d e

i n c r e a s e d the r e b i n d i n g of p a r t i a l l y

h o l o r e c e p t o r to o l i g o ( d T )

cellulose.

Fox et. al. (58)

f o u n d that e s t r o g e n a n d a n d r o g e n r e c e p t o r s b o u n d m o r e ly to DNA c e l l u l o s e al.

in the p r e s e n c e of l y s o z y m e s .

(57) d e m o n s t r a t e d

it

calf purified also strong-

Thanki

that H 2 A , H 2 B a n d H3 w e r e m o s t

t i v e w h i l e the l y s i n e - r i c h HI w a s i n a c t i v e .

binding

celluloses,

w a s o b s e r v e d that b a s i c p r o t e i n s s u c h as l y s o z y m e and thymus histones

the

binding

£t

effec-

Polylysine

and

520 γ-globulin were ineffective.

Addition of individual

histones

to a 200-fold purified mouse kidney estrogen receptor complex resulted in the stabilization of the steroid bound to the receptor.

H2A and H2B were most effective, HI and H3 were

less effective while lysozyme and γ-globulin were ineffective.

Histone H2B was cleaved by cyanogen bromide into a

N-terminal half containing 32% of the residues as basic amino acids and the C-terminal half which contained 20% of its residues as basic amino acids.

Both N-terminal and

C-terminal half molecules were tested for their ability to stimulate the oligo (dT)-cellulose binding of estrogen holoreceptor.

The N-terminal half was more active than the

C-terminal half, while the mixture of N- and C-terminal half produced an intermediate effect (Fig. 3).

_

60

ο*

Μ lormlnnl

α

Ζ m

20 ο

10

20

H2b HALF-MOLECULE ADDED tyiq)

Figure 3. Effect of N- and C-terminal half molecules of H2B on oligo (dT) cellulose binding of estradiol receptor (57).

521 The N - t e r m i n a l half of H2B s t a b i l i z e d

the 4S s p e c i e s of

h o l o r e c e p t o r w h i l e a d d i t i o n of the C - t e r m i n a l half m a r k e d a g g r e g a t i o n of r e c e p t o r p r o t e i n al.

(Fig. 4).

(59) o b s e r v e d that r a b b i t u t e r i n e c y t o s o l

the

led to Kallos

receptors

the et

bound

to the i m m o b i l i z e d h i s t o n e s w i t h the a f f i n i t y o r d e r ; H2B > H2A >> H4 M O

>>> H i .

F o r m a t i o n of a c o m p l e x b e t w e e n

estro-

g e n r e c e p t o r s and h i s t o n e s w a s s h o w n b y a s h i f t in the e l e c t r i c p o i n t to h i g h e r pH v a l u e s . tinct binding site, separate

iso-

The p r e s e n c e of a d i s -

from e s t r o g e n and

polynucleotide

b i n d i n g d o m a i n s , w a s d e t e c t e d by l i m i t e d p r o t e o l y s i s . p a r t i a l l y d i g e s t e d e s t r o g e n r e c e p t o r D N A b i n d i n g of r e c e p t o r was m a r k e d l y r e d u c e d w h i l e s t e r o i d and b i n d i n g w e r e u n a f f e c t e d . T h u s , it a p p e a r s that receptors have a site(s)

histone estrogen

for s e l e c t i v e b i n d i n g of

h i s t o n e s or s i m i l a r c a t i o n i c p r o t e i n s . containing

In the

the

nucleosomal

D i g e s t i o n of

nuclei

radio-labeled estrogen receptors, followed

density gradient centrifugation

i n d i c a t e d that the

a c t i v i t y w a s p r i m a r i l y a s s o c i a t e d w i t h the

by

radio-

mononucleosomal

f r a c t i o n s w h i c h c o n t a i n e d H2A, H 2 B , H3 a n d H4 h i s t o n e s not HI

(60,61).

These observations may

r o l e for the h i s t o n e s receptor

molecules.

indicate a

in the n u c l e a r p o s i t i o n i n g

of

but

specific the

522

(·) No addition (o)+ H2b ES

Γ

(ο) + Ν terminal halfmolecule (·)+ C terminol balf-molecute 4.6 S i

4 6 5

Φι '

A^

\r r 'Ρ \\ ΐ DE

i

; Ά φ

-

9

j\

Ν

r

eexes

Ο

I

/

I I I 10 20 Ο FRACTION

10

20

Figure 4. Stabilization of estrogen holoreceptor by the island C-terminal half molecules of H2B (57).

The effect of histone H2B on oligodeoxynucleotide properties of estrogen receptors also indicated interesting feature.

binding

another

When uterine cytosolic estrogen recep-

tor complexes were bound to the immobilized

oligodeoxynucleo-

tides and subsequently eluted with 0.5 Μ KCl, the receptor complexes obtained were incapable of binding to oligo (dT), oligo (dC) and oligo (dA), while retaining the binding to oligo (dG) at reduced levels.

Addition of H2B to this

preparation fully restored the binding to all the oligo-

523 nucleotide celluloses in the order seen with crude estrogen receptors (Table 2).

This property may reflect the ability

of histones or histone-like cationic proteins to modulate the recognition capabilities of the DNA-binding domain of steriod receptors in general, and estrogen receptors in particular.

Table 2 Rebinding of E 2 R eluted from oligo(dT)- and oligo(dG)cellulose complexes to oligodeoxynucleotide-celluloses

% bound of input E_R

E2R

off

E 2 R off

oligo(dT)-•eellulose

oligo(dG)-•cellulose

Cellulose

-H2B

+ H2B

-H2B

+ H2B

Oligot dG)

18

33

21

32

01 igo(dA)

0

6

0

6

Oligo(dC)

0

12

0

14

Oligo(dT)

0

22

1

23

E 2 R oligo(dG)- or oligo(dT)-cellulose complexes were formed under standard binding assay conditions. E 2 R was achieved by using 0.5 Μ KCl.

Elution of Bound

Samples (0.2 ml) of the

eluates were used in the rebinding assay with fresh oligodeoxynucleotide-celluloses

in a total volume of 0.6 ml so

that the final concentration of KCl was 0.15 Μ.

H2B was

added to the rebinding assay at 50 pg where indicated.

524 S t e r o i d R e c e p t o r R e c o g n i t i o n of S p e c i f i c DNA The d e m o n s t r a t i o n

that steroid hormone

criminate between nucleotide bases

Sequences

receptors could

i n d i c a t e d that

DNA base sequence

r e c o g n i t i o n w a s a p o s s i b l e step in

action.

it r e m a i n e d

However,

for the t u m u l t u o u s

tumor virus

(MMTV) t r a n s c r i p t i o n b e f o r e a d e t a i l e d

interaction was forthcoming.

a d d i t i o n of g l u c o c o r t i c o i d

Studies

to p r o v i r a l c o n t a i n i n g

(62).

analysis

taining cells

(63).

cells short

into g l u c o c o r t i c o i d

con-

E x o g e n o u s M M T V D N A not i n t e g r a t e d

These experiments

into to the

i n d i c a t e d that the v i r a l D N A

s u f f i c i e n t for h o r m o n e r e s p o n s i v e n e s s and that h o s t D N A not i n v o l v e d These

f i n d i n g s set the s t a g e for a d e t a i l e d s e a r c h for

t i o n e n d o n u c l e a s e s a n d r e c o v e r e d a 1.45 kb s e g m e n t the long t e r m i n a l r e p e a t r e g i o n They transfected

plasmid containing dine kinase recipient

(tk)

(LTR)

and a small

restric-

containing coding

this c l o n e d p r o d u c t along w i t h a

the h e r p e s s i m p l e x v i r u s g e n e for

into m o u s e L c e l l s w h i c h w e r e tk

transformants were tk+ when grown

of g l u c o c o r t i c o i d s .

A n Jji v i v o a s s a y

.

in the

thymiThe

presence

is now a v a i l a b l e

d e t e r m i n i n g w h a t p a r t of the M M T V g e n o m e c o n t a i n e d the mone responsive

the

proviral

Fasel et aJL. (65) c l e a v e d the M M T V g e n o m e w i t h

region.

was was

(65,66).

g l u c o c o r t i c o i d r e c e p t o r e f f e c t o r r e g i o n w i t h i n the DNA.

of

hormone

receptor

the h o s t c e l l c h r o m o s o m e a l s o w a s c a p a b l e of r e s p o n s e hormone.

that

Furthermore, specific fractions

M M T V D N A w e r e e n o u g h to m e d i a t e a g l u c o c o r t i c o i d response when transformed

mammary

indicated

i n c r e a s e d the rate of M M T V D N A t r a n s c r i p t i o n a f t e r p e r i o d s of e x p o s u r e

hormone

developments

in the role of g l u c o c o r t i c o i d s a s i n d u c e r s of m o u s e of t h i s

dis-

specific

for hor-

region.

H y n e s a n d her c o w o r k e r s

(66) c o n s t r u c t e d c h i m e r a of the

L T R a n d a HSV tk c o d i n g

segment.

T h e y w e r e a b l e to

MMTV

produce

525 n u c l e o t i d e d e l e t i o n s at d e f i n e d d i s t a n c e s synthesis

i n i t i a t i o n or CAP site

at w h i c h m e t h y l g u a n o s i n e

residue

is a t t a c h e d ) .

f o r m e d c e l l s , RNA s y n t h e s i s w a s c o r r e c t l y LTR promoter termination Among

the d e l e t i o n s ,

-202 were responsive

5' to the

RNA

( p e n u l t i m a t e 5" e n d of m R N A In the

initiated

in the r i g h t w a r d tk c o d i n g

sequence was sufficient

for r e s p o n s e .

B u e t t i and D i g g e l m a n n ( 6 7 )

indicated

- 1 0 5 to - 2 0 4 w a s n e c e s s a r y

for the h o r m o n e

by

from

response. these

found t h a t s e q u e n c e s b e t w e e n - 2 0 4 to - 6 0 0 multiple

w e a k e r s i t e s of c o n t r o l , u p s t r e a m of the p r i m a r y W h e n the - 1 0 5 m u t a n t w a s u s e d level of c o r r e c t l y

response

in the t r a n s f e c t i o n ,

the

initiated transcripts was nearly

the

s a m e as that seen w i t h the e n t i r e M M T V D N A .

The

c o i d r e c e p t o r e f f e c t o r r e g i o n of D N A w a s c l e a r l y from t h o s e n u c l e o t i d e s c o m p r i s i n g promoter.

LTR

Similar experiments

i n c r e a s e d the a m p l i t u d e of r e s p o n s e s u g g e s t i n g site.

LTR

site

that the s e q u e n c e

A l t h o u g h the c o r r e c t t r u n c a t e d L T R w a s s u f f i c i e n t ,

basal

to

to g l u c o c o r t i c o i d s w h i l e t h o s e w h o s e

T h i s i n d i c a t e d that a l i m i t e d r e g i o n of the

investigators

the

region.

t h o s e w i t h the s e q u e n c e s from - 1 3 7

s e g m e n t s e n d e d a t - 5 0 or - 3 7 r e l a t i v e to the LTR C A P were not.

trans-

from

the L T R

glucocortiseparate

transcriptional

F u r t h e r a l t e r a t i o n s of the c h i m e r i c c l o n e

(68)

i n c l u d i n g a d d i t i o n of 4 e x o g e n o u s b a s e p a i r s o r d e l e t i o n 20 b a s e p a i r s at p o s i t i o n - 1 0 7 o r s u b s t i t u t i o n of the

of

rat

s a r c o m a v i r u s L T R for that of MMTV at p o s i t i o n - 5 9 to + 1 0 0 h a d no e f f e c t o n g l u c o c o r t o c o i d A n a l y s i s of the a b o v e s t u d i e s

responsiveness.

indicated that a limited

s e q u e n c e w i t h i n the M M T V L T R w a s n e c e s s a r y

for

glucocorticoid

r e s p o n s i v e n e s s b u t the p o s i t i o n of t h i s s e g m e n t had no

fixed

spatial distance

the

from the t r a n s c r i p t i o n a l p r o m o t e r a n d

r e s p o n s e r e g i o n c o u l d be d i s s o c i a t e d

f r o m a weak b u t

tional promotor, suggesting

that a s p e c i f i c

c o n t a c t w i t h RNA p o l y m e r a s e

II w a s not a n e s s e n t i a l

e n t for the r e c e p t o r e f f e c t .

func-

protein-protein ingredi-

The i m m e d i a t e c o n s e q u e n c e

of

526 receptor

i n t e r a c t i o n w a s p r o p o s e d to be a local c h a n g e

chromatin configuration

a n d / o r p r o d u c t i o n of a f a c i l e

s i t e for R N A p o l y m e r a s e

II o r a c c e s s o r y

in entry

transcriptional

factors. A complementary

l i n e of r e s e a r c h w a s

of P a y v a r and his c o w o r k e r s

(15).

i n i t i a t e d by the

They

found

b i n4 d i n g of rat l i v e r g l u c o c o r t i c o i d h o l o r e c e p t o r , 10

finding

selective purified

f o l d , to be r e s t r i c t e d to s e l e c t e d r e g i o n s of the

pro-

v i r a l D N A , s o m e w i t h i n the L T R r e g i o n a n d some in c o d i n g r e g i o n s of the v i r a l g e n o m e .

S c h e i d e r e i t et^ a_l. (70)

using

the d e f i n e d L T R d e l e t i o n c o n s t r u c t s of the L T R -v HSV Tk chimera

i n t r o d u c e d by H y n e s £ t a ^ .

of 2 s p e c i e s of rat g l u c o c o r t i c o i d 40 Κ m o l e c u l e . binding

(66) s t u d i e d the

h o l o r e c e p t o r , a 90 Κ and a

Both species were alike

to Μ M T V D N A .

binding

in t h e i r

preferential

If the c h i m e r i c D N A c o n t a i n e d

segments

including

nucleotide -202 relative

sequences

5' u p s t r e a m

binding.

If t h e y i n c l u d e d s e q u e n c e s - 5 0 o r l e s s , t h e r e

no b i n d i n g .

to the CAP site o r m o r e

from that p o s i t i o n , there w a s

C o m p l e x e s of the 90 Κ r e c e p t o r :

selective was

DNA w e r e

r e c o v e r e d by i m m u n o p r e c i p i t a t ion u s i n g m o n o c l o n a l

antibodies

d i r e c t e d a g a i n s t the n o n - D N A b i n d i n g p o r t i o n s of the

receptor

a n d it d i d not m a t t e r w h e t h e r p u r i f i e d r e c e p t o r o r a c r u d e cytosol preparation was

used.

These data suggested that non-receptor cytosol w e r e not e s s e n t i a l

components

for s e l e c t i v e r e c e p t o r b i n d i n g b u t

v a t i o n of the g l u c o c o r t i c o i d r e c e p t o r w a s n e c e s s a r y , NaMoC> 4 p r e v e n t e d the i n t e r a c t i o n . w a s the d e t e r m i n a t i o n

Of p a r a m o u n t

actias

importance

t h a t a r e g i o n of the M M T V L T R f r o m - 5 0

to - 2 0 2 w a s the b i n d i n g

site w h i c h w a s the same a r e a

as the h o r m o n e r e s p o n s e r e g i o n in t r a n s f o r m a t i o n

defined

experiments.

H a v i n g e s t a b l i s h e d a s e g m e n t of the L T R D N A as a p r e f e r e n t i a l binding

t a r g e t for g l u c o c o r t i c o i d

holoreceptor,

it

remained

527 for S c h e i d e r e i t a n d B e a t o

(70) to p i n p o i n t

for the r e c e p t o r w i t h i n this s e g m e n t .

the c o n t a c t

c h e m i c a l and e n z y m a t i c p r o b e s , t h e y e s t a b l i s h e d t h a t were

four b i n d i n g s i t e s in a s e q u e n c e

o t i d e s 5' to the C A P s i t e . a)

In the r e c e p t o r :

points

Using a combination from - 7 2 to - 1 9 2

A t t h e s e s i t e s they

of

there nucle-

found:

DNA c o m p l e x , s e v e r a l g u a n i n e

residues

w e r e p r o t e c t e d a g a i n s t m e t h y l a t i o n by d i m e t h y l

sulfate.

At the 4 s i t e s , the p r o t e c t e d r e s i d u e s w e r e f o u n d in a common

sequence: 5 ' T G T

Τ C Τ 3'

3' A C A

A G A 51

T h e p r o t e c t i o n at the 4 s i t e s i n c r e a s e d w i t h r i s i n g concentrations. w i t h i n the

receptor

H y p e r m e t h y l a t i o n of o t h e r b a s e s a l s o

occured

complex.

b)

Binding

to the s i t e s o c c u r e d

c)

M e t h y l a t i o n of a l i m i t e d n u m b e r of g u a n i n e

tive amplification was

independently but

coopera-

possible.

abolished glucocorticoid

residues

holoreceptor binding.

In the

s e n s e s t r a n d the g u a n i n e r e s i d u e at - 1 7 4 and in the a n t i s e n s e s t r a n d t h o s e at - 1 7 1 a n d - 1 8 0 w e r e the most protected against methylation

bases

in the r e c e p t o r :

DNA

complex. d)

No c l e a r cut e f f e c t s of p r o t e c t i o n o r i n h i b i t i o n observed

e)

Among

in a d e n i n e

were

residues.

the four s i t e s , that at the m o s t u p s t r e a m

position

d e m o n s t r a t e d g r e a t e r a v i d i t y for the h o l o r e c e p t o r the o t h e r s .

B u t at the l o w e s t r e c e p t o r

all s i t e s b o u n d f)

concentrations,

receptor.

The p r o t e c t e d g u a n i n e r e s i d u e s w i t h i n the c o m p l e x separated Since

than

from e a c h o t h e r by 10 + 1 n u c l e o t i d e

it is the N7 p o s i t i o n of g u a n i n e w h i c h

l a t e d and it is e x p o s e d of the h e l i x , the

pairs.

is m e t h y -

in G : C p a i r s of the m a j o r

investigators concluded

were

groove

that p o i n t s

of

m a x i m a l c o n t a c t of the r e c e p t o r w e r e o n the s a m e face of

528 the h e l i x at two g u a n i n e r e s i d u e s s e p a r a t e d by 2 full c o n s e c u t i v e A c o m p a r i s o n of o t h e r g l u c o c o r t i c o i d flanking sequence

in the m a j o r

groove

turns. regulated genes

indicated either similar binding

city or sequence homologies.

5'

specifi-

In the c l o n e d g e n e of

h

m e t a l l o t h i o n e i n II u n d e r r e g u l a t i o n b y the h o r m o n e as w e l l c a d m i u m , t h e r e w a s one strong b i n d i n g to - 2 6 5 from the C A P s i t e .

s i t e ; l o c a t e d at - 2 4 5

S e q u e n c e a n a l y s i s of

those

r e g i o n s p r o t e c t e d a g a i n s t D N a s e I d i g e s t i o n by b o u n d corticoid holoreceptor containing

(footprinting)

yielded a

gluco-

sequence

5' T G T T C T 3' c o m m o n in d i g e s t i o n p a t t e r n s of

the

p r o t e c t e d M M T V L T R r e g i o n at two s t r o n g b i n d i n g

sites.

5' f l a n k i n g

tyrosine

r e g i o n s of t r y p t o p h a n o x y g e n a s e and

transaminase

(73,74)

The

h a v e also b e e n s e q u e n c e d y i e l d i n g

the

h o m o l o g o u s a r e a s at p o s i t i o n s r e l a t i v e l y e q u i v a l e n t to

the

transcriptional near

i n i t i a t i o n of M M T V

identical sequence

i.e. 5' T X A G T T C T 3'.

in four g l u c o c o r t i c o i d

b i n d i n g of the h o l o r e c e p t o r .

Yet differences

i n d u c t i o n , m u l t i p l i c i t y of s i t e s , e t c .

tional

f a c t o r s m a y be i m p o r t a n t

in e a c h

in the

interaction with control

the

kinetics addi-

case.

r e g i o n s of d e f i n e d

s e q u e n c e s are those of the c h i c k o v i d u c t egg w h i t e C o m p t o n and h i s c o w o r k e r s

for

indicate that

O t h e r s t e r o i d r e g u l a t e d g e n e s s t u d i e d at the level of receptor

The

regulated

genes strongly suggest a primary recognition template of

as

(74) f o u n d p u r i f i e d

holoDNA

proteins.

progesterone

r e c e p t o r s u b u n i t A , d e r i v e d from m a t u r e h e n o v i d u c t s ,

pref-

e r e n t i a l l y b o u n d to c l o n e d

gene.

f r a g m e n t s of the o v a l b u m i n

T h e y u s e d the r e t e n t i o n of r e c e p t o r b o u n d DNA o n ulose

nitrocell-

f i l t e r s as a m e a s u r e of b i n d i n g w i t h s u b s e q u e n t

elution

and e l e c t r o p h o r e s i s a s a m e a n s of i d e n t i f i c a t i o n of the

DNA

fragment.

the

An interesting

d e t a i l of t h e i r work was t h a t

r e c e p t o r did not d i s t i n g u i s h b e t w e e n s p e c i f i c and

nonspecific

529 DNA at low temperatures; following

incubation at 37°C, the A

subunit bound selectively to a small fragment of the ovalbumin gene compared to a larger plasmid fragment or a 6 molar excess of chicken α globin gene sequences.

The ovalbumin

fragment used was OV 1.7 which contained 1,338 nucleotides of the 5' flanking region and 589 nucleotides of coding tions.

sec-

These authors estimated that there was a 10 fold

difference in affinity of binding to specific as opposed to nonspecific

sequences.

Using an alternative means of assay, Mulvihill and her coworkers (75) found selective binding of progesterone receptor to several cloned segments of the egg white proteins including ovalbumin, conalbumin, ovomucoid, the X and Y pseudogenes.

These workers measured the displacement of

holoreceptor from nonspecific calf thymus DNA cellulose by effective gene segment competitors.

Crude or partially

purified progesterone receptor, subunit A, were about the same in their binding descrimination with maximal occuring at 0.05 - 0.08 Μ KCl or NaCl.

interaction

At lower ionic

strengths, the receptors aggregated and at higher ionic strengths, they dis sociated from the DNA.

The DNA cellulose

assay is a measure of equillibrium binding while the nitrocellulose filter assay the holoreceptor to DNA.

determines the rate of association of Deletion mutants were constructed

from a 1.7 kb cloned segment including the 5' flanking

region

of the ovalbumin gene by successive digestions with exonuclease III and SI nuclease or Bal 31 exonuclease.

These dele-

tions demonstrated 3 binding regions existing with a 40 fold increase in relative binding affinity.

When a computer

analysis, set at a cut off of 60% homology, was made of the 14 kb known sequences of egg white proteins a consensus 19 base pair sequence was identified 42 times in the overall group of genes.

The sequence is:

A T C £ £ A T T £ T C T G £ T T G T A

530 Of the s e g m e n t s c o n t a i n i n g were selective binding tive binding sequence

the h o m o l o g o u s s e q u e n c e , 41 of

t e m p l a tes.

T h e f i d e l i t y of the

is h o w e v e r a m a t t e r of

42

puta-

current

research. In a n o t h e r s t u d y (77) the p u r i f i e d A s u b u n i t of progesterone

oviduct

r e c e p t o r w a s found p r e f e r e n t i a l l y c o m p l e x e d to a

s e g m e n t of the 5' f l a n k i n g r e g i o n of the o v a l b u m i n g e n e - 1 3 5 to - 2 4 7 r e l a t i v e to the t r a n s c r i p t i o n a l

initiation site.

e l u c i d a t i o n of s t e r o i d c o n t r o l of o v a l b u m i n e x p r e s s i o n

suf-

f e r e d b e c a u s e a lack of an jji v i v o t r a n s f e c t i o n a s s a y

of

biological responsiveness

tk

chimeric transformants (78,79)

s i m i l a r to the M M T V L T R - H S V

in tk - L c e l l s .

l i n k e d the 5' f l a n k i n g

including

the t r a n s c r i p t i o n a l

ß - g l o b i n g e n e and a n SV40 (pSV-OG).

Recently, Dean et al.

r e g i o n of the

ovalbumin

s t a r t s i t e to a s e g m e n t of

early g e n e u n d e r

its o w n

r e g i o n - 9 5 to - 2 2 1 w a s n e e d e d

s t e r o i d e n h a n c e m e n t b u t not for b a s a l

l e v e l s of

the

control

In t r a n s f e c t e d p r i m a r y o v i d u c t c e l l s , they

t h a t the 5' f l a n k i n g

The

found

for

synthesis.

A g a i n d e l e t i o n m u t a n t s of the c o n s t r u c t w e r e used to d e f i n e the e f f e c t o r r e g i o n . tive

in i n c r e a s i n g

E s t r o g e n and p r o g e s t e r o n e w e r e

t r a n s c r i p t i o n of t h e s e c h i m e r a s .

c o n c l u d e d o n the b a s i s of

individual

They

hormone effects on a

s e r i e s of d e l e t i o n s t h a t p r o g e s t e r o n e l a p p i n g e f f e c t o r s i t e s o r they w e r e

effec-

and estrogen had

in c l o s e

proximity.

H o w e v e r , this m a y b e m o r e c o m p l e x as the a u t h o r s s t a t e p r e l i m i n a r y note t h a t w h e n the 5' f l a n k i n g r e g i o n of b u m i n is s p l i c e d to a h e t e r o l o g o u s p r o m o t e r and the is t r a n s f e c t e d , insensitive.

over-

it is p r o g e s t e r o n e s e n s i t i v e and

in a

ovalchimera

estrogen

531 F i n a l l y , o t h e r g e n o m i c s e g m e n t s h a v e b e e n used to

transform

recipient cells and have maintained hormonal response these

include

lysozyme gene

«j μ globulin

(80), g r o w t h h o r m o n e

(83), t r y p t o p h a n o x y g e n a s e

s t e r o i d b i n d i n g p r o t e i n (85) s e g m e n t s .

—

(81,82),

(84) and

prostatic

The structural

deter-

m i n a n t s of r e c e p t o r b i n d i n g and h o r m o n e r e s p o n s e s h o u l d forthcoming

in the n e a r

be

future.

Conclusion T h i s c h a p t e r has r e c o u n t e d regarding

the s h i f t of the

steroid hormone receptors

From a questionable

pendulum

interaction with

n o n s a t u r a b l e c h a r a c t e r of the b i n d i n g that receptor binding

to the

and hormone response

the

in the 5'

r e g i o n of n u m e r o u s s t e r i o d r e g u l a t e d g e n e s .

Our

f o c u s h a s b e e n o n the role of s t e r o i d h o r m o n e r e c e p t o r s

as

DNA nucleotide sequence recognition proteins, analogous

to

the m y r i a d of p r o k a r y o t i c g e n o m i c r e g u l a t o r y p r o t e i n s lac r e p r e s s o r , C A M P b i n d i n g p r o t e i n . p o u n d e d b y the e v i d e n c e 1.

The s t e r o i d - and DNA b i n d i n g

Steroid hormone

is c o m -

s i t e s of

ligand

steroid

separated.

B i n d i n g of the r e c e p t o r to D N A b i n d i n g of small

3.

The a n a l o g y

i.e.

that:

r e c e p t o r s are s p a t i a l l y 2.

is m o d u l a t e d by

their

molecules.

receptor action

is s e n s i t i v e

to

i n h i b i t o r s w h i c h m o d i f y c a t i o n i c a m i n o a c i d s at putative DNA binding 4.

and

determination

is b a s e d on

p r e s e n c e of a l i m i t e d , d e f i n e d s e q u e n c e p r e s e n t flanking

DNA.

i m p o r t a n c e b e c a u s e of the n o n s p e c i f i c

the

sites.

S t e r o i d h o r m o n e r e c e p t o r b i n d i n g to DNA is to a g e n t s w h i c h r e s t r u c t u r e a t the t e m p l a t e b i n d i n g

the s e c o n d a r y

site.

sensitive structure

532 Underlying the biological importance of interaction with a particular sequence heterogeneity is the ability of the steroid holoreceptor to discriminate among the nucleotide bases within the helix.

Numerous studies using homologous or

heterologous polyribonucleotides,

polydeoxyribonucleotides

and oligodeoxynucleotides indicating that steroid holoreceptors have common nucleotide base preferences for binding. These are dG or G > dT or U > dC or C >, dA or A.

Perhaps

the real meaning of all the nucleic acid, native and synthetic, binding studies are that receptors possess capabilities of interacting in particular ways with each of these bases in a distinct manner.

An indication of the

potential biological meaning of these affinities is the critical role that the three dG base residues play in the binding of glucocorticoid receptors to sequences flanking the promoters of MMTV and h metallothionein II transcription. Obviously, this clone is insufficient to explain the specificity of interaction for there are numerous regions containing dG residues on the same face of helix at two consecutive turns.

But it does underlie the anchoring role

that dG residues play in the receptor:specific sequence interact ion.

Two other aspects of DNA interaction may come to the fore as purified participants are studied.

First that the DNA

binding site of steroid receptors contain subsites - some stable and others malleable.

The latter probably are of

critical importance in deciphering the specific binding/ effector sequence.

Secondly, the latter malleable sites are

subject to allosteric modulation by neighboring chromosomal proteins.

The pendulum has swung very far from the time when

chromosomal proteins were considered the nuclear acceptor sites for steroid receptors but the protein composition surrounding the DNA binding sequence is probably of crucial importance -- in defining permissive or nonpermissive

533 chromatin targets, stabilizing or destabilizing sequence

steroid

receptor:specific

nucleotide

a c c e s s to b i n d i n g

s i t e s w i t h i n the g e n o m e or m o d u l a t i n g

r e g u l a t o r y a r e a s n e a r the g e n o m e , ments,

interactions,

i.e. e n h a n c e r - l i k e

i n d e p e n d e n t of the r e c e p t o r b i n d i n g

s e n s i t i v e to r e c e p t o r

blocking ele-

sites which

: DNA c o m p l e x a t i o n .

In this

are

regard,

the e v i d e n c e that t i g h t l y b o u n d n o n h i s t o n e p r o t e i n s of o v i d u c t c h r o m a t i n are r e q u i r e d for the s a t u r a t i o n a n d t a r g e t cell s p e c i f i c i t y of p r o g e s t e r o n e Chromatin alteration Z a r e t and Y a m a m o t o

kinetics

receptor

is c u r r e n t l y v i s u a l i z e d as a n

e a r l y c o n s e q u e n c e of s t e r o i d r e c e p t o r (85)

: DNA

chick

binding.

important

interaction.

found that a s i n g l e D N a s e 1 s i t e

was

h y p e r s e n s i t i v e w i t h i n a c h i m e r i c c o n s t r u c t of M M T V L T R - H S V

tk

transfected

to

nuclease

into tk

L cells.

The s e n s i t i v i t y of the s i t e

i n c r e a s e d w i t h e x p o s u r e of the r e c i p i e n t c e l l s

d e x a m e t h a s o n e and r e s c i n d e d w i t h w i t h d r a w a l of the Other sites were more modestly sensitive

to

hormone.

to DNase 1 d i g e s t i o n

w i t h h o r m o n e a d d i t i o n but r e m a i n e d so d e s p i t e h o r m o n e

deple-

tion.

chrom-

T h e s e a u t h o r s d e f i n e d three c l a s s e s of c h i m e r a

a t i n s t r u c t u r e a c o n s t i t u t i v e h y p e r s e n s i t i v e s i t e , as w e l l i r r e v e r s i b l e and r e v e r s i b l e

inducible hypersensitive

The l a t t e r c o i n c i d e c l o s e l y w i t h the r e g i o n of binding

and h o r m o n e r e s p o n s i v e n e s s

receptor

in t r a n s f o r m e d c e l l s .

i m p o r t a n c e of c h r o m a t i n s t r u c t u r e , as a d e t e r m i n e n t or and h i s c o w o r k e r s

(86) that

P l a s m i d s of M M T V L T R and the t r a n s f o r m i n g

The

conse-

q u e n c e of h o r m o n e a c t i o n , m a y be m o r e a p p r o a c h a b l e w i t h f i n d i n g of O s t r o w s k i

as

sites.

the

chimeric

f r a g m e n t of

bovine

p a p i l o m a v i r u s t y p e 1 c a n t r a n s f o r m c e l l s w i t h as m a n y as 200 c o p i e s of the c h i m e r i c m o l e c u l e s p e r d i p l o i d g e n o m e s . are unintegrated, extrachromosomal

e p i s o m e s w h i c h are

These sensi-

t i v e to g l u c o c o r t i c o i d

hormone regulation through sites

the M M T V L T R s e g m e n t .

Of m o s t

i m p o r t a n c e , they e x i s t

m i n i - c h r o m o s o m e s w h i c h p u t a t i v e l y c o n t a i n the

as

accessory

in

534 chromosomal proteins with a role in hormone receptor nuclear action.

This probably represents an important step forward

to the ultimate goal of a complete JJI vitro transcriptional system under steroid hormone control.

Acknowledgement Research work in the authors' laboratories has been supported by grants from the New York State Health Research, Inc. (BRS 35074), National Institutes of Health (AM 23075; HD 18406) and National Science Foundation (PCM 7825517).

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THE

RAT PITUITARY

RECEPTOR

IN

PROLACTIN

ESTROGEN

THE

GENE

RECEPTOR:

REGULATION AND

THE

OF

ROLE

OF

THE

NUCLEAR

TRANSCRIPTION

OF

THE

LOCALIZATION

OF

THE

NUCLEAR

UNOCCUPIED RECEPTOR

James

D.

Shull,

Wade

V.

Welshons,

Mara

E.

Lieberman,

Jack Gorski Departments of Biochemistry and Animal Science University of Wisconsin, Madison, WI

53706

Introduction We

have

recently

examined

estrogen on the synthesis by

nuclei

glands

which

of

are

intact

pituitary

cells.

stimulates

the

the

of prolactin

isolated

male Our

effects

from

rats

or

transcription

of

anterior

rat

cells

centrifugation in

Percoll

of

gradients

cytochalasin to

estrogen

gene

jji vivo

(1-4).

We have

also used a cell enucleation procedure based on density

anterior

that

Prl

through at least two independent mechanisms

equilibrium

B-treated

reexamine

the

receptor nuclei

we

observed

was (5).

that

associated This

and

most with

other

of the

the

In these

unoccupied

fraction

observations

that

have

estrogen contained

led

revision of the classical model of estrogen action

Molecular M e c h a n i s m of Steroid H o r m o n e Action © 1985 Walter de Gruyter & Co., Berlin · N e w York - Printed in G e r m a n y

GH^

subcellular

distribution of the unoccupied estrogen receptor. studies

RNA

pituitary

cultured

illustrate the

administered

(Prl) messenger

the from

results

of

to

(6).

the

540 Estrogen Regulation of Prolactin Gene Transcription Estrogen

has been

shown

pituitary

hormone

through

mechanism

a

Prl messenger RNA that this increase

Prl

to stimulate i^n

which

(11-14).

vivo

the

(7-9)

results

in

synthesis

and

_in

increased

is due at least

an increase in the transcription of the

the (10)

levels

It has more recently been

in Prl mRNA

of

vitro

shown

in part

rat Prl gene

of to

(1-4,

15, 18) .

From Shull and Gorski, Endocrinology,

1984

Figure 1. Stimulatory effect of 17ß-estradiol on prolactin gene transcription. Male rats were injected (10 yg, IP) at the indicated times prior to sacrifice. Anterior pituitary nuclei were then prepared and prolactin gene transcription was assayed (3). Each data point represents the mean and SEM (n=3) for the assay of prolactin RNA synthesized by nuclei prepared from 8-10 animals.

541

to m a l e rats

(6 w e e k s of age) as a single

IP),

in

(48

results to

Similar

72

a

hours)

results

rapid

(within

stimulation

were

Log

observed

Inhibitor

30

injection

minutes)

of

Prl

by

Maurer

gene

and

(10

pg,

prolonged

transcription.

when

he

examined

Concentrotion

From S h u l l and G o r s k i , E n d o c r i n o l o g y ,

1984

Figure 2. Concentration effects of transcriptional inhibitors on RNA s y n t h e s i s by isolated p i t u i t a r y n u c l e i . Anterior p i t u i t a r y nuclei w e r e p r e p a r e d , and the inhibitory e f f e c t s of increasing c o n c e n t r a t i o n s of α - a m a n i t i n (A) a n d a c t i n o m y c i n D (B) on RNA s y n t h e s i s w e r e d e t e r m i n e d . Each d a t a p o i n t r e p r e s e n t s the m e a n and SEM (n=3).

542

A. Sesame Oil ΙΑ ΙΑ

V

-C c>» βοο to < H

600 -

Ο Ο

400

-

w α. 2 200 α. α.

B. 17 B - E s t r a d i o l

i 1111 11II

1000

-

-

None

α-Amon

Act D

None

Inhibitor of RNA

a-Aman

Act. 0

Synthesis

From Shull and Gorski, Endocrinology, 1984 Figure 3. Effects of transcriptional inhibitors on Prl RNA synthesis by isolated pituitary nuclei. Anterior pituitary nuclei were isolated 24 hours after injection of the sesame oil vehicle (A) or 10 pg 17ß-estradiol (Β) and were incubated under normal conditions or in the presence of a-amanitin (α-Aman; 1 yg/ml) or actinomycin D (Act. D; 10 pg/ml). Each bar represents the mean and SEM (n=3) of Prl RNA synthesis in nuclei pooled from 8-10 animals.

the

effect

into

of

a single

ovariectomized

effects

of

injection

female

17B-estradiol

are

(20 y g )

rats

(15).

specific:

of The

17ß-estradiol stimulatory

no effects

estrogen on the transcription

of the evolutionärily

growth

observed

hormone

experiments, quantitating, the

level

Prl-specific

gene

Prl by

of

gene

were

hybridization

mRNA

In

was

assayed

sequences

to

immobilized

precursor by

anterior

cDNA

incorporated pituitary

this

related

(1-3).

transcription

radiolabeled

of

these by

probe, into nuclei

543

From Shull and Gorski, Endocrinology,

1984

Figure 4. Time course of the levels of the nuclear and cytosol forms of the pituitary estrogen receptor following a single injection (10 p g , IP) of 17ß-estradiol. Nuclear (A) and cytosol (B) receptors were assayed as described (3). Each data point represents the mean and SEM of specific binding measurements in individual anterior pituitaries (n=3).

isolated

at

treatment

(3).

well level

the This

characterized of

Prl

indicated

gene

nuclear

and

times

following

transcription

appears

to

transcription

reflect occurring

assay

estrogen has

accurately at

the

been the time

544

the

nuclei

mRNA

are

prepared

sequences

polymerase which

are

II.

nuclear

Briefly,

synthesized

from

Alpha-amanitin

specifically

total

(3).

inhibits

RNA

a DNA

by

Prl-specific

template

at 1 yg/ml, RNA

synthesis

these a

polymerase

65

to

75%

by

RNA

concentration II,

inhibits

(Fig.

2A)

while

the synthesis of Prl mRNA sequences is inhibited by greater than 95% 50

to

(Fig. 3).

60%

synthesis

total of

Actinomycin

nuclear

Prl

mRNA

RNA

D at

10

pg/ml

synthesis

sequences.

Thus

when expressed as parts per million

inhibits

(Fig. Prl

2B)

and

mRNA

total RNA

by the

synthesis

synthesis

is

uneffected (Fig. 3). The

prolonged

stimulation

17ß-estradiol

(Fig.

1)

of

Prl

does

not

gene

transcription

require

a

elevation in the level of the nuclear form of the estrogen receptor level

of

1 hour

(Fig. 4).

nuclear-form

of

pituitary

Figure 4A illustrates that the

receptor

17S-estradiol

by

continuous

peaked

injection,

within

approximately

decreased

approximately

70% from this peak value within 6 hours and returned to its control

value

17ß-estradiol stable

within

mechanism

chromatin

24

regulates which

proteins

or

hours. Prl

perhaps DNA

These

gene

data

suggest

transcription

involves

sequences

that

through

a

a modification

within

or

of

surrounding

the Prl gene (3) . To

investigate

of

intermediary

transcription in

the

possible

proteins

by

in

gene

following

a

single

transcription

saline-treated (Fig. 5B).

the

17ß-estradiol,

cycloheximide-pretreated

hours

requirement

(Fig.

was 5A)

we

animals injection

stimulated and

for

the

induction

of

examined

this

(3). of

When

synthesis Prl

examined

17ß-estradiol,

nearly

2-fold

17ß-estradiol incorporation

injection

(Fig.

in

cycloheximide-treated

Similar results were observed 8 hours

cycloheximide

6).

In

inhibited by greater 3 of [ H]leucine into

these than

gene

induction 3

Prl both

animals following

experiments, 80%

the

acid-precipitable

545

Α.

Β. Cycloheximide

Saline

I1

Ρ ι! mm

HI Oil

E 2 -I7ß

Oil

E.,-170

Treatment From Shull and Gorski, Endocrinology, 1984 Figure 5. The induction of prolactin gene transcription by 17ß-estradiol (10 u g , IP) under conditions of inhibited pituitary protein synthesis: an examination at 3 hours. Sterile saline (A) or cycloheximide (B) was injected 10 minutes before 17ß-estradiol or its sesame oil vehicle. The animals were killed 3 hours after hormone treatment and prolactin gene transcription was assayed as described (3). Each bar represents the mean and SEM (n=3) for the assay of prolactin RNA synthesized by nuclei prepared from 8-10 animals.

material

by

eliminate required

the

the

anterior

possibility

for

the

induction

17ß-estradiol

may

have

sufficient we

quantity

examined

anterior

this

pituitary

in

ρ ituitary t hat

been the

induction cells

of

a Prl

gland protein gene

synthesized

(Fig. (or

transcription in

a

reduced

cycloheximide-treated in in

primary which

7).

cultures

To

proteins) by but

animals, of

cycloheximide

rat had

546

A.

Saline

B. Cycloheximide

500

§

Φ « 400 CO
c ο 3 « α> >

«

fr Oil

l7/3-E2

Oil

Saline

Ι7/3-ε2

Oil

Cyclohex

I7ß-Ez

oil ΐ7β-εΖ

Saline

Cyclohex

Treatment From Shull and Gorski, 1984 Figure 7. Inhibitory effects of cycloheximide on pituitary protein synthesis. Injections are as illustrated and described in Figures 5 and 6. Each bar represents the mean and SEM of the relative level of leucine incorporation in individual pituitaries from a total of five to eight animals.

The

synthetic

estrogen,

16α-estradiol,

has

characterized as being "short acting" as a single of

this

early

hormone

estrogenic

induced

protein

estrogenic

into

immature

responses, synthesis

responses

such

by as

female

such the

as

been

injection

rats

stimulates

water

imbibition

uterus, but

uterine

DNA

not

the and

the

synthesis

late (16).

The rat uterine estrogen receptor has a lesser affinity 16a-estradiol than for 17ß-estradiol a more

rapid

decline

the uterine estrogen

in

the

receptor

level

(16). of

following

the

for

This results in nuclear

a single

form

of

injection

of 16a-estradiol than is observed following an injection of

548

17ß-estradiol

(17).

When the

levels

cytosol

forms

examined

following

an injection of

returned

to

control

In

of

the

their

contrast,

the

significantly equivalent

pituitary

level

injection

for

of

nuclear

within

least

6

17B-estradiol

and

receptor

16«-estradiol, 4

nuclear-form at

the

estrogen

values

of

elevated

of

hours

had

(4,

receptor hours

were

they

18).

remained

following

while

the

an

level

of

cytosol-form receptor remained diminished (4, 18). A

single

injection

of

16a-estradiol

stimulates

the

transcription of the rat Prl gene in a biphasic manner 4,

18).

of

injection

The

paralleling nuclear

initial and

phase was

continued

in duration

form

of

the

observed

through

the

at

elevation

pituitary

within

least

minutes

2 hours,

in the

estrogen

30

(2,

level

thus

of

the

receptor.

The

second phase of stimulated Prl gene transcription was

first

observed

after

the

approximately

level

of

6

hours

nuclear-form

following

receptor

had

injection, returned

control value

(4, 18).

The induction of the

of

Prl

transcription

stimulated

gene

by

to

initial

its phase

16a-estradiol

was

observed in animals pretreated with either cycloheximide puromycin

indicating

mechanism

which

synthesis. the

that this phase

is

independent

is mediated of

In contrast, cycloheximide

induction

stimulated

by

16 α-estradiol

transcription

of

(4,

pituitary

18).

second This

(6

hours

versus

following

8

induction

of

examined the

1

Prl

(Fig. 6).

induction

hour)

hours

of

of

gene

as

no

transcription

effect

effect

was

treatment by

second

phase

protein

although

of

of of

treatment observed when

the

17ß-estradiol

was

These data suggest the possibility the

transcription by 16a-estradiol intermediary

such

cyclohe ximide

blocked

phase

cycloheximide was not simply due to the prolonged

a

protein

pretreatment the

or

through

stimulated

Prl

that gene

requires the synthesis of an other

cycloheximide cannot be ruled out.

unknown

effects

of

549

Our

observations

that

16a-estradiol

stimulates

the

transcription of the Prl gene in a biphasic manner and the

induction

of

the

two phases

differs

cycloheximide

suggest

that this hormone

transcription

through

at

One

mechansim

appears

least

to

pituitary

estrogen

hypothesize

that this mechanism

estrogen-receptor

the

Prl

and

is

includes with

Gorski

to

gene

of

the

attractive

to

an

form

interaction

regulatory have

Prl

mechanisms.

nuclear

It

complex

Durrin

in sensitivity regulates

independent the

receptor.

the

gene.

two

involve

that

regions

localized

of of

regions

near the 5-prime terminus of the Prl gene, which exist in a chromatin

structure

during

a

limited

nuclei

(manuscript

hypersensitive

which

DNAse

is

hypersensitive

digestion

submitted).

sites

correspond

of

to

isolated

It

is

to

regulatory

nicking pituitary

possible

that

these

domains.

We

have hypothesized that the induction of the second phase of stimulated an

Prl

gene

transcription

estrogen-induced

regulator

alteration

of Prl gene

the

responsiveness

to

a

second

of

the

regulator

cells (3,

induction of Prl gene that

elevated

receptor

the

anterior

These

same mechanisms

transcription with

and

of

either

a

second in

pituitary

hypotheses

are

and

affinity

ability

to

animals

duration

injected

allows with

than the

two

receptor,

the being

to

the

maintain is

This

nuclear-form

in the

16a-estradiol

lesser

lesser

function

17ß-estradiol;

an

shorter

the

its

its

by

induces

injection.

of

of

initial phase of stimulated Prl gene transcription which of

levels

of

4).

17ß-estradiol

16a-estradiol,

estrogen

result

level

(18).

It seems likely that these between

the

the

transcription or to an alteration

currently being tested

difference

is

in

following phases

16a-estradiol

would overlap in animals treated with

to

while

17ß-estradiol be the

resolved two

17ß-estradiol.

in

phases

550 Intracellular Location of the Estrogen Receptor Since the direct effects of estrogen involve the of

transcription

not

receptor-steroid complex is recovered in the nucleus of

the

cell

the

after

location the

that

the of

of

at

on

0-4°

C

receptor-steroid was

proposed

of

to

cell,

tissue. has

This

in

(20). for in

received

assigned

based

on

cytosol of

the

wide

of

to the

subsequent nucleus

the

nucleus

recovery

(21,

acceptance

the

labeled

Translocation

the

22).

for

to

extracts

cells

from the cytoplasm

account

the

However,

been

was

receptors

it

estrogen,

autoradiography

complex

interpretation

the

cell.

complex

receptor-steroid

the

receptor

unfilled

and

of

administering

the

most

(19)

briefly

after

unoccupied

cytoplasm cells

nucleus

homogenization

of

appearance of

the

regulation is

surprising

in

of This

both

the

estrogen receptor and the other steroid hormones. Some data also

be

suggest, however, found

in

the

that the unfilled

nucleus,

localization

represents

an

include

appearance

using

the

fraction of receptor

in the

and

that

extraction nucleus

under

In

order

to

avoid

remove

possible

B-induced

cytoplasm

(as

cytoplasts)

of from

cells, without breaking open the cells. and the cell have they

an

fragment

intact plasma

can

be

fused been

containing membrane to used

the and

reform

Enucleation

has

to

show

polymerase,

extracted when the cell

a

data large

nontranslocating

artifacts, cells

we

(24,

used

25)

to

receptor-containing Both the

nucleus

are a

The

of

(see below).

extraction

enucleation

may

cytosolic

artifact.

autoradiography

conditions (23) and other observations

cytochalasin

receptor

the

still viable

that

a

cytoplast

(nucleoplast) alive,

since

cell

(26).

cytosolic

is homogenized,

cytoplasmic in the intact cell, but is instead nuclear

DNA

is not (27).

551

Not

all

cells

cytoplast

will

and

separated.

Freshly

cultures

the

so

we

of

turned

tumor

that

found

fractionation increased

the

were GH^

(28). in

well,

always

pituitary

cells

and

primary

not

enucleated

cell

line,

the

a

usual

cells

rat

receptor cell

to estrogen

The

exhibit the usual characteristics of an

from

estrogen

after

(30).

satisfactorily,

derived

contain

cytosol

synthesis

enucleated, not

(29), and the cells respond

prolactin

if

are

The cells the

and

fractions

dispersed

cells

to

pituitary is

enucleate

nucleoplast

with

therefore

estrogen-responsive

tissue, even though the cells are tumor-derived. We were

able to partially

forming

cytoplasts

that

enucleate

averaged

PREPARATION OF

85%

1/5

of

of

the GH^

the

size

CYTOPLASTS

Percoll, self-forming gradient Welshons, Lieberman and Gorski, unpublished. Figure 8.

Preparation of cytoplasts from GH3 cells.

cells, of

the

552 original

whole

a purified contained fraction more the

cells.

less from

than

cells

from

these

able that

cytoplasts

containing

We

we were

cytoplasts,

contaminating

the

which

removed.

of 1%

which

heterogeneous,

been

Importantly,

preparation

varying called

whole

had

the

The

removed

was

cells

proportions this

of

obtain

sometimes

cells.

been

some whole

to

as well

cytoplasm

cell

+

as had

nucleoplast

fraction.

If the unoccupied estrogen receptor concentration than we

the

in

cytoplasts

concentration

enucleated

GH^

centrifugation

in

the

cells

(Fig.

8),

SUBCELLULAR

is cytoplasmic

should

be

whole

high

cells.

using we

as

then

or

However,

cytochalasin found

the

its

higher when

Β

and

opposite

DISTRIBUTION OF ER

ER/protein

ER/DNA η 3.0

I 50 γ ί

i n

i

c 5 2.0

2 loo Q. σ>

er Ε

ε

er ÜJ

£E Iii 50

1.0

Ö

ö

ε

Ε Q-

WC

C + N CYT

WC

C+N

CYT

From Welshons et a^., Nature, 1984 Figure 9. Estrogen receptor (ER) concentration in whole cells (WC), in cells + nucleoplasts (C + N) and in cytoplasts (Cyt).

553 distribution per

protein

the

cells

9).

The

(5). was

+

The

low

concentration

in

cytoplasts

nucleoplasts,

concentration

of

of

and

estrogen slightly

compared

to

whole

receptor

per

DNA

hand was similar in all fractions

receptor higher

cells on

in

(Fig.

the

other

(Fig. 9), suggesting

that

the receptor was following the DNA content of the cell. the

experiment

detailed

in

Table

1

(from

ref.

receptor per protein was less than 10% of the in

the

whole

estrogen complete

cells.

receptor (Table

The after

2),

recovery

of

was

that

In the

concentration

protein,

enucleation

indicating

5),

DNA

and

essentially

receptor

was

not

selectively lost from the cytoplasts.

The cell + nucleoplast less

protein

fraction

than

average

TABLE 1.

the of

fraction

averaged

original

only

20%

whole

of

the

approximately cells,

so

cytoplasm

1/5

that had

a

been

ESTROGEN RECEPTOR IN ENUCLEATED CELLS

ER/ Prot.

ER/ DNA

Prot./ Cell

DNA/ Prot.

ER/Cell Equivalent

fmol/mg

pmol/mg

pg

ρ g / mg

molecules

Whole cells

113

2.7

363

42

25,000

Cells + nucleoplasts

146

2.9

336

50

30,000

10

1.4

69

Cytoplasts

7.5

2,100*

Estrogen receptor per protein, per DNA and per cell hemocytometer) in whole cells and enucleated cells. cytoplast fraction contained fewer than 1% whole cells.

(by The

*Number of molecules per 5 cytoplasts, since cytoplasts approximately 1/5 the size of whole cells.

are

Welshons et al., Nature, 1984.

554 TABLE 2. RECOVERY OF ESTROGEN BINDING, PROTEIN AND DNA AFTER ENUCLEATION Total Applied or Recovered Estrogen Binding pmol

Protein mg

DNA pg

Before enucleation: Whole cells

2.10

18.5

785

After enucleation: Cells + nucleoplasts Cytoplasts

2.26 0.024

15.5 2.3

777 18

96%

101%

Recovery

109%

Welshons, Lieberman and Gorski, removed.

But

microscopically, it

fraction contained some cells cytoplasm most

of

had the

receptor further that

been

removed.

cytoplasm

content,

contained

less

When

cell

on

that

the

from which much more of

the

To

still

the

fractionated

densities.

unpublished.

a

see

+

if

seen

the

cells

the

full

nucleoplast

density

step

were

estrogen

measured in these fractions,

be

contained

cytoplasm the

could

lacking estrogen

fraction

gradient.

found

at

receptor

the

higher

content

DNA

(per

cell)

There was no evidence

was

that

was

it was found that, while

receptor per protein more than doubled, the receptor tent per

was Cells

constant

(Fig.

10,

removing most of the

the

con-

ref.

5).

cytoplasm

of the cell removed any of the unoccupied receptor. The

enucleation

cells.

Dye

enucleation whole

cell

incorporated enucleation

procedure

exclusion throughout

uptake

of

measuring estradiol

[ H]leucine (Table

did

4).

not

continued

and In

seem

high the

(Table

damage just

receptor 3).

synthesized addition,

to from

content

The

(Table 5), and the steroid receptor

by

fractions

prolactin

incubation

enucleation medium had no apparent effect on the content

the after

after in

the

receptor

in the cell +

555

DISTRIBUTION

OF

ESTROGEN

RECEPTOR

400 Cells +

Nucleoplasts

I

• Cyt]

Ξ

w

c a> ο a. σ> Ε

< ζ Ω

σ> Ε ν er υ ιο ο Ε α.

ο Ε

Λ Cyt.

Ά

Α

Cell

Β

Ά

Fraction

Welshons et al., Nature, 1984 Figure 10. The cell + nucleoplast fraction was further fractionated on a density step gradient. The (intact) cells at higher density steps have had more cytoplasm removed. Arrowhead indicates density of cells before enucleation, and the position cytoplasts would have occupied is indicated in brackets. TABLE 3.

DYE EXCLUSION OF CELLS AND FRACTIONS

% Excluding Dye Initial

Whole cells Cells + nucleoplasts Cytoplasts Mean + standard

97 + 1 98 + 1 98 + 1

Final

95 + 1 93 + 1 73 + 4

error, n's of 6 to 9.

Trypan blue exclusion just after enucleation (Initial) and after measuring the receptor content by whole cell uptake at 37° C (Final). Welshons, Lieberman and Gorski, unpublished.

556 nucleoplast

fraction

just

after

enucleation

was

still

extracted into the cytosol when the cells were homogenized (31).

Therefore,

apparent

effect

on

the

enucleation

the

estrogen

procedure receptor

had

that

no

would

indicate that redistribution of the receptor had occurred.

TABLE 4.

PROLACTIN SYNTHESIS BY GH3 CELLS AND FRACTIONS Leucine Incorporation: ΙΟ" 6 χ DPΜ Per 10 6 Cells or Equivalent

Prolactin Synthesis: % of Total Protein Synthesis

Whole cells

6.2

1,.2 + 0.02

Cells + nucleoplasts

5.7

1,.3 + 0. 06

Cytoplasts

3.0*

3 .4 , + 0.2

Cells, cells plus nucleoplasts, or cytoplasts were incubated with [^Hjleucine to measure general protein synthesis by TCA-precipitable leucine incorporation, and to measure prolactin synthesis using precipitation with antiprolactin antibody. *Assuming 5 cytoplasts per whole cell equivalent. Welshons, Lieberman and Gorski, unpublished.

TABLE 5. EFFECT OF INCUBATION OF CELLS IN ENUCLEATION MEDIUM ON RECEPTOR CONTENT

Whole cell uptake, fmol/mg prot.

Untreated

After Incubation

102 + 2

98+1

Mean + standard error, n=2

Estrogen receptor content was measured in untreated cells and in cells incubated at 37° C for 2 hours in cytochalasin Β plus solvent DMSO in Percoll. Receptor content was not significantly affected. Welshons, Lieberman and Gorski, unpublished.

557 The

estrogen

receptor

using whole cell

[ 3 H]estradiol

of

fractions. uptake

at

Specific

in

hormone.

was measured

in these experiments

(or whole cytoplast or nucleoplast)

the

37°

C

uptake

presence

into

was

of

the

intact,

calculated

100-fold

by

excess

of

that

would

have

been

unlabeled

encountered

homogenized cells or cytoplasts in the 1 or 2 mg that we used

in assays.

The specific

instead

of

37°

C

(Table

had

we

quantities

uptake was more

90% inhibited when the cells or fractions were C

live

subtracting

Whole cell uptake was used to avoid any losses of

receptor

0°

by

uptake

6)

than

incubated

verifying

that

at the

receptors were inside the intact cells.

TABLE 6. TEMPERATURE-DEPENDENT UPTAKE OF ESTRADIOL BY INTACT CELLS OR CYTOPLASTS

Specific Uptake, DPM

37° C

Whole cells Cells + nucleoplasts Cytoplasts

0° C

855 3,570 143

39 120 13

Welshons, Lieberman and Gorski, unpublished. The

specific

Scatchard

uptake

analysis

(Figure 11). per

cell.

The

is observed 1

the

an

linearity the

was

of

as

this

nucleus,

not

it to

is

in

suggest

free

the

in

the

of

intact

solution

plot

binding

receptor

the

and

in

nM sites

intact

(32, 33)

that

concentration cell.

receptor (6),

by

0.34

25,000 binding

Scatchard

the that

saturable,

affinity

cooperative

in extracts when

nM,

interpreted

estradiol

showed

There were approximately

cells contrasts with

above

of

since

We

is bound a

is

have in

soluble

558

SATURATION

ANALYSIS

TOTAL

0

1 2

,c 50

3

4

Estradiol Concentration (nM)

Bound Estradiol (DPMxIO )

Welshons, Lieberman and Gorski, unpublished Figure 11. Saturation analysis of the whole cell uptake of [ 3 H ] estradiol at 37° C by G H 3 cells. receptor

would be expected

binding,

while

hydroxylapatite),

like

the

not show this cooperativity While our nuclear

finding to

lines

nuclear

indicate

of

receptor under

the

estrogen

Recent

are

in

that

autoradiography 2.

in

fact

is

Sheridan

of

(34,

was

even

is

been

several with

found

conditions in

studies

the

with

antibodies

by that

nucleus. to

in the nucleus, though

a

during

(23)

already

monoclonal

35),

receptor

that has

consistent

"nontranslocating" binding

set

does

location,

aK

receptor have detected antibody binding cytoplasm

cells,

extractable et:

immunohistochemical

best-characterized the

intact

(on

(33).

cytoplasmic

evidence 1.

of

receptor

hormone

receptor

that the unoccupied estrogen

homogenization. most

estrogen

contradicts a large body of evidence

interpreted recent

to show the cooperative

immobilized

the the not

preliminary

559

localization

was

reported

nuclear

translocation

receptor

would

compounds

that

the

full

range

also seem of

in

the

(36)). explain

to

bind

the

to

estrogenic

cytoplasm

3.

action

the

(but

Extraction of

receptor

(nuclear)

without of

the

estrogenic

and

stimulate

responses,

yet

are

found bound to the receptor

in the cytosol when the cell

homogenized

interpretation

(37).

homogenization compound

of

the

complex,

extracted

from

addition,

the

unfilled

The like

its

the

nuclear

unfilled

receptors

cell,

the

is

receptor-

unoccupied locus

receptors

that

of for

for thyroid hormone

estrogenic

receptor,

action. vitamin

is

upon

D

and dioxin

is

4.

In

and

the

(similar

to steroid receptors) have been reported to be nuclear

(38,

39, 40, 41).

CELL NUCLEUS

Estrogen

σα.

Figure 12. receptor.

Diagram

of

Induced Proteins

interaction

of

estrogen

with

its

560 Our

in

the

context

suggest

data

that

the

unoccupied

nuclear

in

receptor

the

near

intact the

of

the

cell

site

(Fig.

of

nuclear

nucleus hormone nuclear

when the

elements

and

the

is

cell

receptor

elements

is

cited

receptor

12).

action

absence of hormone, the receptor with

literature

estrogen at

This

all

places

develops

a

After

stronger

and resists extraction.

the

In

the

associated

extracted

homogenized.

mostly

times.

is only loosely usually

above

is

from

the

binding

the

affinity

for

kinetic

data

The

suggest that the receptor may be bound at all times to

its

site

the

of

action,

nucleus. in

the

and

is

not

a

soluble

protein

Although our data may apply only GH^

cell,

mechanisms makes

the

similarity

it tempting

of

the

to speculate

within

to the

receptor

steroid

receptor

that all

steroid

receptors are located in the nucleus.

References 1. Shull,

J.

D.,

(supplement), 292

Gorski,

J.:

Endocrinology

110

(1982).

2. Shull, J. D., Gorski, J.:

Fed. Proc. 42, 206

3. Shull, J. D., Gorski, J.:

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114,

1550-1557

(1984) . 4. Shull,

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D.,

Gorski,

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Μ. E., Gorski, J.:

Nature

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10. L i e b e r m a n ,

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G.,

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Proc.

Nat.

R e e . Prog. Horm. R e e . 2_4, 45-80 Ε.

V.,

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Suzuki,

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23. S h e r i d a n ,

P.

Kawashima,

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W.,

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26. V e o m e t t , G . , P r e s c o t t ,

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M.,

Shay,

P r o c . N a t . A c a d . Sei. 71, 1999-2002 G.,

Spear,

B.

A c a d . Sei. T3, 1136-1139 28. T a s h j i a n , Α . , 47 , 61-70

Τ.,

( 1968) .

N a t u r e 282, 579-582 24. P o s t e , G.:

Notides,

(1968 ).

Ε., J u n g b l u t , P. W . , D e S o m b r e , Ε. R . : Sei. 5ji, 632-638

Acad.

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E n d o c r i n o l o g y 82/ 777-782

21. G o r s k i , J . , T o f t , D., S h y a m a l a , G . , S m i t h , D . ,

27. H e r r i c k ,

256,

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20. S t u m p f , W . Ε.:

25. Bossart,

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J.: R.,

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22. J e n s e n ,

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U.W. P h . D . T h e s i s

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19. T o f t ,

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(1979).

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15. M a u r e r , R. Α.:

18. Shull,

Biochemistry

(1979).

N a t l . A c a d . Sei. 76, 824-828 J.:

Proc.

(1977).

12. R y a n , R., Shupnik, Μ. Α . , G o r s k i , J . : 2044-2048

J.:

(1978).

Bancroft,

(1970 ) .

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Porter,

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R.:

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Levine,

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562 29. Haug,

Ε.,

Naess,

0.,

Gautvik,

Κ.

Μ.:

Molec.

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Endocr. 12, 81-95 ( 1978) . 30. Haug, E., Gautvik,

Κ. M.:

Endocrinology

99,

1482-1489

(1976). 31. Welshons, W. V., Gorski, J.: 32. Notides,

A.

C.,

Lerner,

in preparation.

N.,

Nat. Acad. Sei. 78, 4926-4430 33. Sakai,

D.,

Gorski,

J.:

Hamilton,

D.

E. :

Proc.

(1981). 22,

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3541-3547

(1984) . 34. King, W. J., Greene, G. L.:

Nature 307, 745-747

(1984).

35. McClellan, H. C., West, N. B., Tacha, D. E., Greene, G. L.,

Brenner,

R.

M. :

Endocrinology

114,

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( 1984) . 36. Jensen, E.

R.,

Ε. V., Greene, Nadji,

M. :

G. L·., Closs,

Ree.

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Horm.

E.,

DeSombre,

Res.

3fi,

1-40

(1982). 37. Jordan, V. C., Tate, A.

C.,

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Wolf, M. F., Welshons, W. V.:

S.

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B.,

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38. Walters, M., Hunziker, W., Norman, Α.:

J. Biol.

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H.,

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Koerner, D., Dillman, W. H.: 529-565

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D. R.:

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259,

CHARACTERIZATION

OF DIFFERENT

FORMS OF THE ANDROGEN

RECEPTOR

AND THEIR INTERACTION WITH CONSTITUENTS OF CELL NUCLEI

Eppo Mulder and Albert 0. Brinkmann Department of Biochemistry, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands

Introduction

It is now generally accepted that most, if not all, actions of steroid

hormones

are

mediated

by

an

effect

of

a

steroid

receptor complex on the transcription of specific parts of the genome in the nucleus (1). likely

interacts

with

In this process the receptor

chromatin

or

DNA

to

most

modulate

the

transcription of specific genes (2, 3, 4). Androgen

receptors

distribution.

are

characterized

by

a dipolar

charge

They are acidic proteins with an IEP of 5.8 (5)

and with a net negative charge at neutral or slightly alkaline pH.

Once the steroid has bound

protein,

the

resulting

to its intracellular

complex

undergoes

further

receptor changes,

termed a c t i v a t i o n , that increases its affinity for specific acceptor

sites within the cell nucleus.

Whether this process

o c c u r s e x c l u s i v e l y at the nuclear level is still a m a t t e r of debate but recently strong e v i d e n c e has been presented for a nuclear

locus

action (6, 7).

of

this

important

step

in estrogen

hormone

A c t i v a t i o n of androgen r e c e p t o r s is supposed

to be a c c o m p a n i e d by a change in the ionic p r o p e r t i e s of the complex

resulting

in a higher

isoelectric

point

(5).

dipolar behavior may represent an important characteristic relation to nuclear interaction. are reported and

Activated androgen

to bind to positively charged

nonhistone

basic

proteins

and

to

The in

receptors

chromatin

histones

negatively

charged

Molecular M e c h a n i s m of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

564 ribonucleic acids and nonhistone nuclear proteins (8, 9). Several forms of androgen receptors have been extracted nuclei

but

little

is

known

about

the

significance of these forms (10, 11, 12).

from

physiological

Alternatively the

occurrence of these different nuclear forms might

reflect

partial degradation of the native form during isolation as has been found for androgen receptors present in cytosol fractions of rat prostates

(13).

This chapter

deals with

different

aspects of the transformation process (activation) of androgen receptors to the nuclear binding state and the forms of the receptor

involved

in

this

characteristics

of

nuclear acceptor

sites and

receptor

with

forms

the

process.

interaction in vitro

DNA,

RNA

of

Furthermore

both

the

with

receptor

interaction of different

and

polynucleotides

are

described.

Results and discussion I

Characterization

οf the activation process of

androgen

receptors Activation of receptors can be accomplished by a variety of in vitro manipulations, such as heating, salt treatment, dilution or aging of the receptor preparations (14).

We have studied

this process for androgen receptors and have characterized the different changes that occur in the properties of the receptor during

this

interaction

process

with

respect

with polyanions,

to

sedimentation

dissociation

rate and

rate, charge

distribution. Interaction of androgen receptors from seminal vesicles of the ram with phosphocellul ose. Cytosols containing two distinct [ ^H ]-methy ltr ienolone

binding

proteins

(8S

and

4S)

were

565 dpm

χ ΙΟ"3

85

US

1

1

Α

ΧΑΛ dpm χ 10"2 C

KL

o-l

1

1

,

10

20

30

10

20

30

fraction number

F i g u r e 1_ B i n d i n g o f androgen r e c e p t o r s t o p h o s p h o c e l l u l o s e : a n a l y s i s by s u c r o s e g r a d i e n t c e n t r i f u g a t i o n . The l a b e l l e d c y t o s o l o b t a i n e d f r o m s e m i n a l v e s i c l e s o f t h e ram was s u b j e c t e d t o s u c r o s e g r a d i e n t c e n t r i f u g a t i o n ( f o r 210 min at 1°C in a Beckman V T i 65 r o t o r a t 3 7 0 , 0 0 0 χ g ) e i t h e r d i r e c t l y ( A ) , or a f t e r f i l t r a t i o n o v e r p h o s p h o c e l l u l o s e ( B ) . The r e c e p t o r s i n t h e r e s i d u a l f r a c t i o n p r e p a r e d as f o r p a n e l Β w e r e a c t i v a t e d and a g a i n s u b j e c t e d t o s u c r o s e g r a d i e n t c e n t r i f u g a t i o n b e f o r e (C) and a f t e r (D) f i l t r a t i o n o v e r p h o s p h o c e l l u l o s e .

incubated R1881,

with

affinity

for

sedimenting IB),

complex.

The

converted

the

3-4S

probably

Μ KCl.,

phosphocellulose

is a synthetic,

androgen

form

was

representing 8S

1A).

receptor.)

retained the

sedimenting

t o a 3-4S s e d i m e n t i n g

Sephadex

(fig.

(Methyltrienolone,

n o n - m e t a b o l i ζ able androgen w i t h by

was

form a f t e r

G-25 g e 1 f i 1 t r a t ion

the

high

slower

phosphocellulose

activated form

Only

(fig.

steroid-receptor almost

completely

incubation

and d i l u t i o n

with

(fig.

0.4 1C).

566 This

fraction

complexes, again

containing

originating

incubated

complete

with

retention

phosphoce1lulose

activated

androgen

from the n o n - a c t i v a t e d

phosphoce1lulose. of

the

(fig.

ID).

from p h o s p h o c e l l u l o s e

3-4S

This

sedimenting

extraction.

can be c o n c l u d e d

of

t h e a n d r o g e n r e c e p t o r can be c o n v e r t e d

faster

sedimenting

the n o n - a c t i v a t e d

form with a higher

D i s soc i a t i o n r a t e

οf

androgen

uterine

t issue.

cytosol

a temperature

complex

from a low a f f i n i t y

measurable

by

sedimenting in

at

calf

endogenous

the 5S

receptor,

in

(15,

16).

uterine

enzyme

binding

suppressed

of

(e.g.

obta ined in

the

calf

calf

uterine

steroid-receptor

dissociation the

state,

rate,

DNA-binding receptors with

activity. to

is

form

are

also

relatively

low

receptors

can

These

the e s t r a d i o l

androgens

form

from

to a high a f f i n i t y

a tissue

from

results

activated,

Androgen

tissue,

these

polyanions.

of

of

the

i n t o an

hormone

formation

independently

when

state

by

recovered

affinity for

receptor

change

in a

8S s e d i m e n t i n g

receptors

estrogen

induced

proteolytic

be measured properly

the

as a d e c r e a s e

accompanied present

For

form

From

it

was

resulted

The r e c e p t o r c o u l d be

by s a l t

that

receptor

8S f o r m ,

and

these

progesterone receptors

is

with a 500-fold excess

triamcinolone

molybdate

the

hormone-

androgen

receptor

acetonide). In

fig.

2 the

dissociation

effect

c o m p l e x e s i s shown. receptor

is

present

dissociation)

which

precipitation cytosol

in

capacity complex

the

with

is

at

a high

binds

to

of

(20

mM) on

these

of

from

observed. at low

affinity

DNA- o r

ammonium

the amount o f

transformation

25°C

In the a b s e n c e of m o l y b d a t e most of as

presence

increases

increase of

of

kinetics

state

phosphocellulose.

sulphate

of

the

20 mM m o l y b d a t e 5

to

65%

and

ionic

indicates

that

strengths,

the

the

(slow After

receptor

in

DNA-binding

concom i t t a n t l y

slowly dissociating

This

complex

an

steroid

receptor

molybdate

prevents

but does not

inhibit

567

Figure 2_ Dissociation rate at 25°C of R1881-androgen receptor complex from calf uterus. a) A c t i v a t e d f o r m , in t h e absence of molybdate. b) N o n activated form stabilized with 20 mM molybdate.

no

80 TIME

transformation

120 (min)

by high salt concentration.

have been described

for

the

foreskin fibroblasts (17).

androgen

Similar

receptor

findings

from

human

In sucrose gradients (0.4 Μ KCl)

all forms of the uterine androgen receptor sediment at 4.5S. A change

in sedimentation

value comparable

to the 4 to 5S

transformation as shown for estradiol receptors after receptor activation is not observed for the androgen receptor. These observations demonstrate

that transformation

androgen

with

receptor

to

a

form

polyanions and nuclear chromatin

a high

is accompanied

of the

affinity

for

by a 30-fold

increase in affinity for the steroid.

Anti-androgens

and

the

activation

of

androgen

receptors.

Because the role of the ligand in the transformation step of androgen receptors to the activated (i.e. DNA binding) state is hardly known the effect of androgens and the anti-androgen

568 cyproterone

acetate

on

the

activation

receptor

in the rat p r o s t a t e

In o r d e r

to e x c l u d e

tissue

obtained

from

[3H]-R1881

After

1

h

Metabolic R1881

were performed

castrated

(20 n M )

(18) a n d

the

were

incubation

with

The

show

incubation

3B

a nuclear

measured.

as

any

3.6S

in n u c l e i

after

anti-androgen.

exchange

were

The

Analogous

by e s t i m a t i o n Under

of

these

only w h e n

prostates

receptors

was the

further stability

dissociation

3A

ligand

the

nM

or

nuclear

of

χ

10-3

dissociated

acetate

t h a t of

constant

of

did

[3H]-R1881

was

could

(not be

shown). localized

obtained

cyproterone

the with

acetate

levels

androgen

be

nuclei

tissue with

were

using

receptors

incubated

with

with prostatic

receptor

in

vitro

anti-androgen

the

the

complexes complex

were at

min - -''. a

The

much

1 χ 10-2

min~l.

rate

with

studied 10°C. A s

to

complex For

of

the

shown at

in

this

approximately

receptor a

order

complex.

dissociated

anti-androgen

faster

in

androgen

receptor

R1881-receptor

kinetics

R1881-receptor

at

[3H]-

tissue

these

could

t e m p e r a t u r e w i t h a d i s s o c i a t i o n rate c o n s t a n t of 2.5

and of

the

receptor

conditions

the

this

fig.

results

investigated of

with

purpose

measured.

gradients

testosterone

in c o m p a r i s o n

radioactive

was

radioactivity

acetate

40

nM).

described

(18).

i n t e r a c t i o n of c y p r o t e r o n e

establish

in

extract

sucrose

(testosterone

assay.

measurable

minced

on

37°C

as

prostatic

only

of

salt

at (20

chromatography.

i n c u b a t i o n of t h e p r o s t a t i c

ligands

4 |ΛΜ) f o l l o w e d

that

cyproterone

tritiated

an

in a

peak

radioactive

unlabelled

layer

association

Radioactivity

sedimented Hardly

fig.

ligand

anti-

prostatic

incubated

isolated

minced

by thin

in

and

radioactive

not o c c u r , as w a s checked results

androgen

in vivo of the

[3H]-cyproterone acetate

d e g r a d a t i o n of

during

the

with minced

animals

nuclei

bound

of

[ 3 H ] - c y p r o t e r o n e acetate

and

incubation

previously

process

studied.

any rapid d e g r a d a t i o n

androgen, experiments

with

was

complex

dissociate

rate

569

Ln ( I Bound) 1.81 1.1 χ 1(P s i t e s / n u c l e u s R1881 , k _ 2 = 2 . 5 x 1 0 min

2.8 -

CA

_2

\ k _ 2 = 1 . 0 x 1 0 min

2.0 -

—I

1.2

1 120

60

30

—I

—I— 180

CA

R1881

210

minutes

Figure 3 A . D i s s o c i a t i o n k i n e t i c s a t 10°C o f a n d r o g e n c y t o s o l receptor complexes from r a t p r o s t a t e l i g a t e d e i t h e r w i t h [3H]-R1881 or with [3H]-cyproterone acetate. A represents the s t a b i l i t y o f t h e R 1 8 8 1 - a n d r o g e n r e c e p t o r c o m p l e x under t h e incubation conditions. B. A n d r o g e n r e c e p t o r l e v e l s i n p r o s t a t e n u c l e i 1 h a f t e r i n v i t r o i n c u b a t i o n a t 30°C o f m i n c e d p r o s t a t e s o b t a i n e d f r o m c a s t r a t e d r a t s w i t h 20 nM [ 3 H ] - R 1 8 8 1 o r [ 3 H ] - c y p r o t e r o n e a c e t a t e (CA).

The p r e s e n t receptors castrated acetate.

results

demonstrate

t o a DNA-binding rats

after

A possible

cyproterone

acetate

in

state

that is

vitro

incubation

explanation might

be

complex.

several

studies

cyproterone

specific

R1881

or

for

found

anti-androgen-receptor that

activation

impaired

in

the

with mode

the

Although

it

acetate

dihydrotestosterone

of

androgen

in p r o s t a t e s

cyproterone of

action

instability has can

receptor

from

been

of

the

shown

compete binding

of

in for

sites

570

the relative binding activity of the anti-androgen is low (18, 19, 20). The apparent lack of transformation of anti-androgenreceptor complexes found in the present study might therefore be explained in terms of a low affinity of the ligand and a rapid dissociation of the anti-androgen-receptor complex.

II

Molecular properties of androgen receptors

Multiple forms of the androgen receptor have been described (10,

11,

13).

(obtained containing receptors. between

Sedimentation

by sucrose gradient 0.4 Μ

KCl)

were

values

ranging

centrifugation

observed

for

from in

the

3-4.5S

gradients

transformed

Also for the nuclear receptor, forms with S-values

3 and

cytoplasmic

4S have

receptor

been

reported

involvement

(10,

21).

of proteolytic

For

the

enzyme

activity in the formation of the 3.0 and 3.6S forms in vitro has been described (13).

It can be envisaged that a similar

process might also occur in vivo in the nucleus. the

serine

protease

inhibitor

diisopropyl

Addition of

fluorophosphate

(5

m M ) and the thiol protease inhibitor leupeptin (0.25 m M ) to the buffers during not

isolation of the receptor from nuclei did

affect the sedimentation value

of the androgen receptors

Table I_ Size of androgen receptors obtained from different sources. Estimation of size in buffer with 0.4 Μ KCl after activation of the receptor. Small form

: 2.5-3.0 S; 1.8-2.2 NM; 20-30 kDa Rat prostate (non castrated rat) Ram seminal vesicle.

Intermediate form

: 3.6-4.0 S; 2.8-3.0 nm; 40-50 kDa Rat prostate.

Large form

: 4.5-5.0 S; 4.4-5.0 nm; 80-120 kDa Rat epididymis Calf uterus NHIK tumor cells

571

prepared

in

castrated) prostate

our

laboratory

o r ram s e m i n a l cytosol

is

from

prostates

vesicles

capable

to

(table

catalyse

of

I). the

S e £ a r a _ t i_££

of

differences. complexes steroid state

inhibitors

androgen Surface

receptor 15,

transformation

22,

23).

It

IEP o f

5.8 t o 6.3

more b a s i c opposite

to

receptors receptor

£Ü

steroid

for characterization

Obviously

androgen

and t h i s

the

that

DNA-binding in

for

shown

change

interaction

receptors

become

in s u r f a c e

of

charges

the complex

with

sites.

l i q u i d chromatography

(HPLC) o f

steroid

has been i n t r o d u c e d and was s u c c e s s f u l l y a p p l i e d forms

and

(24,

quantification

25).

In o r d e r

of

different

to c h a r a c t e r i z e

liquid

chromatography

system

for

steroid the

we a p p l i e d a n i o n

c h r o m a t o g r a p h y on a Mono Q column w i t h t h e r e c e n t l y protein

of

c o m p l e x i s a c c o m p a n i e d by a c h a n g e

p r o p e r t i e s of androgen r e c e p t o r s , "fast

£2ΐϋΕ£® receptor

of

c h a r g e d genomic a c c e p t o r

characterization

of the

activation

(5).

R e c e n t l y high p r e s s u r e

ί>££ί£

of

an a c t i v a t e d ,

has been

upon a c t i v a t i o n

m i g h t be i m p o r t a n t

££

changes

have been used a s p a r a m e t e r

(5,

forms in

shown).

£.££££i.££.£

charge

the a n d r o g e n - r e c e p t o r the

(not

(not

contrast

conversion

l a r g e ( 4 . 5 S ) t o i n t e r m e d i a t e and s m a l l r e c e p t o r absence of p r o t e a s e

rats In

ionic

exchange introduced

(FPLC)". T h i s

novel

approach to chromatography o f l a b i l e b i o m o l e c u l e s u s e s

columns

with

a

uniform

separation

and f a s t

increased

pressure

[^H]-R1881 prostate,

All

androgen

in

peak a t

receptor

allow

at only a

the

cytosol

presence

of

receptor

from

20 mM m o l y b d a t e

rat

eluted

0 . 3 2 Μ NaCl f r o m a Mono Q a n i o n e x c h a n g e with

activity the

fast

moderately

6 A ) . T h i s p e a k o f bound r a d i o a c t i v i t y

suppressed

constituting

which

flow c h a r a c t e r i s t i c s

labelled

column ( f i g .

spheres

(26).

prepared

as a s i n g l e completely

monodisperse

peak

a

was eluted

100-fold

molar

recovered at

0.32

in

could

excess the

of

be

R1881.

fractions

Μ NaCl and a

75-fold

572 purification of the androgen receptor was achieved in fraction 28 with a recovery of 71%. On sucrose gradients (high ionic strength) the FPLC peak sediraented as 3.6S entity, while a molecular

weight

of

48,000

was

found

chromatography. The molybdate-stabi1ized

after

ACA-44

gel

form of the androgen

receptor in cytosols prepared from rat epididymis and calf uterus were eluted

from the Mono Q column with a recovery of

85% and at the same ionic strength (0.32 M) as the prostatic androgen receptor. Further analysis of the eluted receptors on sucrose gradients and by ACA-44 gelchromatography

resulted in

Figure 4_ FPLC chromatography of androgen receptor complexes from the cytosol of rat prostates in the presence (A) or absence (B) of 20 mM sodium molybdate. 500 1 cytosol samples were applied on a Mono Q column. Elution was accomplished with a linear salt gradient (0-0.35 Μ NaCl). Each 1 ml fraction was assayed for total radioactivity. In figure Β only specific binding is represented.

DPM

χ

DPM χ

120 -

10

12 .

H-R. H - R , „ „ , + 100 f o l d molar e x c e s s 1881

R

1881

Μ

Μ

NaCl

r •*

40 -

NaCl

8-

- .2

.1

10

20 ml

30

40

2

-

-.4

573 sedimentation coefficients of 4-5S and molecular weights of 90,000

(see

also

table

I).

These

results

indicate

that

androgen receptors prepared in the presence of molybdate show a strong

interaction with anionexchange

resins,

indicating a

net negative surface charge of the molybdate stabilized Furthermore,

a

substantial

purification

of

the

form.

androgen

receptor could be obtained within a reasonable short time (40 min) . Prostate cytosol, prepared

in molybdate-free buffer, was also

analyzed on FPLC. The elution profile is shown in fig. 4B. A three-fold observed

reduction of the peak eluted at 0.32 Μ NaCl was

(recovery

receptor-like

35%).

In addition

androgen binding

were

three other peaks found,

indicating

of the

presence of multiple forms of the androgen receptor in the absence of molybdate. These multiple

forms eluted at a lower

ionic strength might represent androgen receptor which

became

procedure addition

activated

or

during

the

either

the

during

ionexchange

high proteolytic

the

complexes

homogenization

chromatography.

enzyme

activity

In

present

in

prostate cytosol might have caused further degradation of the androgen

receptor

due

to the

relative

instability

activated complex. Even generation of a mero-type

of

the

receptor

cannot be excluded (27; see table I, small receptor form). It is concluded powerful,

that

fast

tool

the for

FPLC-anion

exchange

characterization

system and

purification of steroid receptors. In addition

is a

partial

this technique

could be applied as a rapid procedure for the

quantitative

estimation of androgen receptors in small biological samples.

Εst imat ion

of

molecular

mass

of

androgen

receptors.

defining the molecular properties of the activated androgen

receptors,

several

investigators

have

By

form of used

the

elution volume during gelchromatography and the sedimentation coefficients

in sucrose gradients

for receptor

sizes and

the

574

ability

to

androgen

bind

to

receptor

DNA

for

receptor

proteins

are

activation.

extremely

Because

labile,

often

denaturation of the binding site occurs, which causes the loss of

the

(radioactive)

receptor

impossible.

ligand

and

In addition

makes

androgen

detection receptor

of

levels

the in

c y t o s o l p r e p a r a t i o n s of a n d r o g e n target tissues are 10 to 20 times

lower

than

progesterone molecular

the

generally

receptors.

mass

electrophoresis for

those

of

With

the

found

respect

receptors

application

of

this

to the e s t i m a t i o n

recently

has been successfully

attachment of the ligand

attached

to

conjugated

technique

proteins

is

and of

SDS-PAGE

prerequisite

the

covalent

to the steroid binding domain of the

in w h i c h

synthetic

also

applied. A

receptor m o l e c u l e (affinity labelling). have been published

for o e s t r o g e n

e.g.

Several p r o c e d u r e s

the steroid can be

via

covalently

photoactivation

of

highly

ligands and of ligands containing

groups

which become highly reactive upon irradiation with U.V. light. Chemical

linkages

acetylation steroids.

with

with

protein

bromo-

Affinity

or

might

be obtained

chloro-acetoxy

labelling

not

only

through

derivatives permits

of the

i d e n t i f i c a t i o n of p r o t e i n s p r e s e n t at very low abundancy in cytosolic

preparations

unequivocal

of

identification

stage of a purification

high

complexity,

of steroid

binding

but

also

the

proteins at any

procedure.

We have studied the molecular properties of a DNA-binding form of

the

androgen

affinity

receptor

labelling.

from

A partial

calf

uterine

purified

cytosol

(approx. 40

after times)

DNA-binding a n d r o g e n receptor p r e p a r a t i o n ligated w i t h

the

synthetic

and

androgen

ligand

[^H]-R1881

was

photolysed

s u b s e q u e n t l y p r e c i p i t a t e d w i t h t r i c h l o r o a c e t i c acid. A f t e r extraction

with

ethylacetate

the precipitate

was

solubilized

in S D S - s a m p l e buffer and applied on a 8% P o l y a c r y l a m i d e gel. A f t e r e l e c t r o p h o r e s i s the gel w a s sliced and each gel slice was

counted

for

radioactivity.

The

SDS-PAGE

profile

of

575

10

-τ

20 gel slice number

20 gel Stic« number

Figure A SDS-PAGE p r o f i l e s o f p h o t o l y s e d [3 Η]-R1881-cytοso X p r e p a r a t i o n s f r o m c a l f u t e r u s i n t h e p r e s e n c e o f 2.5 ^M t r i a m c i n o l o n e a c e t o n i d e . I r r a d i a t i o n of c y t o s o l was p e r f o r m e d a f t e r p a r t i a l p u r i f i c a t i o n w i t h ammonium s u l p h a t e p r e c i p i t a t i o n (40%) and D N A - c e l l u l o s e c h r o m a t o g r a p h y . D N A - c e l l u l o s e bound f r a c t i o n i r r a d i a t e d in the p r e s e n c e of 15 nM [ 3 H ] - R 1 8 8 1 ; D N A - c e l l u l o s e bound f r a c t i o n i r r a d i a t e d in the p r e s e n c e of 15 nM [ 3 H] -R1881 + 3 (ΛΜ d i h y d r o t e s t o s t e r o n e . SDS-PAGE profiles of photolysed [3H]-R1881 cytosol p r e p a r a t i o n s from ( W i s t a r ) rat p r o s t a t e a f t e r partial p u r i f i c a t i o n w i t h FPLC a n i o n e x c h a n g e c h r o m a t o g r a p h y , c y t o s o l + 5 nM [ 3 H ] - R 1 8 8 1 ο : c y t o s o l + 5 nM [ 3 H ] - R 1 8 8 1 + 500 nM R1881. photolysed uterus could 98,000

is

[3H]-R1881-androgen shown

be

detected

in f i g .

associated

D molecular

suppressed

additional

200-fold molar

present.

This

with

weight.

completely

finding

receptor

c o m p l e x e s from

5A. One m a j o r peak o f

in

The a

a protein radioactive

cytosol

excess

indicates

of

of

approximately peak

preparation

could where

dihydrotestosterone

that

calf

radioactivity

the s i t e s

to which

be an was the

576 ligand

is

cytosol

attached

20 t i m e s

are

saturable.

higher

f o u n d and t h e s y n t h e t i c progesterone

it

is very

t h e o b s e r v e d peak d o e s r e p r e s e n t D androgen

500-fold

molar

the

cytosol

receptor

excess

of

together

[3H]-R1881

binding

photoaffinity (a

resulted

two

in

the

to

in c a l f the

the of

tissue

Similar

favor

[3H]-R1881

progesterone

labelled

After

rats

as d e s c r i b e d fractions

or

protein

molybdate

at

of

represents

the

section

have

presence

uterine

mM).

native

form the

reported

that

study f o r

recently affinity

Whether

receptor

cytosol.

4 A ) . The

weight

in

The

peak

for

by a

the of

the

size

of

86

kD

receptor

labelling

for

of

the

prostate The

prostate

weight,

high

cytosol

despite

the

leupeptin

the

strain

In was

100-fold

the

PMSF ( 0 . 6 mM) and

from a d i f f e r e n t

(28).

of

receptor.

unanswered.

present

molecular

(20 mM),

molecular

calf

cytosol

androgen

remains

activities

androgen r e c e p t o r o b t a i n e d been

fig.

be s u p p r e s s e d

androgen

molybdate A

from

that

i s s h o w n . One m a j o r peak

which c o u l d

influenced

of

appearing

prostate

(see

dihydrotestosterone.

enzyme

bands

stabilized

with

on a Mono Q column d u r i n g FPLC

calf

labelled

the

proteolytic

second:

f u r t h e r p r o c e s s e d as d e s c r i b e d

46,000 D,

excess

rat

to

block

is concluded

receptor

with

added to

a

[3H]-R1881

98,000 D.

in the p r e v i o u s form of

with

of

first

either

androgen

performed the

was

in o r d e r

w i t h a MW of

was f r a c t i o n a t e d

ρhotoaffiηity

(0.25

the

5B t h e SDS-PAGE p r o f i l e

detected

might

were

27-29 w e r e

DNA-binding figure

of

is a protein

irradiation,

castrated

molar

form

the p r e s e n c e

receptor;

It

for that

and not

cytosol,

receptors

progestagen)

specifically

studies

were

to e s t a b l i s h

uterine

progesterone

synthetic

DNA-binding

uterine

levels

t h e androgen r e c e p t o r

a t 1 1 0 , 0 0 0 D and 8 1 , 0 0 0 D r e s p e c t i v e l y . uterine

calf

triamcinoloneacetonide

with

labelling

[3H]-R5020

important

r e c e p t o r . Two arguments

a 98,000

in

receptor

l i g a n d R1881 has a h i g h a f f i n i t y

receptors,

the p r o g e s t e r o n e

Since

progesterone

of

preparation

was m o r e p u r i f i e d

prostate rats used and

has in the

577 ligand

used

was

b r o m ο a c e t ο χ y d i h y d r ο t e s t o s te rone.

m e t h o d o l o g i c a l d i f f e r e n c e s might p r o b a b l y e x p l a i n extent

the

However,

discrepancies the

r e c e p t o r of activity

observed

observed.

molecular

proteolytic

of

46

kD

activity

in

the c o r t i c o i d

receptor

different

regions,

affinity,

one r e g i o n

region

that

is

a

might

androgen r e c e p t o r a s i m i l a r

enzymes.

weights

one

indicate

that

to

for

degradation

interaction

The a c t i v a t e d

androgen

for

for

but

including

receptor

chromatin,

interact

cibacron

blue

b u f f e r s were

of

displays

natural

2'5'-ADP

these

androgen

with and

and

by

proteolytic

is given

the

Under

d o e s not d i s s o c i a t e presence

the

an a f f i n i t y

RNA, is

of

a

molecule

obtained

for

rat prostates

from

added

with

to

nuclei, of

not

(10,

13,

low for

ionic

extraction

from of

are summarized

the

phosphate, strengths

the e x t r a c t i o n

DNA

cellulose reagents

binding

androgen

androgen in t a b l e

and the

region

binding

receptors II.

salt

inhibitors,

f r o m t h e r e c e p t o r , an i n d i c a t i o n

apart

30-35).

by high

these

chromatin

only

polyanions

Pyridoxal

effective

influence

separate

receptor

the

phosphoce1lulose,

disrupted

receptor.

heparin,

the

in

receptor

synthetic

sepharose

polyanions

shown t o be v e r y

receptors

sepharose.

binding

and a t h i r d

c o n c e n t r a t i o n s and i n a d d i t i o n by v a r i o u s o t h e r which

for

three

9.

nucleosomes,

and

with

as

sites

several

sepharose

Interactions

the

activated

steroid

such a s t r u c t u r e

Ill

of

the

affinity

in f i g .

nuclear acceptor

proteolytic

considerable

for

showing

DNA-binding

prone

Character!sties

with

androgen

can be p o s t u l a t e d

next s e c t i o n s and summarized

heparin

the

a receptor molecule with

region

with

most

tissue

structure

(29):

Further e v i d e n c e

DNA,

for

98 kD in a t i s s u e w i t h l o w e n d o g e n i c

and

These

to a certain

of

ADPsteroid for

the

in

the

site.

Data

from n u c l e i

of

578 Table II Extraction of labelled androgen receptors from nuclear pellets o b t a i n e d f r o m rat p r o s t a t e s i n c u b a t e d in v i t r o w i t h testosterone. extraction medium

fmoles receptor/100 mg tissue

buffer KCl, 0.4 Μ cibacron blue, 0.4 mM heparin, 0.2 mg/ml pyridoxal phosphate, 10 mM

8 53 45 60 53

Prostates obtained from one day castrated rats were incubated with 2.10 Μ [^H]-testosterone and in a parallel experiment with an additional amount of 2.10 - 6 Μ non-radioactive testosterone. A thoroughly washed nucler pellet was prepared and extracted for 1 h at 4°C. The amount of radioactive steroid extracted was estimated and corrected for nonspecifically bound steroid with the values obtained from the parallel incubation.

Cibacron

blue

localized

probably

acts

as

a

substance

cation exchange properties and

some

showing

both

hydrophobic

interactions. The lower optimal concentrations of heparin and Cibacron blue compared to pyridoxal phosphate required

for

interference with receptor binding to nuclear chromatin might indicate the involvement of a spatial arrangement of more than one negatively charged group separated by a less polar region. The effect of pyridoxal phosphate

might primarily

involve

binding of this molecule to an essential ε-amino group of the receptor

as was

first shown

for the corticoid

receptor by

Litwack and co-workers (36). The similarities for

the

in the effects of the various reagents used

extraction

elution of receptors suggests

of

receptors

from

that DNA-binding

from

DNA-cellulose is involved

nuclei

and

and related

for

the

matrices

in receptor binding

in

the nucleus, but do not necessarily imply that DNA is the sole m o l e c u l a r structure responsible for binding of the androgen

579

Figure Digestion with micrococcal nuclease of nuclear chromatin obtained from rat prostate nuclei with [ 3 H ] - D H T labelled androgen receptors. The sucrose density gradient profiles show both distribution of [ 3 H ] - D H T labelled receptors and different nucleosome fractions (the peak in the dotted line indicates the position of the mononucleosomes, sedimenting slightly faster than the marker with a sedimentation value of 11.2 S).

receptor to chromatin. In

studies

integrity

with of

nucleases

the

linker

Rennie

(30) observed

regions

of

the

that

nucleosomes

the in

chromatin is necessary for steroid binding and that most of the androgen receptor is associated

with fractions containing

large oligomers of nucleosomes. In addition we observed small amounts of 10S,

androgen-receptor

suggesting

complex

release of androgen

sedimenting

between

receptors partly

9-

coupled

to a chromatin fragment (fig. 6). Recent

studies

of

specific binding

Davies

by prostate

androgen receptors.

and

Thomas

(37)

showed

chromatin of partially

The highest binding capacity was observed

for oligonucleosome aggregates of up to twelve obtained

from

active genes.

the

tissue purified

fraction

enriched

in

nucleosomes

transcriptionally

580 IV

Interaction

of

androgen

receptors

with

DNA,

RNA

and

forms of

the

polyribonucleotides In our

studies

androgen ("3S",

we h a v e c o m p a r e d

receptor

castrated their

rats)

from

ability

to

On the

other

single

stranded

prostates,

interact

with

in t a b l e

different

to

with

the

"4S"

for

the

small

("4S",

from

with respect

polyanions. (fig.

DNA-sepharose

DNA) was r e d u c e d

compared

i.e.

I,

s t r o n g l y w i t h ADP-sepharose

hand b i n d i n g

particularly

rat

r a t s ) and i n t e r m e d i a t e

f o r m s as d e s c r i b e d

forms i n t e r a c t e d

when

obtained

from non-castrated

two d i f f e r e n t

Both

7).

(containing

mainly

the "3S" r e c e p t o r

receptor.

This

s i g n i f i c a n t when s m a l l amounts o f

to

form

effect

was

i m m o b i l i z e d DNA

w e r e u s e d and was n o t due t o d e g r a d a t i o n o f DNA, b e c a u s e

the

"3S"

The

receptor

preparation

specificity

of

the

preparations competitive

with

poly(UG)

are

sepharose of illustrates inhibits

The

the

of

of

form

to

high

investigated

"4S"

in

for

e.g.:

fragments

obtained

binding

from

of

a the

RNA and

binding

to

ADP-

the r e c e p t o r . F i g . 8

in

DNA-binding

calf

for

thymus DNA

the

hardly for

the

receptor.

receptor

perform

in

binding

( f i g . 8).

the

difference

obtained

sufficient

was s t r i k i n g

restriction

of

the "3S" f o r m , but c o m p e t e s w e l l

affinity

code f o r p r o s t a t i c were

was

androgen

inhibition

Double-stranded

the

polyribonucleotides for

using

significant

on the

form

and p o l y ( U ) ,

receptor

competitors

forms.

binding

The

(38).

the

complex to ADP-sepharose

be p r e p a r e d

purified (39).

assay,

potent

sites

"3S"

could

DNAse a c t i v i t y

b o t h " 4 S " and " 3 S " f o r m s o f

two r e c e p t o r binding

of of

nucleotides

binding

steroid receptor

was f r e e

interaction

a series of

the

of

poly(UG),

protein

regions

of

receptor

poly(AU),

genomic

with

clones

(39).

the

rat in

competition

"3S"

and c o n t r a s t e d of

from

quantities

partially experiments

for

certain

poly(I), the from

These

genes

prostates

a

low

genes

gene

which

poly(G) affinity which

fragments

contain

the

581

fmol bound

D i f f e r e n t i a l b i n d i n g o f two f o r m s o f t h e a n d r o g e n r e c e p t o r to A D P - a n d O N A - s e p h a r o s e .

20 '

ADP-s

1

DNA-s

4S

Figure 7_ Interaction of androgen r e c e p t o r s (25 f m o l e s ) e i t h e r in 3S- o r 4 S - f o r m w i t h 2 ' , 5 ' - A D P sepharose or DNA c e l l u l o s e . Each matrix c o n t a i n s a p p r o x i m a t e l y 10 pg of ( p o l y ) n u c l e o t i d e .

ADP-s

•

DNA-s

3S

Figure £ C o m p e t i t i o n e x p e r i m e n t s w i t h two d i f f e r e n t f o r m s of the androgen r e c e p t o r , ( f r o m 3 8 ) . BQ/B r e p r e s e n t s the r a t i o of androgen r e c e p t o r complex bound t o A D P - s e p h a r o s e in the absence (Bo) and presence (B) of a c o m p e t i t o r .

582 presumed hormone receptor binding site, located 5'-upstream from the transcription initiation site (40).

Our data suggest

that the "3S" form of the androgen receptor lacks the specific domain or conformation necessary for interaction with DNA, but retains a high affinity for certain forms of RNA. As discussed in the section on multiple receptor forms, the small

(3S) and

intermediate

(4 S) receptor

fragments of the transformed 4.5S receptor. speculate

that

forms

might

be

It is tempting to

in vivo transformation of the receptor to its

DNA-binding form is followed by processing to a form that has lost the ability to recognize specific binding sites on double stranded binding

DNA site

and

is

transported

through

its

away

binding

from

the

to RNA.

In

chromatin tissues

of

normal, mature animals the receptor would then be present for a substantial form.

part

in the intermediate

(4S) and small

(3S)

In this respect it is interesting to note that for rat

prostate nuclear receptors both a 3S form (11, 12, 21) as well as a 4S form (10, 12) have been reported.

Summary and conclusions Androgen

receptors

isolated

in a non-activated

(i.e. non-DNA-

binding) state can be converted in vitro to an activated (DNAbinding) state by treatment Activation of androgen increase

in affinity

with concentrated

receptors

salt

is accompanied

solutions.

by a 30-fold

for the androgen ligand and by a high

affinity of the complex for polyanions and nuclear

chromatin.

Mainly three different forms of androgen receptors could be isolated,

which

sedimentation certain

could

be distinguished

coefficients,

of their binding

polyribonucleotides,

estimated

by

denaturating

and

gelchromatography conditions

on

after

of and

their

basis of

to DNA, RNA and molecular

electrophoresis

affinity

their

labelling.

mass under It

is

583

TRANSFORMED ANDROGEN RECEPTOR

• 11 + 11 + I I I

hypothesized consists

of

that three

size

(region

high

affinity

fragment and

is

which

S)

kDa ( 1

S)

contains RNA.

formed

with

of

the

region the

regions

(*. 6S)

the monomeric androgen r e c e p t o r different

for

formof

different

kDa (3

15

80-120 kDa

contains

combination largest

I)

25

of

I,

regions

the s t e r o i d Together

and I I I

site

of

a

in t h e is

larger 45,000

Finally,

A model

receptor

in

and has a

II

approx.

domain.

results

molecule

smallest

region

weight

receptor.

androgen

the

binding

DNA-binding II

which

with

a molecular

androgen the

of

the

monomeric,

relating

shown

infig.

the 9.

Acknowledgements We

thank

Drs.

W.

de

Boer

and

J.A.

Foekens

for

their

584 contributions to this study and Dr. H.J. van der Molen for his many

helpful

discussions.

We

are

indebted

to Schering

(Berlin, FRG) for the supply of radioactive and

AG

radioinert

cyproterone acetate.

References 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Yaraamoto, K.R., Alberts, B.M.: Ann. Rev. Biochem. 45, 721746 (1976). O'Malley, B.W., Means, A.R.: Science 183, 610-620 (1974). Davison, B.L., Mulvihill, E.R., Egly, J.M., Chambon, P.: Cold Spring Harbor Symp. Quant. Biol. 47, 965-976. Yamamoto, K.R., Payvar, F., Firestone, G.L., Maler, Β.Α., W r ä n g e , 0, C a r 1 s t e d t - D u k e , J., G u s t a f s s o n , J.-A°., Chandler, V.L.: Cold Spring Harbor Symp. Quant. Biol. 47, 977-985 (1983). Mainwaring, W.I.P., Irving, R.: Biochem. J. 134, 113-127 (1973). King, W.J., Greene, G.L.: Nature 307, 745-747 (1984). Welshons, W.V., Lieberman, M.E., Gorski, J.: Nature 307, 747-749 (1984). Mainwaring, W.I.P., Symes, E.K., Higgins, S.J.: Biochem. J. 156, 129-141 (1976). Tymoczko, J.L., Liang, Τ., Liao, S.: In: O'Malley, B.W., Birnbaumer, L. (eds) Receptors and Hormone Action, Vol. 2, Academic Press, New York, pp. 121-156, 1978. Mainwaring, W.I.P.: The Mechanism of Action of Androgens, Springer-Verlag, New York 1977. Liao, S., T y m o c z k o , J.L., C a s t a n e d a , E., L i a n g , T: Vitamins and Hormones vol. 33, Academic Press, New York, pp. 297-317, 1975. M u l d e r , E., V r i j , Α., F o e k e n s , J.A.: M o l e c . C e l l . Endocrin. 23, 283-296 (1981). Wilson, E.M., French, F.S.: J. Biol. Chem. 254, 6310-6319 ( 1979) . Grody, W.W., Schräder, W.T., O'Malley, B.W.: Endocr. Rev. 3, 141-163 (1982). W e i c h m a n , B.M., Notides, A.C.: J. Biol. Chem. 252, 88568862 (1977). Bailly, Α., Le Fevre, B., Savouret, J.F., M i l g r o m , E.: J. Biol. Chem. 255, 2729-2734 (1980). Kovacs, W.J., Griffin, J.E., Wilson, J.D.: Endocrinology 113, 1574-1581 (1983). Brinkmann, Α.Ο., Lindh, L.M., Breedveld, D.I., Mulder, E., Van der Molen, H.J.: Mol. Cell. Endocrinol. 32, 117-129 (1983). Raynaud, J.P., Bouton, M.M., Moguilewsky, M., Ojasoo, T. Philibert, D., Beck, G., Labrie, F., Mornon, J.P.: J. Steroid Biochem. 12, 143-157 (1980).

585 20. Z a k a r , Τ . , T o t h , Μ.: J . S t e r o i d B i o c h e m . 17, 287-293 (1982) . 21. R e n n i e , P . S . , Van D o o r n , E., B r u c h o v s k y , N . : M o l e c . C e l l . E n d o c r . 9, 145-157 ( 1 9 7 7 ) . 22. H o l b r o o k , N . J . , B o d w e l l , J . E . , J e f f r i e s , M., Munck, Α . : J . B i o l . Chem. 258, 6477-6485 ( 1 9 8 3 ) . 23. B r i n k m a n n , Α . Ο . , B o l t - d e V r i e s , J . , De B o e r , W., L i n d h , L.M., M u l d e r E . , Van d e r M o l e n , Η.J.: J. Steroid. B i o c h e m . , in p r e s s ( 1 9 8 5 ) . 24. P a v l i k , E.J. Van N a g e l j r . J . R . , M u n c e y , M., D o n a l d s o n , E.S., H a n s o n , M., K e n a d y , D., R e e s , E.D., T a l w a l k a r , V . R . : B i o c h e m i s t r y 21, 139-145 ( 1 9 8 2 ) . 25. H u t c h e n s , T.W., W i e h l e , R.D., S h a h a b i , Ν . Α . , Wittliff, J . L . : J . C h r o m a t o g r . 266, 115-128 ( 1 9 8 3 ) . 26. S o d e r b e r g , L . , B e r g s t r o m , J . , A n d e r s s o n , K.: Protides B i o l . F l u i d s P r o c . C o l l o q . 30, 629-634 ( 1 9 8 3 ) . 27. S h e r m a n , M.R., P i c k e r i n g , L . A . , R o l l w a g e n , F.Μ., M i l l e r , L . K . : F e d . P r o c . 37, 167-173 ( 1 9 7 8 ) . 28. C h a n g , C . H . , L o b l , T . J . , R o w l e y , D . R . , T i n d a l i , D.J.: B i o c h e m i s t r y 23, 2527-2533 ( 1 9 8 4 ) . 29. C a r l s t e d t - D u k e , J . , O k r e t , S . , W r a n g e , O, G u s t a f s s o n , J . A ° . : P r o c . N a t l . A c a d . S e i . USA 79, 4 2 6 0 - 4 2 6 4 ( 1 9 8 2 ) . 30. R e n n i e , P . S . : J . B i o l . Chem. 254, 3947-3952 ( 1 9 7 9 ) . 31. D a v i e s , P . , T h o m a s , P . , B o r t h w i c k , N.M., G i l e s , M.G.: J . E n d o c r . 87, 225-240 ( 1 9 8 0 ) . 32. L i a o , S . , S m y t h e , S . , T y m o c z k o , J . L . , R o s s i n i , G . P . , C h e n , C . , H i i p a k k a , R . A . : J . B i o l . Chem. 255, 5541-5551 ( 1 9 8 0 ) . 33. M u l d e r , E., F o e k e n s , J . Α . , P e t e r s , M.J., Van d e r M o l e n , H . J . : FEBS L e t t e r s 97, 260-264 ( 1 9 7 9 ) . 34. M u l d e r , E., V r i j , L . , F o e k e n s , J . Α . : S t e r o i d s 36, 6 3 3 - 6 4 5 ( 1980) . 35. L i n , S . , Ohno, S . : Eur. J . Biochem. 124, 283-287 ( 1 9 8 2 ) . 36. C a k e , M.H., D i S o r b o , D.M., L i t w a c k , G.: J . B i o l . Chem. 253, 4886-4891 ( 1 9 7 8 ) . 37. D a v i e s , P . , T h o m a s , P . : J . S t e r o i d B i o c h e m . 20, 57-65 ( 1984 ) . 38. M u l d e r , E., V r i j , A . A . , B r i n k m a n n , Α . Ο . : B i o c h e m . B i o p h y s . R e s . Commun. 114, 1147-1153 ( 1 9 8 3 ) . 39. M u l d e r , E., V r i j , A . A . , B r i n k m a n n , Α.Ο., Van d e r M o l e n , Η.J., P a r k e r , M.G.: B i o c h i m . B i o p h y s . A c t a 7 8 1 , 1 2 1 - 1 2 9 (1984) . 40. P a r k e r , M., H u r s t , H., P a g e , M.: J . S t e r o i d . B i o c h e m . 20, 67-71 ( 1 9 8 4 ) .

DIFFERENTIAL SENSITIVITY OF SPECIFIC GENES IN NOJSE KIDNEY TO ANEROGENS AND ANTIANDROGENS

James F. Catterall, Cheryl S. Watson, Kinmo K. Kontula, Olli A. Jänne, C. Wayne Bardin The Population Council and The Rockefeller University, New York, N.Y. 10021

1230 York

and

Avenue,

Introduction Early studies on the action of testosterone emphasized t h a t t h i s steroid has a variety of actions on almost every organ in the bocfy. Those on the reproductive t r a c t were called androgenic while those on other organs were called anabolic actions. Androgen receptors were f i r s t identified in reproductive tissues such as prostate and seminal vesicle, and studies which showed t h a t antiandrogens could compete with testosterone or i t s metabolite, 5c(-dih/drotestosterone (DHT), for receptor binding s i t e s were the f i r s t t o clearly associate androgen receptors with the action of t e s tosterone and DHT. Hie ultimate proof, however, that androgen receptors were an essential link between testosterone and the androgen-induced g e n o type was the i d e n t i f i c a t i o n of receptor mutants in the r a t and mouse which were insensitive to mary of the e f f e c t s of testosterone and other androgens (1-4). Studies on these animals also emphasized that both the androgenic and the anabolic e f f e c t s of testosterone were mediated via androgen receptors (5).

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

588

The general concept of androgen action that has evolved holds that interaction of testosterone or EHT with the androgen receptor changes i t s conformation which allows i t to bind to an "acceptor s i t e . " In this manuscript the term acceptor is used to describe a functional site in chromatin to which the steroid-receptor complex binds. Current studies suggest that a portion of the acceptor s i t e i s formed ty a unique segment of DNA at the 5 '-end of hormonally responsive genes (6,7). Since the same gene is not responsive in all tissues, formation of acaeptor sites i s dependent upon organ specific differentiation, this iirplies that there are specific factors which are necessary for steroid-induced gene expression in some t i s sues. Interaction of the steroid-receptor complex with the acaeptor site results in the expression of genes which ultimately determines the hormone-induoed phenotype. Since the overall male phenotype for a given species i s relatively uniform, i t was assumed that androgen receptor interaction with acceptors resulted in the synthesis of a relatively uniform and characteristic group of mRNfts and proteins for a given organ, and that number of steroid receptor complexes on chromatin would determine the amount of each mRiR and protein produced. It was not known, however, how androgen receptor complexes were partitioned between the individual acaeptor sites of different genes. One possibility was that the receptor had the same affinity for each acceptor in the same way that each molecule of testosterone had equal acaess to activate each receptor binding site. If this were the case, then one might predict that the dose of testosterone (viiich ultimately relates to the nunber of receptors bound to chromatin in a given organ) required both to initiate and to produce maximal responses would be similar for most genes. Alternatively, androgen receptor complexes might exhibit differential a f f i n i t i e s for several acceptor sites. If this were the case, then the number of receptors required to initiate and to produce maximal activity would vary between different genes in the same cell. This latter possibility also predicts that the potency of a given androgen might not be strictly related to i t s affinity for a given receptor (5). Much of the work on androgen induced gene expression has involved analysis of the abundant gene products stimulated fcy androgens in the rat prostate (8,9) and seminal vesicle (10-12). However, no attempts were made in

589

these

studies to correlate specific gene expression with androgen receptor

dynamics or to compare the androgen sensitivity of these

different

genes,

the mouse kidney has become an irrportant syst an for the stud/ of the androgenic control of individual gene expression.

Gene-specific

complementary

DNA (cDNA) probes are now available for four mouse kidney mRNAs: two abundant species, kidney designated

MK908 (15)

androgen-regulated

protein

(KAP) (13,14),

an mRNA

and the non-abundant mRNfts for ornithine decarboxy-

lase (ODC) (16,17) and ρ-glucuronidase (ß-GLUC) (18,19). In the present review, we summarize studies in which we used cDNA probes for KAP, ODC and p-GLUC in establishing the patterns of gene-specific responses to testosterone in the mouse kidn^r. The availability of these cDNAs to one group of investigators permitted the simultaneous measurement of the mRNA accumulation in the kidne/s in response to a variety of stimuli. In addition, nuclear androgen receptor concentration has been correlated with the expression of each individual gene (20). The results reveal distinct responses of the three genes to physiological levels of testosterone, variations in nuclear receptor concentration, duration of hormone treatment, and to antiandrogens. Uie results are consistent with the hypothesis that androgen-responsive genes have differential sensitivities to the testosterone-androgen receptor complex.

Recent Advances in the Measursnent of Bound Nuclear Androgen Receptor Recent studies suggested that steroid receptors are nuclear However,

in

the absence

proteins (21).

of specific hormone, t h ^ are rapidly lost from

nuclei and are found in cytosol

when cells

are

disrupted

in hypotonic

buffers. When the appropriate steroid ligand is bound to the receptor, the conformation of this protein changes so that i t will bind to stituents.

con-

In this state, receptors are not readily lost into cytosol when

cells are broken. nuclear

nuclear

In this review these receptors are referred to as "bound

receptors." I t is these receptors that are believed to be bound to

acceptor sites and are correlated with hormone action.

590

The concentration of bound androgen receptor in renal nuclei of following

testosterone

treatment I d .xixp is

low

androgen-responsive tissues such as the prostate.

the mouse

relative

In addition,

to

other

apparent

receptor concentrations have varied under different conditions for preparation and extraction of nuclei.

For this reason, a rigorous analysis of the

various components of the nuclear receptor assay was undertaken in order to maximize i t s specificity and sensitivity (20). evolved

for

these

The assay

conditions

that

studies were based upon the observations that a large

fraction of bound nuclear androgen receptors was lost when nuclei were isolated

in

aqueous buffers and that another fraction of the bound receptors

was excluded from the final assay since i t could not be extracted ty KCL. To overcome these difficulties, nuclei were isolated hexylene

glycol (22)

in

the

presence

of

and bound nuclear receptor extracted with 5 mM j y r i -

doxal 5'-phosphate (20,23,24).

Nuclear receptor concentration was deter-

mined fcy incubating extracts, in triplicate, with a saturating concentration (22.5 nM) of [3H]methyltrienolone for 18 hrs at 4°C. total

Non-specific and

binding were determined in the presence and absence of a 1000—fold

molar excess of testosterone, respectively. f ran those

unbound ty

Bound ligands

adsorption to hydroxylapatite.

were

separated

Using these assay

conditions, 3-4 times more bound nuclear receptors are measured than when aqueous buffers are used for nuclear isolation and KCL is used for extraction.

Cloned cDNA Probes for mRNAs that Exhibit Unique Responses to Androgens For studies of differential gene expression in mouse kidney,

specific hybridization probes. which

codes

response

to

androgens

in

we have measured concentrations of three mRNAs with gene Hie KAP mRNA is an abundant gene product

for a protein (Mr=20,000) of unknown function.

The other two

are low abundance mRNAs which code for the androgen-induced renal p-GLUC and ODC.

enzymes,

591

Complementary DNA piasmids were prepared for each mRNA using two different approaches.

Since

KAP mRNA was shewn to be approximately 100-fold more

abundant than ODC or p-GLUC mRNA, KAP mRNA was purified

by

extraction

of

total mRNA from kidneys of androgen-treated fanale mice followed ty sucrose density phy (14).

gradient

centrifugation and

0.02%, respectively, of the c e l l ' s purified

oligo(dT)-cellulose

chromatogra-

CDC and p-GLUC mRNAs, which represent approximately 0.05% and from renal

mRNA after

followed by chromatography on protein modification of

androgen treatment,

were

polysemes fcy imminoadsorption to specific antibodies A-Sepharose (16,18).

This was a

the imrrunoadsorbent chromatographic method of Shapiro and

Young and resulted in a 300-fold purification of the ODC and p-GLUC mRt&s. The additional steps introduced into the procedure (16,18) (including passing of polysomes through protein A-Sepharose prior use

to antibody

addition,

of the ribonuclease inhibitor, RNasin, and inclusion of a high concen-

tration of heparin in the buffers) increased the purity of the two mRNAs. Purified mRJRs were assayed fcy translation _i_D vitro and used as for

preparation of

cDNA plasmid libraries.

ferential colory hybridization. and

templates

Ihese were screened by d i f -

In the case of KAP cDNA clones, the "plus"

"minus" probes were prepared from KAP mRNA-enriched gradient fractions

and mRNA from female mouse kidney, respectively.

A similar assay

for CDC

and p-GLUC cDNA clones was devised using probes prepared from protein ASepharose-bound ("plus") and drop-through tions.

In each case,

("minus")

polyscmal

colonies that preferentially bound the plus probe

over the minus probe were selected for further analysis. tion

of

RNA frac-

Final identifica-

the cDNA clones was made fcy hybridization-selected translation j j j

vitro, which correlated individual cDNA plasnids with

each

specific mRNA

activity.

Acute Response to Testosterone Following a single injection of a large dose of testosterone (10 mg), bound nuclear androgen receptor concentration increased to reach a maxinun at one hr.

By 2 hrs the receptor level was s t i l l seven times the control level or

592 182 fmoles/mg DNA (Fig. 1 ) . the receptor concentration in the nuclei then decreased ty approximately 50% over the next 48 hrs. The measurements of specific mRNAs indicated that there was no change at or before 2 hrs when receptor levels were highest. Thereafter, the response of these three mRNAs t o a single dose of testosterone varied greatly, emphasizing the d i f ferential effect of the hormone on genes in the same tissue. CDC and KAP mRNAs responded rapidly, increasing 2 . 1 - and 1.6-fold in 6 hrs, respectively . Hcwever, KAP mRNA was alnost maximally stimulated at this interval in that i t increased to only 2-fold by 48 hrs while CDC mRNA continued to accumulate to over eight times the untreated level fcy 24 hrs. p-GLUC mRNA remained unaffected until between 12 and 24 hrs of treatment when the f i r s t increase was detected; thereafter, i t rose to 1.7 times the control value a t 48 hrs. In another stud/, p-GLUC mRNA concentration reached maximal levels after 14 days of continuous androgen administration. These results indicated that for p-GLUC the lag before the f i r s t detectable response was evident and the time to reach the maximal response were strikingly d i f ferent from those of the other two genes. Hie magnitude of the increases in mRNA concentrations in these studies was determined ty densitanetric scans of autoadiograns of transfer blot hybridizations. This semiquantitative method revealed relative increases, rather than absolute values. Hcwever, t h i s type of analysis i s valid when comparing the proportional responses of several genes in the same tissue to hormones and/or drugs (Fig. 1). the results shown in Figure 1 indicate a differential responsiveness among the

three

genes

in

the

presence

of maximal levels of nuclear androgen

receptors after a single dose of testosterone. that,

in

addition

to

the

initial

response i s in part dependent on the receptor

complex

Previous studies

suggested

receptor concentration, the androgen length

of

time

remain tightly bound in nuclei (24).

that

the

androgen

I t was, therefore,

of interest to determine the relative expression of the three genes in presence

of

constant

submaximal

nuclear receptor levels.

the

To do t h i s we

determined the response of each mRNA to the physiological testosterone concentration present in normal male mice.

593

TIME AFTER TESTOSTERONE (h)

Figure Is Changes in mRNA accumulation and nuclear androgen receptor concentration a f t e r a single doee of testosterone. Testosterone (10 mg) was administered ty intraperitoneal injection of 0.2 ml of ethanol/sesame oil (1/9? v/v) t o female NCS mice. At the indicated times poly (A)-mRNA was prepared from kidney extracts and analyzed ty electrophoresis and blot transfer hybridization. Autoradiographs of each transfer f i l t e r were scanned with a densitometer (Shimadzu model CS-910). The untreated female level was assigned the value of 1. Androgen receptor measurements were made ty an exchange assay on nuclear extracts prepared from identically treated animals. Nuclear androgen receptor, (AR^) values represent the mean of 3 experiments. Kidneys from four animals were pooled a t each time point in a l l experiments. Abbreviations: KAP, kidney androgen-induced protein; ODC, ornithine decarboxylase; p-GLUC, ρ-glucuronidase.

594

The Effects of Endogenous Testosterone The relative differences in mRNA concentrations in intact females and intact males are shown in Table I. In each case, physiological amounts of testosterone secreted ky the t e s t e s of the male mice were s u f f i c i e n t t o cause an increase in mRNA concentration over the values in female animals. Hcwever, there was a major difference among the three genes as measured ky the abundance of t h e i r mRNAs. The autoradiograms of the Northern blots from which the data were derived were exposed t o achieve comparable s i g nals; t h i s required 24- and 13-fold longer exposure times for ρ-GLöC and ODC mRNAs, respectively, than for KAP mRNA. Table I Relative concentration of three mRNAs in intact female and male mice. Each mRNA concentration i s expressed as a percent of t h a t obtained a f t e r maximal stimulation with testosterone for 7 days. Gene KAP CDC p-GLOC

Female % 35 10 5

Relative mRNA Concentration Male % 100 30 35

Table I also indicates the extent t o which the individual RNAs could be increased with a maximal dose of exogenous hormone. Once again, variation among the gene products was evident. While p-GLUC and CDC mRNA concentrations were further increased ty exogenous testosterone administration, KAP mRNA was unaffected. Hie concentrations of androgen receptor in the nuclei of female, male and testosterone-treated male mice are shewn in Table I I . The receptor level in females was approximately 50% of that of intact males, whereas chronic testosterone treatment increased the level in males f i v e - f o l d . These l a t t e r nuclear receptor values represent the maximal concentrations attainable in mouse kidney and are associated with f u l l expression of CDC and p-GLUC genes. I t remains t o be elucidated, hewever,

595

whether

maximal

accumulation of these two mRNAs indeed requires t h i s high

nuclear receptor concentration. ferential

sensitivity

concentrations.

Taken together, these

suggest

of

at

a

nuclear androgen receptors, as i t cannot be further

stimulated by pharmacological steroid concentrations physiological

dif-

of the three genes t o lew nuclear androgen receptor

Maximal expression of the KAP gene appears to occur

low concentration with

data

hormone levels.

bound nuclear receptors resulted in

above

that

achieved

By contrast the same concentration of CDC and p-GLUC mRNft concentrations

which were only 30 and 35 percent of maximal. Table I I Concentrations of bound androgen receptors in renal nuclei fran intact female, intact male, and testosterone-inplanted male mice. Animal group Intact females Intact males Testosteroneinplanted males'3

Androgen receptor concentration 3 150 + 60 280 + 100 1,540 + 390

(13) c ( 6) (5)

The receptor concentrations (mean + SD) are expressed as molecules/cell assuming that receptors are evenly distributed within the kidney with 6 pg DNA/cell. a

^The animals were treated with testosterone-containing inplants (release rate, 200 yg steroid/day) for 7 days. c

Aninals per group are shown in parenthesis.

KAP mRNA i s Induced by Testosterone in Androgen Insensitive Tfn/Y Mice The androgen insensitive Tfn/Y mouse i s resistant to almost a l l of the actions of testosterone even when high doses are administered (25,26). In t h i s respect, i t differs fran the Tfm rat which has a reduced quantity of presumably normal androgen receptors and responds in a dose-dependent manlier to a 200-fold higher amounts of testosterone than normal rats (27-

596

29).

the Tfn/Y mouse also has a low concentration of androgen receptor but

i t i s disputed whether these are normal or mutant receptors (30-32). The relative concentrations of the three mRNAs were determined after

testosterone

treatment

of

Tfn/Y mice.

treatment increased the KAP mRNA concentration that

found

in

the

before

Unexpectedly, testosterone approximately

kidneys of untreated animals.

2-fold

These

observations

respond t o the lew

suggested

concentration

of

that

androgen

over

Neither CDC nor p-GLUC

mRNA concentrations were significantly affected fcy testosterone animals.

and

the

in

these

KAP gene was able to

receptor

in

Tfn/Y

mice,

whereas the CDC and p-GLUC genes were not. These results do not resolve the issue as to whether the receptors in Tfn/Y mice are normal. I f the receptors in these animals are the same as wildtype, the results could be interpreted as indicating that the KAP gene responds to a lewer concentration of bound nuclear receptors than almost ary other gene in the mouse gencme. Alternatively, i f the receptors in Tfn/Y mice are abnormal, then the acceptor for KAP must be sufficiently different frcm that of other genes to accommodate the mutant receptor.

Differential Inhibition of Individual mRNAs fcy Flutamide IVie results reported above complex

indicate

accumulation, expression

these results also

is

testosterone-androgen

suggest

that

this

differential

very

variation

in

gene

If this i s the case, then one would

gene responses when the availability of receptor com-

plexes i s changed in the presence of an antiandrogen, is

receptor

due to the different responsiveness of each gene to a given

amount of androgen-receptor complexes. expect

that

interacts with dissimilar genes to produce unique patterns of mRNA

sensitive

to

that i s , a gene that

low concentrations of androgen-receptor complexes

should be more resistant t o antiandrogens since there would always be a few receptors antagonist. containing

occupied

ty testosterone even in the presence of large doses of

Accordingly, Silastic

female

animals were

treated

with

flutamide-

rods (releasing either 150 jug/day or 650 jjg/day) with

597

Table III Inhibition of testosterone-induced mRNA accumulation fcy flutamide Treatment group T*3 Τ + Fl Τ + F2

iLeJLative_SQ^J^ajAor^ .KAP

ODC

ß-GLUC

4.5 3.7 3.4

5.0 2.9 0.7

11.6 5.1 1.0

*Hie mRNA concentration i s expressed relative to the untreated female level (1.0) of each group.

a

^Female NCS mice were inplanted with Silastic rods containing testosterone (T, release rate: 40 jug/day) alone or in combination with similar inplants of flutamide (Fl, 150 ;ug/day; F2, 650 Mg/day). Renal poly(A)-mRNA was prepared after 8 days of treatment. Table IV. SuraTary_of Jielative _Gene^Sgeci f i c JResgonses in _Mouse JÜdney.^ Animal/treatment group

KAP

ODC

g-GLoc__

Female Male Male + Τ

++ ++++ ++++

+ ++ JJJJ

+ ++ ±±++

Duration of Treatment 12-24 hrs 2 days 14 days

++++ ++++ ++++

+++ ++++ +Ü+

+ ++ ++++

+ +

+ _+

++++ +++ +++

++++ ++ +

Tfn/Y Tfn/Y + Τ Female + Τ Female + Τ + Fl Female + Τ + F2

++-H++ +_.

^Ihis table i s presented as a brief summary of the qualitative differences in sensitivity of the three gene markers to various conditions of androgen exposure. The qrmbols do not represent quantitative changes. T, testosterone; Fl, F2, flutamide, 150 and 650 /jg/day, respectively. and without testosterone-containing rods (releasing 40 jig/day). Hie results dicwn in Table I I I suggest that the sensitivity of the three mRNAs

598

to flutamide was essentially the inverse of their terone.

sensitivity

to

testos-

KAP mRNA, which was shewn t o be unusually responsive to testos-

terone, was l e a s t affected ty the non-steroidal antagonist. mRNAs were both

ODC and p-GLUC

much more sensitive to flutamide inhibition in that l e s s

than 10-15% of the testosterone-induoed mRNA concentrations remained treatment

with

the higher dose of antiandrogen.

after

Flutamide alone resulted

in no significant variation in the concentrations of

ary

mRNA (data

not

shewn).

Summary and Conclusions 1. Androgens stimulate the epithelium of the proximal tubule c e l l s of the mouse kidney to increase the s/nthesis of several proteins (5). Cloned complementary DNA probes have recently been prepared and characterized for the mRNAs for three of these proteins: p-GLUC (18,19), ODC (16,17), and KAP (14). Previous studies had shown that p-GLUC and ODC enzyme a c t i v i t i e s were regulated differently by testosterone in t h i s tissue (24,33) in that ODC responded t o testosterone with more rapid kinetics than p-GLÜC. KAP induction kinetics have not been demonstrated as the protein has been ident i f i e d only fcy translation j j j j ö t r P (13). KAP mRNA i s a relatively abundant species while those of p-GLUC and ODC were of the non-abundant mRNA class. Hie availability of these three gene-specific probes allowed us t o measure a unique spectrum of responses t o testosterone. 2. In order to fully characterize the regulation of renal

gene

expression

fcy testosterone, i t was necessary to measure the concentration of the bound androgen receptor in nuclei under the same conditions as the sion

studies.

A reliable

gene

expres-

method for the measurement of bound androgen

receptor in mouse renal nuclei was established (20). 3. In t h i s stuc^ a general trend of androgen with

the

following

ranking:

KAP mRNA »

sensitivity

was

established

ODC mRNA > p-GLUC mRNA. The

results suggest gene-specific variations in response were related t o tion

of

exposure

to

dura-

testosterone, physiological levels of testosterone,

599 concentration of bound nuclear androgen receptors, and treatment by antiandrogens (Table IV). 4. As the bound androgen receptor concentration in kidney nuclei was maximal ty 1 hr a f t e r a single steroid do9e, i t i s clear that subsequent biological responses which occurred over the next 4-48 hrs were determined in part by factors other than the i n i t i a l receptor concentration. Indeed, our previous studies have suggested t h a t , under these conditions, i t i s the product of nuclear receptor concentration and the residence time of the receptor that predicts the biological response (24). This product appears t o be, however, dissimilar for the three mRNAs studied. In accordance with t h i s notion, Watson .et ü l . (34) hypothesized that a minimum number of receptor molecules must bind in the region of the [Gus] genetic complex in chromatin in order t o achieve an induction response. This hypothesis i s now extended t o include d i f f e r e n t i a l kinetics of interaction between androgen receptors and chromatin binding s i t e s on separate genes consistent with the data presented here. Furthermore, i t has been suggested that effector molecules such as steroid hormone receptors bind t o nuclease sensitive domains in chromatin (35,36). Differential responsiveness of individual genes t o a hormone may reside in the size and/or complexity of the "active" domain with which the receptor must interact. 5. A related hypothesis was developed in order t o explain the differences in expression of the conalbumin and ovalbumin genes in response t o steroid hormones (37). In t h i s case, multiple binding s i t e s for the steroid receptor were postulated for the l e s s sensitive gene which thus requires longer duration of treatment or higher receptor concentration t o become f u l l y induced. The same d i f f e r e n t i a l responsiveness could also be explained if the acceptor of various genes bound receptor complexes with d i f f e r e n t effectiveness. The r e s u l t s reviewed here provide evidence in support of such a hypothesis based on gene-specific sensitivity t o the androgenreceptor complex. The KAP gene, which i s the most hormonally "sensitive" gene studied, i s maximally induced in males fcy physiological testosterone levels. Thus, the KAP gene m^ require the fewest nrntber of bound receptor-androgen complexes for activation because i t s acceptor has either multiple binding s i t e s or a high a f f i n i t y for receptors.

600 6. Ihe results reviewed here shew that androgen-resistant Tfir/Y animals exhibit a condition in which receptor concentration appears adequate with respect to the KAP gene, but too 1CM to have e f f e c t s on ODC or p-GLUC mRNA, even with pharmacological doses of androgens. The induction of KAP mRNA in Tfn/Y animals suggests that a l l or part of the androgen receptor in this variant (30-32) is biologically active. The results do not exclude the possibility that these androgen receptors ma/ be qualitatively different from wild-type receptors (38), but s t i l l capable of effecting the response of KAP mRNA. Further experiments will be required to distinguish this from other p o s s i b i l i t i e s . 7. Hie antiandrogen flutamide was also androgen

sensitivities

of

the

used to

three

evaluate

the

dissimilar

gene products in mouse kidney. We

reasoned that expression of the most sensitive gene should be affected to a lesser

extent than those requiring higher concentrations of nuclear andro-

gen receptors for f u l l expression. sensitivity

On the basis of

differential

of the three genes observed in other experiments, the KAP gene

was predicted to exhibit the least inhibition fcy flutamide, while gene was expected to be l e s s affected than that of p-GUJC. tions were verified providing further evidence that the stimulated

androgen the CDC

ihese predic-

KAP gene

can be

a t very low functional receptor levels, consistent with a lewer

required threshold of androgen receptor binding to chromatin s i t e ( s )

asso-

ciated with this gene.

Acknowledgement This work was supported fcy NIH grants HD-13541 and 3 F05 TW3192-0151.

We

wish to thank Ms. Susan Richman for preparing the manuscript.

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5.

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Compton, J.G., Schräder, W.T., and O'Malley, B.W.: Proc. Natl. Sei. USA _8£), 16-20 (1983).

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Gustafsson, J.A., Carlstedt-Duke, J . , Okret, S., Wikstrom, A.C., Wrange, 0 . , Payvar, F . , and Yamamoto, K.: J. Steroid Biochem. 2Qr 1-4 (1984).

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R.P.,

and

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McDonald, C., Williams, L . , McTurk, P., Fuller, F . , Mclntoäi, Ε., and Higgins, S . : Nucleic Acids Res. 11,917-930 (1983).

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17.

McConlogue, L . , Gupta, Μ., Wu, L . , and Coffino, P . : Proc. Natl. Acad. Sei. USA jJl,540-544 (1984).

18.

Catterall, J.F. and Leary, S.L.: Biochemistry 22,6049-6053 (1983).

19.

Palmer, R., Gallagher, P.M., Bcyko, W.L., and Ganschcw, Natl. Acad. Sei. USA _8£>,7596-7600 (1983).

20.

Isanaa, V . , Pajunen, A . E . I . , Bardin, C.W., and Janne, O.A.: nology 111,833-843 (1982).

Kistler,

NY Acad.

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22.

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Pajunen, A.E.I., Isomaa, V.V., Janne, Biol. diem. 257,8190-8198 (1982).

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Bardin, C.W., Bullock, L.P., Sherins, R. J . , Mowszowicz, Blackburn, W.R. s Ree. Prog. Hormone Res. 25,65-109 (1973).

26.

Sclienkein, I . , Levy, Μ., Bueker, E.D., and Wilson, J.D.: ogy .24,840-844 (1974).

27.

Sherins, R. J . , Bullock, L., G ^ , V.L., Variha-Perttula, Τ., din, C.W.: Endocrinology .28,763-770 (1971).

J.W.: Biochem. O.A.,

Bioptys.

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and Bardin,

2fi. Grosanan, S.H., Axelrod, Β., and Bardin, C.W.: Life (1971).

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Comm.

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and

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and Bar15,175-180

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30.

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33.

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34.

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35.

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36.

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37.

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38.

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M-J,

and

Chem. 257,13018-13027 G.S.:

MOLECULAR PHARMACOLOGY ANTITUMOR V. Craig

PROPERTIES

OF TAMOXIFEN;

IN A N I M A L S A N D

AN Α Ν Τ Ι E S T R O G E N

WITH

MAN

Jordan

D e p a r t m e n t of H u m a n O n c o l o g y , W i s c o n s i n C l i n i c a l Center, U n i v e r s i t y of W i s c o n s i n , Madison, W I USA

Cancer 53792

Introduction In 1 9 5 8 ,

Lerner and coworkers

cal p r o p e r t i e s of M E R (Fig.

1).

25,

The compound

the

(1) d e s c r i b e d

(1-3).

Clinical

antiestrogenic

activity

and the o c c u r r e n c e alternatives.

trials with MER

25 h a s

(5).

clomiphene

Although

animals, The

the d r u g

commercial

is n o w r o u t i n e l y

used

derivatives tested

however,

f o c u s of

breast cancer. (ICI

46,474)

effect

for

only

for

than MER

25,

Nafoxidine

(10,11)

in

animals

properties

in

in s u b f e r t i l e w o m e n a mixture

of

(enclomiphene)

for the

(6).

estrogenic geometric

i n d u c t i o n of

of

triphenylethylene 1970's

ovulation

were

tamoxifen

have

investigators

from contraception

upon the clinical

however,

a search

been

antiestrogenic/antifertility

in t h e e a r l y

research

potency

(7).

synthesized the

its

low

(4) c a u s e d

Clomid®,

Many structural activity,

the

is m o r e p o t e n t

induces ovulation

in a n n o v u l a t o r y w o m e n

and

25 c o n f i r m e d

has antifertility

and antiestrogenic

antiestrogen species

laboratory

some estrogenic properties

preparation

(zuclomiphene) isomers,

(MRL 41)

in

however,

of C N S s i d e e f f e c t s

but unlike MER

in a l l

properties

in p a t i e n t s ,

Clomiphene

pharmacologi-

first non-steroidal

is a n t i e s t r o g e n i c

tested and also has a n t i f e r t i l i t y animals

the

(U-11,100A)

to

(8,9) a n d

b o t h s h o w n to h a v e a c o u r s e of a d v a n c e d

is a v a i l a b l e

b e c a u s e of t h e l o w r e p o r t e d

shifted

hormone-dependent tamoxifen

beneficial

breast

for b r e a s t

cancer,

cancer

incidence of side e f f e c t s

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

therapy (12).

604 /C2H5

/C2H5 och2ch2n

OCH2CH2N c

2h5

enclomiphene och2ch2n

ch30

tamoxifen

nafoxidine

Ο

D

NCHjCHJO

trioxifene

S\ keoxifene

Fig. 1. Structure of non-steroidal antiestrogens described in the text.

Tamoxifen (Nolvadex®) is available for the treatment of breast cancer in more than 70 countries throughout the world; in dollar sales alone, it is one of the most important drugs used in cancer therapy.

This fact has stimulated interest in the

discovery of new, and perhaps more specific, antitumor agents.

Trioxifene is antiestrogenic in laboratory animals

(13,14) and possesses antitumor activity in animals (15) and man (16), but the drug is not available for general clinical use.

Keoxifene (LY 156758) and the related compound LY 117018

have weak estrogenic activity in laboratory animals and a high binding affinity for the estrogen receptor (17-19).

These

drugs have a short duration action because of their polar

605 n a t u r e , w h i c h m a y f a c i l i t a t e their, r a p i d c o n j u g a t i o n excretion

(20).

and

T h i s p r o p e r t y may e x p l a i n the low p o t e n c y

the L Y - c o m p o u n d s as a n t i t u m o r a g e n t s in a n i m a l s

In this c h a p t e r I w i l l r e v i e w the s t u d i e s u n d e r t a k e n l a b o r a t o r y d u r i n g the p a s t d e c a d e , to d e s c r i b e the

in my

molecular

p h a r m a c o l o g y of t a m o x i f e n and to s u p p o r t the use of the as an a n t i t u m o r

General

of

(21).

drug

agent.

pharmacology

Tamoxifen has a particularly plexing, pharmacology

i n t e r e s t i n g , and o f t e n

in l a b o r a t o r y a n i m a l s .

its p r e d o m i n a n t l y e s t r o g e n i c p r o p e r t i e s

per-

Tamoxifen

exhib-

in m o u s e u t e r i n e

It is p o s s i b l e , h o w e v e r ,

and

vaginal assays

(22-24).

refractoriness

in the o v a r i e c t o m i z e d m o u s e v a g i n a s u c h that

cannot r e s p o n d to e x o g e n o u s e s t r o g e n s t i m u l a t i o n weeks following a large subcutaneous (25).

During

this p e r i o d

a n d there is a n i n c r e a s e vagina, h o w e v e r ,

u t e r i n e g r o w t h is fully

contrast tamoxifen

for

fully

stimulated,

vaginal cornification assays

The

in r e s p o n s e

is a p a r t i a l a g o n i s t w i t h

to

antiestrogenic

(22,26,27).

s t i m u l a t i o n of the cell t y p e s .

Tamoxifen

an increase

in the s i z e of the luminal e p i t h e l i a l c e l l s

very little

increase

levels

(14,27-29).

Stromal and myometrial cells

a p p a r e n t l y not a f f e c t e d by t a m o x i f e n . increase the u t e r i n e (30-32).

a

causes but

in m i t o t i c a c t i v i t y or w h o l e u t e r i n e

hydroxylated metabolite,

4-hydroxytamoxifen

DNA

are

T a m o x i f e n and

level of p r o g e s t e r o n e

Paradoxically,

In

Allen-Doisy

T h e rat u t e r u s is a c o m p l e x o r g a n and t a m o x i f e n p r o d u c e s differential

it

many

l e u k o c y t i c for 3-4 w e e k s .

in rat u t e r i n e w e i g h t a s s a y s and

a

tamoxifen

in v a g i n a l w e t and d r y w e i g h t .

is u n a b l e to c o r n i f y

e s t r a d i o l and the s m e a r r e m a i n s properties

i n j e c t i o n of

to induce

its

(Fig. 2) a l s o receptors

in v i v o

t a m o x i f e n and 4 - h y d r o x y t a m o x i f e n

do

606

tamoxifen

4-hydroxytamoxifen /CHj

R

CH3 4-methyltamoxifen CI 4-chlorotamoxifen

Fig. 2. The m e t a b o l i c a c t i v a t i o n of t a m o x i f e n to 4 - h y d r o x y t a m o x i f e n and the f o r m u l a e of the t a m o x i f e n d e r i v a t i v e s r e s i s t a n t to h y d r o x y l a t i o n . not increase progesterone cells

in c u l t u r e ,

receptor

i n d u c t i o n by r a t

but b o t h c o m p o u n d s

estradiol-stimulated

progesterone

reversibly

uterine

inhibit

receptor induction

(33).

Metabolism It is p o s s i b l e that the s p e c i e s d i f f e r e n c e s

in the

cology of t a m o x i f e n are the r e s u l t of d i f f e r e n c e s b o l i c t r a n s f o r m a t i o n of the d r u g .

pharmain the

As y e t , no q u a l i t a t i v e

f e r e n c e s have been found b e t w e e n the m e t a b o l i s m of

metadif-

tamoxifen

607 in rats, m i c e and c h i c k e n s

(34).

t a m o x i f e n in all t h r e e s p e c i e s

The p r i n c i p a l m e t a b o l i t e

is 4 - h y d r o x y t a m o x i f e n .

the a d m i n i s t r a t i o n of 4 - h y d r o x y t a m o x i f e n does n o t r e s u l t the a p p e a r a n c e of a d d i t i o n a l

major metabolites

(34).

p h a r m a c o l o g y of the p a r e n t

is the r e s u l t of the

actions of 4 - h y d r o x y t a m o x i f e n

and t a m o x i f e n

metabolites not yet described). hydroxytamoxifen

(35).

occurs

the

individual

(and any

other

H o w e v e r , the p o t e n c y of

is g r e a t e r than t a m o x i f e n b e c a u s e 50 times g r e a t e r than that of

4-

4-hy-

d r o x y t a m o x i f e n h a s a b i n d i n g a f f i n i t y for the e s t r o g e n tor a p p r o x i m a t e l y

in

The

m e t a b o l i c a c t i v a t i o n of t a m o x i f e n to 4 - h y d r o x y t a m o x i f e n in the rat and m o u s e so that the final e x p r e s s i o n of

recep-

tamoxifen

S t u d i e s w i t h d e r i v a t i v e s of t a m o x i f e n s u b s t i t u t e d

methyl or h a l o g e n

of

Indeed

in the 4 (para) p o s i t i o n of t a m o x i f e n

with (Fig.

2) w h i c h are u n a b l e to be m e t a b o l i c a l l y a c t i v a t e d ,

show a

lower p o t e n c y

tamoxifen

in u t e r i n e w e i g h t t e s t s in vivo than

a l t h o u g h the r e l a t i v e b i n d i n g a f f i n i t i e s equivalent

in v i t r o

are

(36).

The k n o w n m e t a b o l i t e s of t a m o x i f e n shown in Fig.

3.

in a n i m a l s and m a n are

The h i g h a f f i n i t y m e t a b o l i t e

4-hydroxy-

t a m o x i f e n is a m i n o r m e t a b o l i t e of t a m o x i f e n in p a t i e n t s whereas

the m a j o r m e t a b o l i t e

is N - d e s m e t h y l t a m o x i f e n

(37),

(38,39).

M e t a b o l i t e Y is a m i n o r m e t a b o l i t e of t a m o x i f e n in p a t i e n t s (40,41) and this is b e l i e v e d

to be formed from

t a m o x i f e n v i a an i n t e r m e d i a t e M e t a b o l i t e Ζ tamoxifen)

(39).

Metabolite

D, M e t a b o l i t e Ε and t a m o x i f e n

oxide have only been described animals

(42,43).

genic activity

in v i t r o or in

Metabolite D expresses

in r a t

(35) o r m o u s e

and is a w e a k a n t i e s t r o g e n .

N-desmethyl-

(didemethylated

little or no

estro-

(44) u t e r i n e w e i g h t

This c a t e c h o l d e r i v a t i v e

tests

of

t a m o x i f e n h a s , h o w e v e r , b e e n s h o w n to be u n s t a b l e

in v i t r o

because

affinity

it is r e a d i l y o x i d i z e d

(45).

The b i n d i n g

M e t a b o l i t e D for the e s t r o g e n r e c e p t o r is e q u i v a l e n t to of e s t r a d i o l .

Metabolite

N-

laboratory

E, t a m o x i f e n w i t h o u t the

of

that

dimethyl-

608

OCH2CH2N

metabolite Ε t OCH2CH2N

,CH3 'CHJ

*

tamoxifen N - o x i d e

HO

metabolite D OCH2CH2N

// CH Ι

OCH2CH2OH

4 - h y d r o x y tamoxifen (monohydroxy tamoxifen, metabolite B)

metabolite Y t

OCH2CH2NH2

Ν - d e s m ethyl tamoxifen metabolite X metabolite Ζ

Fig. 3.

The m e t a b o l i t e s of t a m o x i f e n in a n i m a l s and

aminoethyl vitro

side chain,

(45,46).

is fully e s t r o g e n i c in vivo

man.

(14) a n d

in

609 M e c h a n i s m s of a c t i o n in v i v o T h e fact t h a t n o n - s t e r o i d a l a n t i e s t r o g e n s w i l l c o m p e t i t i v e l y ο inhibit the b i n d i n g of [ J H ] e s t r a d i o l to e s t r o g e n r e c e p t o r s d e r i v e d f r o m rat u t e r i

(47) f o c u s e d a t t e n t i o n u p o n

receptor mediated mechanisms. 4-hydroxytamoxifen

The a n t i e s t r o g e n s

inhibit estradiol-stimulated

estrogen

LY 117018 increases

and in

immature rat u t e r i n e w e t w e i g h t but the i n h i b i t i o n can be r e v e r s e d by i n c r e a s i n g d o s e s of e s t r a d i o l uterotrophic reversibly

(48).

Similarly

the

a c t i v i t y of t a m o x i f e n in the m o u s e u t e r u s can be

i n h i b i t e d by M E R 25

predominantly

(49).

These results

indicate a

e s t r o g e n r e c e p t o r m e c h a n i s m for the

expression

of both the a n t i e s t r o g e n a n d e s t r o g e n i c a c t i o n of

tamoxifen.

Tamoxifen

(27,50) and 4 - h y d r o x y t a m o x i f e n

(31,50) cause

l o c a l i z a t i o n of o c c u p i e d e s t r o g e n r e c e p t o r c o m p l e x e s the n u c l e a r c o m p a r t m e n t of rat u t e r i n e c e l l s .

a

within

Antiestrogenic

p r o p e r t i e s are p r o b a b l y e x p r e s s e d t h r o u g h a c o m p e t i t i v e ance of a g o n i s t and a n t a g o n i s t at sites w i t h i n the n u c l e u s essary

receptor complexes

(27,50).

(though p r o b a b l y an a d v a n t a g e )

estrogen receptor pool completely effects.

Apparent differences

bal-

interacting

It is, h o w e v e r , not to o c c u p y the

to p r o d u c e

nec-

cytosolic

antiestrogenic

in the n u c l e a r o c c u p a n c y

times

of e s t r o g e n s and a n t i e s t r o g e n s are r e f l e c t e d by d i f f e r e n c e s the levels of a v a i l a b l e

cytosolic estrogen receptors

a d m i n i s t r a t i o n of c o m p o u n d s

(51,52).

plexes with estrogen receptors the b l o o d .

Antiestrogens

in the n u c l e u s but these

receptors.

is c l e a r e d

h a v e a long b i o l o g i c a l

as such c o n t i n u e to m a i n t a i n

following

Estradiol rapidly

lost o v e r the n e x t few h o u r s as the s t e r o i d

half

low l e v e l s of a v a i l a b l e

comare from

life

and

cytosolic

This e f f e c t has been s h o w n to be a p r o p e r t y

t h e i r d u r a t i o n of a c t i o n and not t h e i r m e c h a n i s m of

in

of

action

(53). T h e s y n t h e s i s of r a d i o l a b e l l e d

4 - h y d r o x y t a m o x i f e n has

per-

m i t t e d the d i r e c t s t u d y of the d i s t r i b u t i o n and t a r g e t l o c a t i o n of an a n t i e s t r o g e n .

[ Η]4-hydroxytamoxifen

tissue

binds

to

610

estrogen receptors

in the u t e r u s , but a l s o to

s p e c i f i c s i t e s in the u t e r u s and liver

antiestrogen

(54).

Antiestrogen

b i n d i n g s i t e s w e r e f i r s t d e s c r i b e d by S u t h e r l a n d a n d

coworkers

(55) and have b e e n s h o w n to be l o c a t e d in the m i c r o s o m a l t i o n of rat t i s s u e s

(56).

M a n y d i f f e r e n t c l a s s e s of

bind to the a n t i e s t r o g e n b i n d i n g site t h e r e f o r e ,

frac-

drugs

their

precise

role in the m o l e c u l a r m e c h a n i s m of a c t i o n of a n t i e s t r o g e n s controversial.

Indeed,

c o m p o u n d s can be i d e n t i f i e d that

to the a n t i e s t r o g e n b i n d i n g s i t e in v i t r o b u t d o n o t e i t h e r e s t r o g e n i c or a n t i e s t r o g e n i c p r o p e r t i e s

and e s t r o g e n s

in v i v o

that are u n l i k e l y to be

cally a c t i v a t e d to c o m p o u n d s w i t h a h i g h a f f i n i t y estrogen receptor tamoxifen).

(e.g., t a m o x i f e n c o n v e r t e d

The c o m p o u n d s

to

for

rat u t e r i n e g r o w t h and

"translocated"

to the n u c l e a r

In the m o d e l

ligands w i t h e i t h e r h i g h or low a f f i n i t y form a complex finity

receptors

compartment.

that has the u n o c c u p i e d

a l r e a d y in the n u c l e a r c o m p a r t m e n t .

receptor

receptor (Fig.

in the n u c l e u s to p r o d u c e b i o l o g i c a l

the low a f f i n i t y

ligand d i s s o c i a t e s

can

effects.

the h i g h

ligand r e c e p t o r c o m p l e x r e m a i n s w i t h the n u c l e a r

tion w h e r e a s

4)

for the r e c e p t o r

is d i s r u p t e d and f r a c t i o n a t e d ,

Ε

proges-

t h e s e d a t a to s u p p o r t a n e s t r o g e n

model for the u t e r i n e cell

W h e n the t i s s u e

the

4-hydroxy-

t e r o n e r e c e p t o r s y n t h e s i s but the c y t o s o l i c e s t r o g e n are a p p a r e n t l y not

with

metaboli-

(4-methyltamoxifen, Metabolite

and ICI 47,699) all s t i m u l a t e

W e have interpreted

bind

exhibit

(49).

R e c e n t l y w e h a v e o b s e r v e d some i n t r i g u i n g e f f e c t s antiestrogens

is

affrac-

from the

re-

ceptor during homogenization.

T h e u n o c c u p i e d r e c e p t o r thus

leached out into the c y t o s o l .

We have recently

t h e s e o b s e r v a t i o n s are

suggested

inconsistent with a cytoplasmic

tion for the e s t r o g e n r e c e p t o r w i t h a r e q u i r e m e n t l o c a t i o n to p r o d u c e a b i o l o g i c a l

response

for

that

locatrans-

in the n u c l e u s .

We

have a r g u e d that if the r e c e p t o r can be l e a c h e d out of the c l e u s w h e n a low a f f i n i t y unoccupied

receptor

ligand d i s s o c i a t e s t h e n p e r h a p s

is i n i t i a l l y

located

is

nuthe

in the n u c l e u s and

is

611

only extracted tissue

into cytosol during homogenization of the

(57,58).

A in vivo

Estrogenic Responses

i

High Affinity Ligand H

Unoccupied Receptor

Β CELL DISRUPTION in vitro

Fig. 4. A functional model for estrogen action. High affinity ligand (H) enters the cell and binds to the estrogen receptor (R) in the nucleus to produce an activated complex HR* and estrogenic responses. During cell disruption, this complex is retained in the nucleus. Low affinity ligands (L) enter the cell and bind to nuclear receptor to produce an activated complex LR*. During homogenization in vitro, the LR* complex dissociates and unoccupied R, and presumably ligand, leaks out of the nucleus into the cytosolic fraction.

612

Mechanism of action studies in vitro The potential mechanisms of action of antiestrogens are illustrated in Fig. 5.

In general, antiestrogens have been found

to inhibit the synthesis of estrogen regulated proteins (46, 59-62) and alter the rate of proliferation of hormone responsive cells (63-65).

Antiestrogens apparently induce a G^

block in the cell cycle (65,66).

B i n d i n g to AEBS?

Dynamics of N u c l e a r Receptor Complex

In this regard, it is

SECRETION PROTEIN SYNTHESIS

I

\

PROGESTERONE RECEPTOR 24K and 3 6 K PROTEINS

52K PROTEIN PROLACTIN GONADOTROPINS Effect on E s t r o g e n Regulated Protein Synthesis

Altered Receptor Interaction E f f e c t o n Cell Proliferation CALMODULIN

C i Block

CYTOPLASM

E f f e c t on Nuclear Receptor Form

AE

Fig. 5. Potential mechanisms of action of antiestrogens in vitro.

(AE)

interesting to note that tamoxifen has been found to be one of the most potent inhibitors of calmodulin mediated enzyme systems (67).

613 ο [ Η]antiestrogen have resulted

Studies with

tion of a b i n d i n g of c e l l s

(56).

component

The component

antiestrogen binding

site

interaction

(65) but this

The s t r u c t u r a l

fraction

s p e c i f i c i t y of l i g a n d s has b e e n

deter-

binding

The role of b i n d i n g s i t e s

(68).

convincing

M o s t s t u d i e s have

and no involved

actions.

f o c u s e d upon the i n t e r a c t i o n of

antiestro-

g e n s w i t h the e s t r o g e n r e c e p t o r in o r d e r to d e t e r m i n e any d i f f e r e n c e s

b e t w e e n e s t r a d i o l - and a n t i e s t r o g e n

whether

receptor

c o m p l e x e s c a n be o b s e r v e d to a c c o u n t for the a c t i o n of estrogen antagonists.

The a v a i l a b i l i t y of t r i t i a t e d

t r o g e n s has f o c u s e d a t t e n t i o n u p o n a s e a r c h for cal d i f f e r e n c e s complexes.

Similarly, precise assay systems

receptor

in v i t r o

r e s e a r c h w i l l be b r i e f l y

site.

olized

to the 8S e s t r o g e n r e c e p t o r

(69,70), h o w e v e r , [^H]estradiol.

the i n t e r a c t i o n ligand

of

is w e a k

S i n c e t a m o x i f e n c a n be

vi_vo^ to the a n t i e s t r o g e n i c

w h i c h has a h i g h a f f i n i t y

the

Each of these a r e a s of

antiestrogens

[^H]Tamoxifen binds directly the rat u t e r u s

and

reviewed.

Studies with radiolabelled

compared with

to

r e l a t i o n s h i p s of e s t r o g e n s

a n t i e s t r o g e n s has e n a b l e d the d e v e l o p m e n t of a m a p of estrogen receptor binding

the

anties-

physicochemi-

b e t w e e n e s t r o g e n and a n t i e s t r o g e n

d e s c r i b e the s t r u c t u r e - a c t i v i t y

for

in the

in c o n t r o v e r s i a l

e v i d e n c e has been p r e s e n t e d that they are

in e i t h e r a g o n i s t or a n t a g o n i s t

geo-

for the A E B S

side c h a i n is v e r y i m p o r t a n t

to the A E B S

of

like LY 117018 h a v e a

mined; an alkylaminoethoxy

m e c h a n i s m of a c t i o n of a n t i e s t r o g e n s

the

is s o m e w h a t

(49,56) and the e s t r o g e n i c cis

m e t r i c i s o m e r of t a m o x i f e n has a h i g h a f f i n i t y (49).

identifica-

is g e n e r a l l y r e f e r r e d to as

(AEBS)

a m i s n o m e r as s o m e p o t e n t a n t i e s t r o g e n s low a f f i n i t y

in the

l o c a t e d in the m i c r o s o m a l

metab-

4-hydroxytamoxifen

i n t e r a c t i o n w i t h the e s t r o g e n

recep-

614 tor, it is o n l y n a t u r a l this s u p e r i o r r e s e a r c h

that s t u d i e s s h o u l d h a v e f o c u s e d

M o n o c l o n a l a n t i b o d i e s r a i s e d to the e s t r o g e n r e c e p t o r

are

k n o w n to i n t e r a c t at d i f f e r e n t s i t e s o n the p r o t e i n . D547

Antibody

(raised to the e x t r a n u c l e a r r e c e p t o r f r o m M C F - 7

which apparently

i n t e r a c t s at a site far r e m o v e d

tamoxifen receptor complexes

receptor complex.

li-

4-hydroxy-

from h u m a n b r e a s t tumor

T h e r e are no d i f f e r e n c e s w h e t h e r D 5 4 7 is

w i t h the r e c e p t o r b e f o r e

cells)

from the

g a n d b i n d i n g s i t e , b i n d s e q u a l l y w i t h e s t r a d i o l and (71).

upon

probe.

cytosols

pre-incubated

the ligand or i n c u b a t e d w i t h

the

In c o n t r a s t a p o l y c l o n a l a n t i b o d y r a i s e d

the calf u t e r i n e e s t r o g e n r e c e p t o r

in the g o a t a p p e a r s

a b l e to d i s c r i m i n a t e b e t w e e n the c o n f o r m a t i o n a l in the r e c e p t o r by e s t r o g e n s and a n t i e s t r o g e n s

to be

shapes (72).

induced Based

u p o n the s t r u c t u r e s of e s t r o g e n s and a n t i e s t r o g e n s we

have

p r o p o s e d the f o l l o w i n g

the

h y p o t h e t i c a l m o d e l to d e s c r i b e

observed experimental results Estradiol

(Fig.

to

6).

first b i n d s w i t h the r e s t i n g r e c e p t o r

in the

nucleus

by an i n t e r a c t i o n of the C^ p h e n o l i c g r o u p w i t h a p h e n o l i c a c c e p t o r site on the p r o t e i n . f o l l o w e d by a c h a n g e

The initial b i n d i n g s t e p

in the t e r t i a r y s t r u c t u r e of the

that locks the s t e r o i d

into the r e c e p t o r and as a

d e v e l o p s the i n t r i n s i c e s t r o g e n i c a c t i v i t y of the These changes

s o c i a t i o n rate of the s t e r o i d mation)

i.e., u n t r a n s f o r m e d

by a l t e r a t i o n s

following activation

receptors have a rapid

tion of the l i g a n d c o m p a r e d with t r a n s f o r m e d (73,74).

The antiestrogen

tamoxifen)

4-hydroxytamoxifen

complex. receptor

in the

dis-

(transfordissocia-

complexes (monohydroxy-

b i n d s w i t h h i g h a f f i n i t y via the i n t e r a c t i o n of

p h e n o l i c g r o u p w i t h the p h e n o l i c s i t e o n the However,

result

in the i n t e r a c t i o n of the s t e r o i d and

have been described experimentally

is

protein

the t e r t i a r y

n e c e s s a r y to d e v e l o p

changes

receptor.

in the r e c e p t o r that

intrinsic activity

p r e v e n t e d by the a l k y l a m i n o e t h o x y

side

are

in the c o m p l e x , chain.

are

the

615

Ligand Binding Activation Estradiol

IIHIIIIIIIIII

Resting

Receptor

Resting

Receptor

Complex

Ill III

D e v e l o p m e n t of Intrinsic Activity

Formation

Manohydroxy tamoxifen Complex Formation

Low Intrinsic

Activity

Fig. 6. H y p o t h e t i c m o d e l s to d e s c r i b e the b i n d i n g of e s t r a d i o l or 4 - h y d r o x y t a m o x i f e n w i t h the ligand b i n d i n g s i t e o n the e s t r o g e n . E s t r o g e n c a n induce a c o n f o r m a t i o n a l c h a n g e in the r e c e p t o r to lock the ligand into the r e c e p t o r w h e r e a s the a n t i e s t r o g e n p r e v e n t s these c h a n g e s from o c c u r r i n g .

S t u d i e s with the p o l y c l o n a l

a n t i b o d y r a i s e d to the

r e c e p t o r tend to s u p p o r t the t e r t i a r y c h a n g e m o d e l

estrogen (72).

i n c u b a t i o n of a n t i b o d y w i t h c y t o s o l i c e s t r o g e n r e c e p t o r s h u m a n b r e a s t t u m o r s i m p a i r s the s u b s e q u e n t b i n d i n g of estradiol

and r e d u c e s

tein interaction. to the r e c e p t o r the a n t i b o d y .

However,

the b i n d i n g of

is u n i m p a i r e d Similarly,

pro-

4-hydroxytamoxifen

by e q u i v a l e n t c o n c e n t r a t i o n s

the i n t e r a c t i o n of the a n t i b o d y 4-hydroxytamoxifen-estrogen

p r o p o s e d m o d e l to e x p l a i n these o b s e r v a t i o n s

The

is i l l u s t r a t e d

T h e p o l y c l o n a l a n t i b o d y m a y i n t e r a c t w i t h the r e c e p t o r to p r e s e n t the

of with

receptor

c o m p l e x e s d o e s not a f f e c t the b i n d i n g of the l i g a n d s .

filled or r e s t i n g

from

[^H]-

the a f f i n i t y of the 1 i g a n d - r e c e p t o r

p r e f o r m e d e s t r a d i o l - and

Fig. 7.

Pre-

un-

conformational

in

616

Fig. 7. H y p o t h e t i c a l m o d e l to d e s c r i b e the b i n d i n g of e s t r a diol or 4 - h y d r o x y t a m o x i f e n to the e s t r o g e n r e c e p t o r . The i n t e r a c t i o n of the p h e n o l i c site (PS) o n the r e c e p t o r p r o t e i n induces a high a f f i n i t y b i n d i n g (HAB) to s i t u a t e the ligand in the c o r r e c t p o s i t i o n . T h e i n t e r a c t i o n of the p o l y c l o n a l a n t i b o d y w i t h the e s t r o g e n r e c e p t o r is i n d i c a t e d by Ab. The a n t i b o d y ( i e s ) i n t e r a c t s w i t h an u n k n o w n s i t e to a f f e c t the c o n f o r m a t i o n a l c h a n g e s that o c c u r at the ligand b i n d i n g s i t e . c h a n g e s that s u b s e q u e n t l y o c c u r to lock e s t r a d i o l receptor.

Thus,

the s t e r o i d

into

falls out of the b i n d i n g

H o w e v e r , the a n t i e s t r o g e n c a n still w e d g e

into the

the site.

exposed

617 binding site because

it d o e s not r e q u i r e f u r t h e r

conforma-

tional c h a n g e s to p r o d u c e h i g h a f f i n i t y b i n d i n g .

We

further

s u g g e s t that o n c e the c o n f o r m a t i o n c h a n g e d o e s o c c u r to the s t e r o i d

into its b i n d i n g

r e v e r s e the

process.

The u l t i m a t e

site, the a n t i b o d y

lock

is u n a b l e

fate of e s t r o g e n and a n t i e s t r o g e n r e c e p t o r

to

com-

p l e x e s w i t h i n the n u c l e u s of c a n c e r c e l l s is unknown,

however,

studies with radiolabelled

provoca-

tive r e s u l t s .

ligands have provided some

H o r w i t z and M c G u i r e

estradiol-estrogen

(75) p r o p o s e d that

r e c e p t o r c o m p l e x e s are d e s t r o y e d

nuclear

(or p r o -

cessed) d u r i n g the f i r s t few h o u r s of n u c l e a r o c c u p a n c y . contrast, antiestrogen-estrogen

receptor complexes

do not p r o c e s s

The d i f f e r e n c e

but a c c u m u l a t e .

In

apparently

in the

nuclear

b i o c h e m i s t r y of the r e c e p t o r c o m p l e x e s

is e m p h a s i z e d by the

observation

complexes have a

that a n t i e s t r o g e n - r e c e p t o r

ferent sedimentation plexes

(76).

We have recently confirmed

r e p o r t s by c o m p a r i n g

[^H] 4 - h y d r o x y t a m o x i f e n

There

tamoxifen-estrogen readily extracted.

the s e d i m e n t a t i o n

receptor

(1 to 6 hr) h o u r s and the Further work

is r e q u i r e d to

Nevertheless,

and

rat in the complexes

4-hydroxy-

r e c e p t o r c o m p l e x e s a c c u m u l a t e and

true loss of r e c e p t o r .

original

is c e r t a i n l y a d e c r e a s e

this r e s u l t r e f l e c t s d i f f e r e n t i a l

com-

[ ^H]estradiol

in M C F - 7 b r e a s t c a n c e r and G H 3

nuclear estradiol-estrogen

d u r i n g the f i r s t few

whether

(77) these

the n u c l e a r r e c e p t o r of

pituitary tumor cells. salt e x t r a c t a b l e

dif-

c o e f f i c i e n t that e s t r a d i o l - r e c e p t o r

are

determine

salt e x t r a c t i o n

or

there are d i f f e r e n c e s

c o e f f i c i e n t s of e s t r a d i o l - and

in

4-hydroxy-

t a m o x i f e n - e s t r o g e n r e c e p t o r c o m p l e x e s f r o m M C F - 7 c e l l s that d o not alter d u r i n g a 24 hr p e r i o d .

A l t h o u g h the

o b s e r v e d in M C F - 7 c e l l s m a y be r e l a t e d to the c o n d i t i o n s used,

results experimental

these d i f f e r e n c e s are not o b s e r v e d

p i t u i t a r y t u m o r c e l l line

(77,78).

in the

GHj

618 Structure-activity

relationships

P r i m a r y c u l t u r e s of rat p i t u i t a r y c e l l s r e s p o n d to

physio-

l o g i c a l c o n c e n t r a t i o n s of e s t r a d i o l by a s p e c i f i c i n c r e a s e prolactin synthesis

(79).

a c t i o n has b e e n v a l i d a t e d

This m o d e l s y s t e m for

antiestrogens.

inhibit

for the e s t r o g e n r e c e p t o r .

related

4-Hydroxy-

is 30 times m o r e p o t e n t than t a m o x i f e n , h o w e v e r ,

e n s u r e that t a m o x i f e n

is n o t m e t a b o l i c a l l y a c t i v a t e d

in v i t r o , s e v e r a l p a r a s u b s t i t u t e d

t i v e s of t a m o x i f e n

(4-methyl,

(59).

4 - c h l o r o , Fig. 2) have

derivabeen

The s u b s t i t u t i o n does not a f f e c t b i n d i n g

ity for the e s t r o g e n r e c e p t o r and the d e r i v a t i v e s of inhibit e s t r a d i o l - s t i m u l a t e d tration-related manner.

prolactin synthesis

Although

t a m o x i f e n to be m e t a b o l i z e d

in a

concenfor

it is

c l e a r l y not a r e q u i r e m e n t for a n t i e s t r o g e n a c t i v i t y . e s t r o g e n a c t i o n in the p i t u i t a r y

cells

is, h o w e v e r ,

p e t i t i v e and r e v e r s i b l e w i t h the a d d i t i o n of e x c e s s

affin-

tamoxifen

it is an a d v a n t a g e

to 4 - h y d r o x y t a m o x i f e n ,

to

to

4-hydroxytamoxifen tested

and

prolactin synthesis, with potency

to the b i n d i n g a f f i n i t y tamoxifen

activity

estrogens

T a m o x i f e n and 4 - h y d r o x y t a m o x i f e n

estradiol-stimulated

estrogen

for the study of s t r u c t u r e

r e l a t i o n s h i p s w i t h i n g r o u p s of n o n - s t e r o i d a l

in

Antiboth

com-

estradiol

(59). A s e r i e s of k n o w n e s t r o g e n s and a n t i e s t r o g e n s has b e e n to e s t a b l i s h s t r u c t u r e - a c t i v i t y

relationships.

The

relative

p o t e n c y of e s t r o g e n s to s t i m u l a t e p r o l a c t i n s y n t h e s i s diethylstilbestrol

ξ estradiol

the d i m e t h y l a m i n o e t h a n e

> ICI 77,949

side c h a i n )

isomer of e n c l o m i p h e n e ) .

(cis

e s t r o g e n s to inhibit e s t r a d i o l - s t i m u l a t e d The c o m p o u n d LY 126412

without

(_cis_ g e o -

prolactin

> trioxifene

was

geometric

T h e r e l a t i v e p o t e n c i e s of

w a s 4 - h y d r o x y t a m o x i f e n ξ LY 117018 Ξ tamoxifen.

(tamoxifen

> ICI 47, 699

m e t r i c isomer of t a m o x i f e n ) Ξ z u c l o m i p h e n e

tested

antisynthesis

> enclomiphene

(trioxifene without

the

side c h a i n ) d o e s not i n t e r a c t w i t h e s t r o g e n r e c e p t o r s up to

619 test c o n c e n t r a t i o n s of 10 estrogenic properties

Μ or exhibit estrogenic or

using the p r o l a c t i n s y n t h e s i s

A m o n g the t r i p h e n y l e t h y l e n e s , trans g e o m e t r i c

anti-

assay.

c o m p o u n d s that h a v e c i s and

i s o m e r s are e x t r e m e l y

i m p o r t a n t for the

devel-

o p m e n t of a l i g a n d - r e c e p t o r m o d e l b e c a u s e the i s o m e r i c

mole-

cules e n c o m p a s s e s t r o g e n i c and a n t i e s t r o g e n i c a c t i o n s .

The

trans isomers

( t a m o x i f e n a n d e n c l o m i p h e n e ) are

w i t h zero i n t r i n s i c a c t i v i t y w h e r e a s 47,699 a n d z u c l o m i p h e n e ) a c t i v i t y of

the cis

antiestrogens

isomers

are e s t r o g e n s w i t h a n

(ICI

intrinsic

1.

T o d e s c r i b e the i n t e r a c t i o n of the g e o m e t r i c i s o m e r s w i t h estrogen receptor,

the trans s t i l b e n e - l i k e

t a m o x i f e n and e n c l o m i p h e n e site w i t h low a f f i n i t y

structure

c o u l d sit l o o s e l y at the

b i n d i n g so that the p h e n y l

the

of binding

ring

substi-

tuted w i t h the p - a l k y l a m i n o e t h o x y side c h a i n is p r o j e c t e d from the b i n d i n g

site

(Fig.

8).

The e s t r o g e n i c

ligands,

z u c l o m i p h e n e a n d ICI 4 7 , 6 9 9 w i t h t h e i r low a f f i n i t y e s t r o g e n r e c e p t o r can c r e a t e a trans s t i l b e n e - l i k e w i t h the p a r a s u b s t i t u t e d p h e n y l ring. the a m i n o e t h o x y (Fig. 8).

the

In this b i n d i n g

and, as a r e s u l t ,

i n t e r a c t i o n t h r o u g h the

state,

a n t i e s t r o g e n r e g i o n of the

no i n h i b i t i o n of e s t r o g e n a c t i o n .

tiary c h a n g e s that are n e c e s s a r y to d e v e l o p a h i g h for the c o m p l e x can o c c u r

(80).

tuted phenyl ring

The

ter-

intrinsic

into t h r e e

categories

Antiestrogens have a side

c h a i n e x t e n d i n g a w a y f r o m the b i n d i n g site, p a r t i a l have a bis p h e n o l i c s t r u c t u r e

side

receptor

unimpeded.

O v e r a l l c o m p o u n d s can be c l a s s i f i e d based upon their s t r u c t u r e

site

ether

T h e r e w o u l d be no i n t e r a c t i o n of the

chain with a hypothetical

activity

for

structure

side c h a i n w o u l d lie next to the p h e n o l i c

o n the r e c e p t o r , w i t h a w e a k oxygen

away

and a g o n i s t s h a v e an

(or no p h e n y l ring at

all).

agonists unsubsti-

620

liLLililiiliiilU

trans monohydroxytamoxifen

estradiol-17/

Β

trans geometric i s o m e r s

cis geometric i s o m e r s

Fig. 8. H y p o t h e t i c a l m o d e l s for e s t r o g e n i c and a n t i e s t r o g e n i c ligand b i n d i n g to the e s t r o g e n r e c e p t o r . E s t r a d i o l - 1 7 8 is a n c h o r e d at a p h e n o l i c site (PS) w i t h high a f f i n i t y b i n d i n g (HAB). t r a n s M o n o h y d r o x y t a m o x i f e n h a s the same h i g h a f f i n i t y b i n d i n g but this a n t i e s t r o g e n i c ligand b i n d s to the r e c e p t o r s i t e so that the a l k y l a m i n o e t h o x y s i d e c h a i n can i n t e r a c t w i t h a h y p o t h e t i c a l a n t i e s t r o g e n r e g i o n (AER) on the p r o t e i n . Comp o u n d s w i t h o u t a p h e n o l i c h y d r o x y l h a v e low a f f i n i t y b i n d i n g (LAB). The trans a n d cis g e o m e t r i c isomers r e f e r to A) t a m o x i f e n ( R = C H 3 , R 2 = C 2 H 5 ) and e n c l o m i p h e n e ( R = C 2 H 5 , R = C 1 ) ; B) ICI 47,699 ( R = C H 3 , R = C 2 H 5 ) and z u c l o m i p h e n e ( R = C 2 H 5 , R2=C1). T h e g e m i n a l bis p a r a h y d r o x y p h e n y l c o m p o u n d s bisphenol)

that are p a r t i a l a g o n i s t s

interesting.

(e.g.,

in v i t r o are

particularly

Belleau's macromolecular perturbation

(45), w h i c h was o r i g i n a l l y p r o p o s e d to e x p l a i n

theory

agonist,

p a r t i a l a g o n i s t a n d a n t a g o n i s t a c t i v i t y of d r u g s at the c a r i n i c c h o l i n e r g i c r e c e p t o r , m a y be used to e x p l a i n agonists

in t e r m s of the e s t r o g e n r e c e p t o r m o d e l .

to B e l l e a u ' s h y p o t h e s i s ,

mus-

partial

According

an a g o n i s t b i n d s to the r e c e p t o r

induces a specific conformational perturbation

(SCP).

An

and

621 a n t a g o n i s t b i n d s to the r e c e p t o r and p r o d u c e s a conformational perturbation intrinsic activity.

non-specific

(NSCP) but the c o m p l e x has

B e t w e e n these e x t r e m e s a p a r t i a l

zero agonist

binds to the r e c e p t o r and p r o d u c e s an e q u i l i b r i u m m i x t u r e a g o n i s t and a n t a g o n i s t r e c e p t o r c o m p l e x e s . definitions

to the e s t r o g e n r e c e p t o r

Applying

of

these

(Fig. 9), e s t r a d i o l

binds

w i t h h i g h a f f i n i t y to the r e s t i n g r e c e p t o r and i n d u c e s a S C P which results site.

in the ligand being

4-Hydroxytamoxifen

locked into the

(antagonist) wedges

r e c e p t o r and p r o d u c e s a N S C P .

Bisphenol

binding

into the

(partial

resting

agonist)

i n t e r a c t s at the l i g a n d b i n d i n g site b u t w h i l e s o m e of r e c e p t o r s can be i n d u c e d to lock the l i g a n d into the

the

protein,

o t h e r l i g a n d i n t e r a c t i o n s are o n l y a b l e to induce a N S C P the

complex.

T h e s t u d y of the s t r u c t u r e - a c t i v i t y an u n d e r s t a n d i n g of s e v e r a l predict pharmacological 1.

in

f e a t u r e s that are d o m i n a n t

activity

(Fig.

A phenolic hydroxyl equivalent diol

is e x t r e m e l y

relationships has

provided and

10).

to the C3 p h e n o l of

estra-

i m p o r t a n t for h i g h a f f i n i t y b i n d i n g

the e s t r o g e n r e c e p t o r .

This s t r u c t u r a l

to

feature permits

v a r i e t y of " s p a c i n g g r o u p s " to o c c u p y the r e c e p t o r

a

binding

site. 2.

Alkylethers have a decreased affinity

for the

receptor

(but an i n c r e a s e d d u r a t i o n of a c t i o n in v i v o ) . 3.

S u b s t i t u t i o n of the p h e n y l ring e x t e n d i n g a w a y from the binding

site g o v e r n s p h a r m a c o l o g i c a l

activity.

w i t h o u t s u b s t i t u t i o n are e s t r o g e n s b u t a p a r a predicts partial agonist activity

in v i v o .

side c h a i n p r e d i c t s a n t a g o n i s t a c t i v i t y

Compounds hydroxyl

E x t e n s i o n of a

in v i t r o .

622

Bisphenol

Fig. 9. A d a p t a t i o n of B e l l e a u ' s m a c r o m o l e c u l a r p e r t u r b a t i o n theory to d e s c r i b e the i n t e r a c t i o n of a g o n i s t , a n t a g o n i s t s and p a r t i a l a g o n i s t s w i t h the e s t r o g e n r e c e p t o r (ER). The p h e n o l g r o u p o n the ligand i n t e r a c t s w i t h the p h e n o l i c site o n the E R (closed t r i a n g l e ) and p r o d u c e a h i g h a f f i n i t y i n t e r a c t i o n if the g e o m e t r y of the l i g a n d is c o r r e c t . E s t r a d i o l (Ej), o n a g o n i s t , i n d u c e s a s p e c i f i c c o n f o r m a t i o n a l p e r t u r b a t i o n (SCP) w h e r e a s 4 - h y d r o x y t a m o x i f e n (OHTAM), a n t a g o n i s t , o n l y i n d u c e s a n o n - s p e c i f i c c o n f o r m a t i o n a l p e r t u r b a t i o n (NSCP). Bisphenol (partial a g o n i s t ) p r o d u c e s a m i x t u r e of SCP and N S C P in the ER.

623

Area of Antagonist Activity

^ O H

Estrogenic Activity (Agonist)

Low Affinity RO

HO High Affinity

Partial Agonist

y Spacing / Groups /

Fig. 10. A general ligand model to describe the structural requirement to control biological activity in vitro.

Antitumor actions in animals The dimethylbenz(a(anthracene

(DMBA)-induced rat mammary car-

cinoma model first described by Huggins et al. (81) has been used extensively to study hormone-dependent cancer and to evaluate potential therapies for breast cancer. The early definition of hormone dependency was based on regression of tumors after ovariectomy and hypophysectomy

(81).

Since estrogen stimulates tumor regrowth in ovariectomized rats but not in animals which are also hypophysectomized

(82),

the pituitary gland clearly plays a central role in the growth and homoestasis of DMBA-induced tumors.

Since

'hormone-

responsive' DMBA-induced tumors have cytosol estrogen

624 r e c e p t o r s a d i r e c t s t i m u l a t i o n of t u m o r g r o w t h by e s t r o g e n possible.

However,

the

interesting observations

tin a d m i n i s t r a t i o n o r i n c r e a s e s perphenazine

stimulate

increases

in e s t r o g e n r e c e p t o r

in this m o d e l are a m a r k e r of h o r m o n e

Tamoxifen tumors

inhibits

(84-88).

estro-

respon-

action.

the g r o w t h of e s t a b l i s h e d

DMBA-induced

T o e x p l a i n the m e c h a n i s m of a c t i o n of

tamoxi-

fen in this m o d e l m o s t i n v e s t i g a t o r s have c o n s i d e r e d an a c t i o n w i t h t u m o r e s t r o g e n r e c e p t o r s to be of importance.

Terenius

(89) first p r o p o s e d

antiestrogens DMBA-

induced rat t u m o r s and that this m i g h t be their p r i m a r y antiestrogens

inter-

fundamental

that

d i r e c t l y b l o c k the b i n d i n g of e s t r o g e n to h u m a n a n d n i s m of a c t i o n .

by

concen-

(83) m a y i n d i c a t e that

s i v e n e s s r a t h e r t h a n a m e d i a t o r of h o r m o n e

mecha-

Many studies have subsequently confirmed

i n h i b i t the b i n d i n g of

is

prolac-

in s e r u m p r o l a c t i n c a u s e d

t r a t i o n s as w e l l as t u m o r g r o w t h gen r e c e p t o r s

that

[^H]estradiol

to

that

DMBA-

i n d u c e d t u m o r t i s s u e s d e t e r m i n e d b o t h in v i v o and in v i t r o (85,90,91). b i n d i n g of

At the s u b c e l l u l a r [^H]estradiol

from D M B A - i n d u c e d

tumors

level,

tamoxifen

inhibits

to the 8S e s t r o g e n r e c e p t o r (90).

The i n t e r a c t i o n of

w i t h the e s t r o g e n r e c e p t o r s y s t e m of D M B A - i n d u c e d been s t u d i e d e x t e n s i v e l y

t a m o x i f e n may the p i t u i t a r y

tumors

tumor,

i n h i b i t t u m o r g r o w t h by a n u m b e r of o t h e r 11.

The a b i l i t y of t a m o x i f e n to

g l a n d has been c o n s i d e r e d e a r l i e r .

cluded, therefore,

It is

in the h o r m o n a l m i l i e u

affect con-

However,

there

is little d o u b t that the

antagonist,

perphenazine, which

by

and

both pituitary

g l a n d can h a v e a n o v e r r i d i n g e f f e c t since a d m i n i s t r a t i o n

of

stimulates

prolactin secretion, will reverse tumor regression tamoxifen.

mecha-

that tamoxifen causes tumor regression

d i r e c t b i o c h e m i c a l e f f e c t s w i t h i n the t u m o r cell are

the d o p a m i n e

has

in v i v o .

multiple mechanisms; alterations important.

obtained

tamoxifen

In a d d i t i o n to a d i r e c t a c t i o n o n the D M B A - i n d u c e d n i s m s as s h o w n in Fig.

the

induced

by

625

Interferes W i t h G o n a d o t r o p i n Release . Ovary Hypothalamo Pituitary Axis Inhibit

Inhibit Prolactin Release

Oestradiol Synthesis

^ D M B A - Induced Mammary Tumour

Inhibit Oestradiol Binding

IS2I Oestradiol Η Gonadotropin mm Prolactin

Fig. 11. S i t e s p o t e n t i a l l y s e n s i t i v e to a n t i e s t r o g e n in the D M B A - i n d u c e d rat m a m m a r y c a r c i n o m a m o d e l . In a d d i t i o n to e f f e c t s o n e s t a b l i s h e d that t a m o x i f e n p r e v e n t s or r e t a r d s tumor induction with DMBA. a large dose of t a m o x i f e n

t u m o r s there

the e v e n t s

inhibits

T a m o x i f e n m a y be e x e r t i n g of c a r c i n o g e n e s i s to m a k e the tissue as a n a n t i e s t r o g e n . be i m p o r t a n t insult.

(92), a n d DMBA

the a p p e a r a n c e of t u m o r s

to the c a r c i n o g e n ,

Alternatively,

for c a r c i n o g e n e s i s

the

the local a c t i v a t i o n of

the c e l l u l a r b i o c h e m i s t r y

to al-

process DMBA

sufficiently rather

than

since prolactin appears

then the i n h i b i t i o n of

(q.v.) m i g h t p r e v e n t the

of

(86).

its e f f e c t by i n h i b i t i n g

refractory

tin r e l e a s e by t a m o x i f e n

in

(5 mg χ 2 s.c.), w h i c h is k n o w n

by p r e v e n t i n g

o r p e r h a p s by a l t e r i n g

is e v i d e n c e

involved

The simultaneous administration

p r o d u c e a long t e r m a n t i e s t r o g e n i c e f f e c t most completely

action

to

prolac-

carcinogenic

626 O t h e r d r u g r e g i m e n s h a v e p r o v i d e d d a t a of g r e a t r e l e v a n c e adjuvant therapy

in the c l i n i c a l s i t u a t i o n .

Four w e e k s

DMBA a d m i n i s t r a t i o n t u m o r i n i t i a t i o n has o c c u r r e d b u t no pable

tumors can be d i s c e r n e d .

to

after pal-

At this time a s h o r t c o u r s e

t r e a t m e n t w i t h t a m o x i f e n for 30 d a y s w i l l d e l a y the

of

appearance

of tumors and r e d u c e the c u m u l a t i v e n u m b e r of t u m o r s

found

(93,94).

classical

A l t h o u g h m o r e p o t e n t as an a n t i e s t r o g e n

pharmacological

models,

4-hydroxytamoxifen

is less

t h a n t a m o x i f e n w h e n g i v e n for 30 d a y s in this tumor model

than c o n t i n u o u s

treatment

effective

DMBA-induced

(95); this m a y be due to d i f f e r e n c e s

In this m o d e l , s h o r t c o u r s e s of t h e r a p y are, less e f f e c t i v e

in

in

half-life.

in g e n e r a l ,

(93,94,96).

example, continuous daily treatment with

much

For

4-hydroxytamoxifen,

s t a r t i n g 4 w e e k s a f t e r DMBA a d m i n i s t r a t i o n , w i l l

dramatically

r e d u c e the total n u m b e r of t u m o r s a p p e a r i n g , w h e r e a s the d o s e s g i v e n as a s h o r t c o u r s e without effect

for 4 w e e k s are

same

virtually

(96).

C o n t i n u o u s t r e a t m e n t w i t h a n t i e s t r o g e n s s h o u l d not be s i d e r e d to be a n a l o g o u s

to o v a r i e c t o m y .

Ovariectomy

con30 d a y s

a f t e r D M B A d e l a y s the a p p e a r a n c e of t u m o r s but e v e n t u a l l y to 50% of a n i m a l s d e v e l o p

tumors

(97).

In c o n t r a s t ,

up

continu-

ous treatment with tamoxifen prevents tumor appearance

in

a b o u t 90% of a n i m a l s e v e n a f t e r

greater

200 d a y s

(93,97).

The

e f f i c a c y of t a m o x i f e n m a y be due to its a b i l i t y to

antagonize

all e s t r o g e n w h e t h e r p r o d u c e d by the o v a r y o r formed by t i z a t i o n of a d r e n a l a n d r o g e n s . appearing

in o v a r i e c t o m i z e d

C e r t a i n l y m a n y of the

aroma-

tumors

rats are h o r m o n e - r e s p o n s i v e

a d d i t i o n a l t h e r a p y w i t h t a m o x i f e n r e d u c e s the t u m o r

since

growth

(97).

In s u m m a r y t h e s e d a t a i n d i c a t e t h a t a f t e r c a r c i n o g e n e s i s i n i t i a t e d by DMBA,

the s u b s e q u e n t a p p e a r a n c e of t u m o r s

v e n t e d by a d m i n i s t r a t i o n of t a m o x i f e n a n d If t r e a t m e n t c e a s e s the t u m o r s d e v e l o p . induced tumor model has limitations

is

is p r e -

4-hydroxytamoxifen. However,

(i.e.,

it d o e s

the not

DMBA-

627 metastasize and, unlike human mammary tumors, is dependent on prolactin) and so other potential models are being developed. N-nitrosomethylurea

(NMU) will also produce mammary tumors in

rats (98) but the degree of hormone dependency is influenced by the schedule of NMU administration

(99).

At this time the

precise hormone dependency of the tumors is unclear but they do appear to grow in response to estrogen and prolactin. Treatment with tamoxifen prevents growth of NMU-induced and reduces the tumor incidence

tumors

(100).

Treatment with tamoxifen in an adjuvant setting 2 weeks after NMU administration for 5 weeks completely inhibits tumor incidence but when therapy stops the tumors subsequently appear (101).

However, continuous therapy with tamoxifen (500 yg or

100 pg daily) will completely prevent the appearance of tumors (101).

These studies have important implications for the

clinical application of tamoxifen for the adjuvant therapy of breast cancer.

Antitumor action in patients Tamoxifen is an effective, non-toxic palpiative treatment for advanced breast cancer (10-12).

The mode of action is con-

sidered to be via the estrogen receptor because a) inhibits the binding of

tamoxifen

3

[ H]estradiol to estrogen receptors

derived from breast tumors (102), b)

estrogen receptor con-

taining breast cancer cells have their growth in vitro retarded by the antiestrogen

(63) and, c)

patients with an

estrogen recetor positive tumor are more likely to respond to tamoxifen therapy than those with an estrogen receptor negative tumor

(103).

The addition of tamoxifen to chemotherapy regimens (104) or the use of tamoxifen alone following mastectomy (105) is generally found to improve the disease free interval in node

628 positive breast cancer.

However, the one or two years of

tamoxifen therapy may not be optimal.

If tamoxifen is tumor-

istatic rather than tumoricidal then continued therapy may be necessary to control recurrence in patients with sensitive disease.

In 1977 Dr. Douglass Tormey of the Wisconsin Clinical Cancer Center initiated a pilot clinical study of long term

(greater

than 5 years of therapy) tamoxifen therapy in patients having up to 2 years of adjuvant chemotherapy following mastectomy. The study compared three groups of patients (no tamoxifen, 2 years of tamoxifen or continuous tamoxifen) has shown a disease free survival advantage for the group receiving continuous tamoxifen therapy

(106).

The additional aims of the study

were to determine whether tamoxifen could be given safely for more than 5 years and to determine whether continual therapy with tamoxifen would result in metabolic tolerance.

We were

particularly interested to establish that tamoxifen was neither inactivated nor converted to estrogens during the years of therapy.

The results of the HPLC assays using post-

column fluorescence activation (107) are illustrated in Fig. 12.

It is clear that tamoxifen does not develop meta-

bolic tolerance or induce the appearance of estrogenic metabolites.

These data have been used to establish the

Eastern Cooperative Oncology Group protocols EST 5181 and EST 4181 to test the value of 5 years of tamoxifen in a broad based, randomized clinical trial.

629 // CHEMOTHERAPY

TAMOXIFEN

' y •

· ·

• . •·· 1

.1 150

,,

ι 300

β β •

ι 450

1

•• ν-'·· ', · ·% ·*· ·· ··. Ι·· ·*

1 1

*>« · ·

1

t/ Λ ι 600 750

•

·

· · •

ι

ι 900

ι

ι 1050

ι

ι

ι

ι ι

ι ι ι ι ι 1200 1350 1500 1650 1 8 0 0

1950

D A Y S OF T A M O X I F E N T H E R A P Y ( l O r n g b i d )

N-desmethyltomoxifen

Metabolite Y

DAYS OF T A M O X I F E N T H E R A P Y ( 1 0 m g b i d )

Fig. 12. The level of tamoxifen and its metabolites Ndesmethyltamoxifen and Metabolite Y during long-term adjuvant therapy with tamoxifen (10 mg bid).

630 Acknowledgements The studies described

in t h i s c h a p t e r w e r e s u p p o r t e d

the p a s t 12 y e a r s by ICI Pic

(Pharmaceuticals

E n g l a n d ; the Y o r k s h i r e C a n c e r R e s e a r c h C a m p a i g n , Stuart Pharmaceuticals, Wilmington, I n s t i t u t e s of H e a l t h and

during

Division), England;

D e l a w a r e ; and

(USA) g r a n t s P 3 0 - C A - 1 4 5 2 0 ,

National

P01-CA-20432

ROl-CA-32713.

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Eur. J. Cancer J^, 205-206

Breast Cancer Res. Treat. J. Chromat. 272,

STUDIES ON GLUCOCORTICOID RECEPTORS IN NORMAL AND NEOPLASTIC RODENT AND HUMAN LEUKOCYTES:

STRUCTURE, DEGRADATION,

KINETICS OF FORMATION AND ACTIVATION * Nikki J. Holbrook , Jack E. Bodwell, Dirk B. Mendel, Allan Munck Department of Physiology, Dartmouth Medical School, Hanover, NH, USA * Present address:

Laboratory of Pathology, National Cancer

Institute, Bethesda, MD, USA

Introduction Over the past several years a major goal of our laboratory has been to characterize corticoid-receptor cell

under

functional studies

the various

complex

physiological and

was

kinetic the

chromatographic

which

forms of the gluco-

exist

conditions,

within

and

to define

relationships.

development

procedure

for

receptor complexes in cytosols.

of

a

the

Central rapid

analyzing

to

intact their such

minicolumn

glucocorticoid-

This procedure has enabled

us to perform studies which would otherwise be impossible. In this review we will attempt to summarize findings from several

of

our

recent

glucocorticoid-receptor

studies

aimed

at

characterizing

complexes in normal and neoplastic

cells both biochemically and functionally.

Results Characterization complexes.

of

rat

thymus

glucocortioid

As a prelude to more detailed

receptor

studies of the

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

638 cytoplasmic glucocorticoid-receptor complexes we first characterized by standard physicochemical procedures the nonactivated (non-DNA-binding) and activated or transformed (DNA-binding) complexes formed in rat thymus cells (1). For this purpose we used buffers containing sodium molybdate (2, 3) to block activation and stabilize the complexes. In

rat

acetonide

thymus (TA)

cells at

incubated

0°C,

the

with

cytosolic

[3H]-triamcinolone complexes

almost entirely of a non-activated species. activated and nonactivated activated

species

activated

to nonactivated

consist

At 37°C, both

complexes are present with the

predominating.

However,

the

ratio

complexes varies with

of

different

steroids as will be discussed below.

Traces of mero-recep-

tor

proteolytic

and

related

complexes

products of the native

(small

receptor

complex which

cleavage

retain the

steroid binding site) are also found, but these complexes may have been formed rapidly after the cells are broken. TABLE 1 CHARACTERISTICS OF GLUCOCORTICOID-RECEPTOR COMPLEXES FROM RAT THYMUS CELLS

COMPLEX PROPERTI ES NONACTIVATED

ACTIVATED

MER0-RECEPT0R

BINDS TO DNA

NO

YES

NO

ELUTION FROM DEAE CELLULOSE (mM KCl)

200

50

DOES NOT BIND

STOKES RADIUS (nm)

8.3

5.0

2.3

SEDIMENTATION COEFFICIENT

9.2

H]-TA AT 10 TIMES FINAL CONCENTRATION IN KRBg-HEPES AT 37"C WAS ADDED AT TIME 0 TO A THYMUS CELL SUSPENSION IN KRBg-HEPES AT 37'C, TO CIVE A FINAL CONCENTRATION OF ABOUT 15 nM. AT THE IMHCATED TIMES, ALI0U0TS OF CELL SUSPENSION WERE REMOVED FOR ASSAY OF HR, HR', AND HR'n. NONSATURABLE BIWINC, DETERMINED FROH A PARALLEL INCUBATION KITH 1 UM TA, GAVE CORRECTIONS (INDEPENDENT OF TIME) OF 1280 CPM FOR HR'n AND OF LESS THAN 2« FOR HR ANO HR'.

With the results in Figure 2, we can ascribe most of this delay to the time required for generation of of HR', the

644 presumed

intermediate between HR and HR'n, rather than to

the time required

for translocation and binding of HR' to

while the formation of HR1 clearly lags behind

the nucleus:

that of HR, no obvious delay is detectable in formation of HR'n relative to HR'. To further distinguish the rates of activation and nuclear binding these

we

performed

experiments,

incubated

for

glucocorticoid

'temperature a

cell

several to

jump

experiments'.

suspension

hours

at

which

0-3°C

form HR was warmed

had

with

rapidly

In been

labeled

to a given

temperature by dilution with 10 volumes of warmed buffer, left

for

the desired

amount

of

time, then

cooled,

lysed

rapidly by further dilution with cold t-lgCl2 and assayed for HR,

HR'

and

HR'n.

Such

experiments

warming step at 37°, 25°, formation

of complexes decreased

temperatures, no delay discernible

at any

were

and 15°C.

done

significantly

in formation of HR'n

temperature.

with

the

Though the rate of

Thus, we

with

lower

from HR' was

concluded

that

nuclear binding is much faster than activation, with a time constant of less than 10 sec. Our results with minicolumns also provided convincing support for our earlier suggestions (14) that triamcinolone acetonide and dexamethasone give higher steady state ratios of HR' to HR than Cortisol and corticosterone.

Cyclic

model

of

relationships.

receptor

kinetics

and

agonist-antagonist

To account for the results just outlined we

devised a cyclic model of receptor kinetics that accounts quantitatively for most of our own results and for results of others.

The model

(Fig. 3) assumes

reaction with rate constants k^ and

(i) a

reversible

for formation of

nonactivated complex HR from hormone Η and receptor R; (ii) an irreversible

reaction with rate constant

kn

for acti-

645

vation

of

HR

to

HR';

(iii)

a

rapid

reversible

(essentially an equilibrium) between HR' and bound

form HR'n;

reaction

its nuclear-

(iv) an irreversible reaction with rate

constant k ^ (the same as that for dissociation of HR) for regeneration of R from HR' and HR'n. coids are assumed required

to differ

only

Different glucocorti-

in

.

All

constants

for predicting kinetic behavious can be measured

experimentally, so that the model can be tested without ad hoc assumptions.

FIC. 3.

CYCLIC MODEL OF RECEPTOR KINETICS (13). HORMONE, H, IS ASSUMED TO REACT REVERS IBLY WITH FREE RECEPTOR, R, TO FORM A NONACTIVATED COMPLEX, HR, THAT THROUGH AN IRREVERSIBLE REACTION PRODUCES THE ACTIVATED COMPLEX HR·. HR' RAPIDLY EQUILIBRATES WITH THE NUCLEAR-BOUND COMPLEX, HR'n. HR' AND HR'n BOTH REGENERATE R THROUCH IRREVERSIBLE REACTIONS.

With measured values for TA and Cortisol the model not only predicts accurately the kinetic results in Fig. 2, but gives steady-state ratios of HR' to HR for the two steroids in quantitative agreement with those found experimentally even though

the

activation

steroid-specific

control

reaction of

has

no

activation.

provision The

for

different

646 ratios turn out to be dynamic consequences of the choice of for the rate of the regeneration reactions. The cyclic model predicts that steroids with high be partial antagonists.

should

Mathematically it can be shown (un-

published) that the cyclic model gives biological agonistantagonist

relations

essentially

indistinguishable

those of the steric-allosteric model (15). antagonism

should

be

from

According to the

cyclic

model,

predictable

from

k

alone.

However, the model does not exclude other mechanisms

of antagonism, such as allosteric equilibria or formation of covalent complexes; it provides an additional mechanism. Since the model includes irreversible steps, it also predicts that normal receptor function requires energy.

That

prediction is consistent with earlier studies in which we found

metabolic

proposed

a

evidence

receptor

for

cycle

an

energy

with

an

requirement,

ATP-dependent

and step

(possibly phosphorylation) for regenerating R (16).

Effects of ATP and pyrophosphate

(PP^) on activation

stability of cytosolic glucocorticoid-receptor complexes.

and Λ

number of studies have suggested a possible role for ATP in the

activation

function

(i.e.

of

steroid-receptor

translocation

activated complexes. reported

that ATP

or

complexes

nuclear

or

in

the

binding)

of

the

In particular, Moudgil and John (17)

at 5-10 mM

promotes

liver cytosolic complexes at 4°C.

activation

of

rat

In agreement with that

observation we have found that ATP promotes activation of rat thymus complexes at 4 C C however,

our

findings

(18) .

indicate

that

Somewhat

surprisingly,

this

is

effect

not

limited to ATP, as other nucleoside triphosphates and PP^ at the same concentrations are similarly effective.

Thus it is

unlikely that the activation seen in vitro is related to the

647

energy dependence of glucocorticoid-receptor

complex form-

ation seen in vivo (16). Two

additional

findings

of

interest

were

noted

when

we

looked further into the effects of PP^ and ATP on rat thymus receptor

complexes

(18) .

We were

initially

surprised

to

find that if we used these compounds to activate cytosolic complexes we achieved much higher levels of DNA binding than by warming cytosols to 25°C, with virtually no mero-receptor formation.

In fact, more than 95% of the total complexes

were converted to the DNA binding form, and that level was maintained for up to 24 hours.

This is in marked contrast

to warmed cytosols, in which by 24 hours more than 80% of total

complexes

are

converted

to

mero-receptor

forms.

Further analysis by gel filtration in the presence of ATP or PP^

revealed

conversion

to

that

these

compounds

mero-receptor.

do

However,

complex was not the 5.6 nm native

indeed the

prevent

DNA-binding

form but had a Stokes

radius of 3.1 nm. While the 5.6 and 3.1 nm DNA-binding complexes could not be distinguished by our standard minicolumn procedure, they could be separated using minicolumns in which the order of the DEAE and DNA columns were reversed, as the intact 5.6 nm DNA-binding complex binds to DEAE while the 3.1 nm complex does not. The 3.1 nm complex was subsequently shown to be formed in a time-dependent manner from the 5.6 nm form. It accumulates in ATP-treated cytosols because its conversion to meroreceptor is prevented. The protection against mero-receptor formation is dose-dependent and requires concentrations similar to those that activate complexes.

We also found that ATP and PP^ in the same

dose range interfere with binding of activated complexes to DNA-cellulose.

648 The ability of ATP and PP^ to activate glucocorticoid-receptor complexes and weaken binding of activated complexes to DNA-cellulose is most likely due to the charge properties of the

DNA-binding

domain

of

the

receptor.

Since

binding domain appears to be positively charged

the

DNA-

(19, 20) it

is reasonable to suppose that the strongly negative PP^ and ATP can both unmask DNA-binding sites on the receptor and weaken

binding

of

those

sites to DNA, by competing

negatively charged groups. prevent

formation

accounted

of

with

The ability of ATP and PP^ to

mero-receptor

can

for by the same properties.

probably

also

be

This hypothesis is

supported by the fact that the DNA-binding site appears to contain lysine residues (19) and lysine seems to be present at

the

sites

for

enzymatic

cleavage

that

yields

mero-receptor (21).

Sulfhydryl

groups

on

the

thymus

glucocorticoid

receptor

complex.

Glucocorticoid receptors in rat thymus and liver

cytosols

are

sensitive

to

sulfhydry1-modifying

reagents.

Treatment of unbound receptors with such reagents prevents binding

of

complexes

glucocorticoid (22-24).

to

However,

form once

glucocorticoid-receptor the

glucocorticoid

has

bound to the receptor, these reagents usually have little effect on the integrity

of the complex

(25-27).

We have

suggested that there is a sulfhydryl group in or near the DNA-binding domain of the activated thymic complex that must remain

reduced

for

the

suggestion originated highly

specific

methanethiosulfonate

complex

to

bind

to

DNA.

from the observations that:

sulfhydryl-modifying

reagent

This i) the methyl

(MMTS) and 5,5'dithiobis(2-nitrobenzoic

acid) (DTNB) substantially inhibit binding of the activated complex

to

DNA-cellulose

sulfhydryl-modifying

(27);

ii)

when

these

reagents are removed from the complex

with reducing agents such as dithioerythritol, the complex regains the ability to bind DNA

(27).

Not all sulfhydryl

649 modifying reagents produce these results.

The commonly used

reagents iodoacetamide and N-ethylmaleimide are ineffective at

inhibiting

DNA

binding

(27)

and

preventing

activated

liver complexes from binding to nuclei (26). A

central

question

that has concerned

us

is whether

the

sulfhydryl group involved with the inhibition of DNA binding after modification by MMTS and DTNB is the same sulfhydryl group whose modification

prevents the glucocorticoid

binding to the receptor.

In other words, is there a sulf-

hydryl group domains,

or

common to both is

there

a

the DNA- and

separate

group

from

steroid-binding

for

each

domain?

Evidence for the latter situation has come from our studies involving chromatography of receptor complexes on reactive sulfhydryl matrices. Reactive sulfhydryl columns (28) function as shown in Figure 4A.

A

thioanion

on

the

glucocorticoid-receptor

complex

(GRC) undergoes a thio-disulfide interchange reaction with the disulfide of the matrix, thereby

covalently

the complex to the matrix (I).

I Motrin - S-S-R + "S-GRC —

Matrix - S-S-GRC + "S-R

1

II Matrix-S-S-GRC + "S-R —'Matrix -S-S-R' + "S-GRC

DSCT — 0 0 0 Ι-0-C-N-CH-CH-N-C-CH-CH-C-N-CH-CH-S-S^Ö) 2 Η 2 2Η 2 2 Η ι V COO" C00" DSTT

ο

9 μ

2

2u

FIC. H] STEROID APPLIED TO COLUMNS

H» STEROID (CPH) RETAINED OW COLUMNS DNACELLULOSE DSTT DSCT

Γ Ή 1 Τ Α LABELED CVT0S0L5 UNMARKED

»796

162

-

133

«ARMED

3696

2359

-

2334

UNMARKED

4941

327

4938

WARMED

4807

2717

4940

UNWARNED

1412

76

1334

0

»ARMED

3890

3611

3880

3307

[3H1DM LABELED CYT0S0LS

* GLUCOCORTICOID RECEPTORS MERE LABELED IN INTACT TOYHUS CELLS MITH EITHER 20 nH [ ^ T R I AMCINOLONE ACETONIDE (TA) OR 50 nH [ S H]DEXAMETHASONE 21 MESYLATE (DH) f o r 2 HOURS AT 0 * C A W THE RESULTANT CYT0S01 EITHER MARMED FOR 15 MIN AT 25*C OR KEPT ON ICE. CYTOSOLS WERE THEN BOUND TO DNA CELLULOSE, DSTT, OR DSCT (291.

Two pieces of evidence suggest that the interaction of the complex with the DSCT matrix is through a sulfhydryl group in or near the DNA-binding domain. First, soluble DNA inhibits the binding of the activated complex to both the DSCT matrix and DNA-cellulose by a similar amount (29);

651

second, treating purified complexes with sulfhydryl modifying reagents inhibits binding to DNA cellulose to the same extent that DSCT-binding is inhibited (29). Evidence to support the idea that these are separate sulfhydryl groups associated experiments binding

in which

domain

receptor

with

was

the

with

the two domains comes

sulfhydryl

covalently

dexamethasone

group

blocked

in the

by

21-mesylate,

steroid

labeling

and

the

from the

complex

then allowed to react with the DSCT matrix.

If a common

sulfhydryl

the

existed

between

the

two

domains,

complex

should not bind to the DSCT matrix, but if there were 2 separate sulfhydryls it should bind.

As shown in Table 3,

the complex did react with the DSCT matrix, indicating the probable existence of a sulfhydryl group located near the DNA-binding domain. The sulfhydryl group associated with the DNA-binding domain appears to be located in a portion of the complex resistant to degradation.

We have digested the activated complex with

immobilized trypsin, thermolysin, subtillisin and endogenous thymic protease (29) to a point where DNA cellulose binding was lost, but there was only a moderate decrease in binding to the DSCT matrix. be

retained

though

Thus, this sulfhydryl groups appears to

on mero-receptor

the DNA

binding

and

function

related is

lost.

complexes, Using

even

similar

types of experiments Harrison et al. (30) also demonstrated the existance

of more

than one

activated

trypsin

digested

and

sulfhydryl complexes.

group

on both

However,

they

were unable to associate these sulfhydryls with functional areas of the complex.

Stability of cytosolic glucocorticoid complexes in leukemic cells. Glucocortiocids have been used in the treatment of leukemia for over 30 years. The benefit derived from

652 Steroid

therapy, however, varies widely within the parti-

cular subtype of di sease. lack

of

glucocorticoid

It is possible that in some cases responsiveness

is

the

result

of

subtle alterations either in the properties of the receptor itself

or

in

McCaffrey leukemia kemia

its regulation.

et

al.

(31)

subtypes,

(ANLL),

In

reported

particularly

appear

to

support

that

of

this

notion

from

some

acute nonlymphocytic

leu-

have

receptors

altered

characteristics

compared to those from normal lymphoid tissues when analyzed by

DNA

and

DEAE

chromatography.

They

hypothesized

that

these abnormal characteristics would correlate with lack of patient response to therapy. We have surveyed a number of leukemia specimens using the minicolumn procedure to determine the relative proportion of activated and nonactivated complexes in cytosols before and after warming cytosols to 25°C (cell-free activation) What

we

found

leukemia

was

cells

that

(CLL)

cytosols

contained

of

chronic

(32).

lymphocytic

glucocorticoid

receptor

complexes in proportions similar to those seen with normal lymphoid tissue.

At 0°C, most complexes were in the non-

activated

with

state,

mero-receptor

forms.

little In

evidence

contrast,

for

activated

specimens

of

or ANLL

classification displayed a much higher proportion of meroreceptor complexes and much lower proportion of DNA-binding complexes

than

CLL

or

normal

specimens.

Of

particular

interest was the question of whether these differences were due to degradation taking place after the cells were broken, or reflected

intrinsic differences

in receptors

from ANLL

cells. We undertook extensive studies to determine the cause of the lability from

of glucocorticoid

ANLL

accumulated broken.

cells rapidly

(33). in

receptor We

ANLL

complexes

found cytosols

that after

in

cytosols

mero-receptor cells

were

The accumulation was most rapid in cytosols which

653 contained

activated complexes or under conditions used to

produce activated complexes, but also occurred in cytosols containing only nonactivated forms. the

lability

of

cytosolic

It was of interest that

receptors

within

the

ANLL

specimens were related to the differentiation state of the cells. of

That is, cytosols of ANLL specimens with properties

monocytoid

differentiation

[M^

French-American-British

(FAB) class] in general contained more stable complexes than specimens primarily of myelocytic differentiation (M^ - M^ FAB classes) . cytosols

of

patterns

Consistent with this observation,

polymorphonuclear

cells

isolated

from

normal

blood were much more labile than those of monocytes. Most important, if cells were first incubated at 37°C with radiolabeled

steroid

to form activated

complexes in vivo,

then broken and analysed on minicolumns, there was in all cases clear evidence for activated or DNA binding complexes. From no experiments did we obtain evidence to suggest any difference in the native receptors of ANLL and CLL cells. We have concluded that the high proportion of mero-receptor in ANLL specimens was due to in vitro proteolysis of otherwise normal receptor complexes.

Further

support for this

conclusion is given by the studies below. We first hypothesized that the differences in lability of cytosolic preparations were due to the relative amounts of proteolytic enzymes capable of degrading the receptors.

If

so, then mixing labile and stable cell preparations prior to lysis should allow us to demonstrate the presence of such receptor-degrading enzymes. stable

preparations

in

In other words, complexes from

mixed

cytosols

should

no

longer

appear stable, but should be converted to mero-receptor by the proteolytic enzymes from the labile cell preparations. Unexpectedly, what the mixing experiments rather

than

the

labile

cytosols

causing

showed was that degradation

of

654

complexes from stable cytosols, the reverse was true; the stable

cytosols

cytosols

stabilized

(Figure 5).

stabilizing proportion

receptor

complexes

Further experiments

in

showed

labile

that the

effect was dose-dependent with respect to the of

characterize

stable the

cytosol

stabilizing

used.

Further

factor

from

attempts

human

to

leukemic

cells were hampered by the lack of availability of tissue. For this reason we looked for such a factor in other cell types.

These studies are discussed below.

pHlTA-ANLL + TA-ANLL

|3H|TA-CLL pHjTA-ANLL · TA-CLL • TA-CLL

| 3 H|TA-CLL * TA-ANLL

ANLL-CLL COMBINATIONS FIC. 5.

DISTRIBUTION OF CLUCOCORTI CO ID-RECEPTOR COMPLEXES IN WARMED CYTOSOLS OF MIXED ANLL AND CLL CELL PREPARATIONS. CELLS OF ANLL AND CLL SPECIMENS WERE INCUBATED WITH [3H]TRIAMCINOLONE ACETON I DE ([JH]TA) OR UNLABELED TRIAMCINOLONE ACETON I DE (TA) FOR 2 HOURS AT 0"C. EQUAL ALIQUOTS OF SUSPENSIONS WERE MIXED AT A RATIO OF 1:1 IN VARIOUS COMBINATIONS; FROM THESE MIXTURES, CYTOSOLS WERE PREPARED. Th€ CYTOSOLS WERE WARMED TO 25°C FOR 15 MIN AND ANALYZED ON MINICOLUMNS.

Calpastatin, a stabilizer of glucocorticoid-receptor complexes. In addition to human CLL cells the stabilizing factor is also present in many tissues including WEHI-7 mouse thymoma cells and rat liver and spleen cells (34). Interestingly, it is not present in rat thymus cells which contain relatively labile cytosolic complexes (8,34). Table 4 summarizes the properties of stabilizing factor partially purified from rat liver cytosol. As shown in the table, it appears to be very similar to calpastatin, a naturally occur-

655 ring

inhibitor

of

a

family

of

neutral

proteases called calpains (35-37). a physiological statin

and

relationship

Whether or not there is

between

glucocorticoid-receptor

further investigations.

calcium-activated

the calpains, complexes

must

calpaawait

An important practical consequence

of our findings however, is that the addition of calpastatin to thymus cytosols and to cytosols such as those from cells of

patients

with

ANLL

greatly

enhances

the

stability

of

complexes.

table k

PROPERTIES OF THE GLUCOCORTICOID-RECEPTOR STABILIZING FACTOR (CRSF) AND CALPASTATIN PROPERTY HEAT STABLE (100°C, 30 MIN) ELUTION FROM OEAE CELLULOSE (mM NaCl)

APPROXIMATE MOLECULAR WEIGHT OF NATIVE AGGREGATE

GRSF YES

CALPASTATIN YES

110

110 (100)

280,000

280,000 (280-300,000)

ISOELECTRIC pH

5.1

EFFECTS ON ACTIVATION OF GLUCOCORTICOID RECEPTOR COMPLEXES

NONE

5.1 Ct.55) NONE

VALUES ARE FROM B0DWELL, J.E., H0LBR00K, N.J. AND MUNCK, A. (UNPUBLISHED) FOR RAT LIVER CALPASTATIN. VALUES IN PARENTHESIS FOR NaCl CONCENTRATION AND MOLECULAR WEICHT ARE FROM MURACHI ET AL. (36), AND FOR ISOELECTRIC pH ARE FROM TAKANO AND MURACHI (37) FOR HUMAN ERYTHROCYTE CALPASTATIN.

Acknowledgements This research was supported by research grants AM03535 and CA17323 from the National Institutes of Health, and by the Norris Cotton Cancer Center Core Grant U. S.

Public

Health

Service.

(CA23108) from the

D.B.M. is a fellow of the

656 Albert

J.

Ryan

Foundation.

N.J.H.

was

supported

by

a

fellowship from the Leukemia Society of America.

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6.

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14.

Munck, Α., Foley, R. : (1980) .

15.

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16.

Munck, Α., Wira, C.H., Young, D.A., Mosher, K.M., Hallahan, C., Bell, P.A.: J. Steroid Biochem. 3, 567 (1972) .

17.

Moudgil, V.K., John, J.K.: (1980) .

18.

Holbrook, N.J., Bodwell, J.E., Munck, Α.: Chem. 25£, 14885 (1983) . DiSorbo, D.M., Phelps, D.S., Litwack, G.: Endocrinology 106, 922 (1980).

19.

Biochem. J. 190, 79 9 J. Biol.

20.

Schmidt, T.J., Litwack, G.: (1982) .

21.

Moran, M.C., Tuazon, F.B., Stevens, Y.-W., Sherman, M.R.: Fed. Proc. 60%),

the

synthetic At

to

60%,

inhibits the

time

progestins

whose

effects

progestins,

physiological

10

translocate

with

synthetic

1 or

partially

of R 5 0 2 0

progesterone,

in the m e d i u m .

50

either

coincides

Other

does

unlike

that,

concentrations these

1 hr p u l s e

blocked. as

have

treatment

extensively

4 days

is

in

approximately

a brief

growth,

rapidly metabolized

Ten days

more

least

replenishment cell

We

physiologic

concentrations or

are

proliferation

7).

Even

know

accumulation

It w o u l d be

changes

ER,

cell

the

cytoplasmic

attenuated

below.

or

inhibit

suppresses

elements,

not

antipromotional

of T 4 7 D ( , n c e l l s .

(Figure

do

changes

are

R5020-treated

T h i s m a y be the

or

contain

therapy.

estradiol

cells

consistent

cells.

further

We

filamentous

degenerative

cytostatic

inhibition

absence

progestins

are

although and

ultrastructural

after

Growth

nM

the

described

regressing

the

this,

cells

cytoskeletal

t o n o f i 1 a m e n t s.

differentiated

reflection see

of

regressive

common

inclusions, which

not f o u n d in the c o n t r o l are

a

biosynthesis.

Electron micrographs large

hormones

hormone

it

is

concentra-

t i o n s (0.1 μ Μ ) , e s t r a d i o l , a n d r o g e n s , g l u c o c o r t i c o i d s , a n d 1 , 2 5 d i h y d r o x y v i t ami η

D3

indicate

have

that

the

effects

proliferation

can

be

that

antiestrogens

no of

direct, and

effect

on

progestins and

cell

independent

progestins

can

growth.

on m a m m a r y target

of

Our

tumor

estrogen, different

data cell and cell

681 populations. in

T h i s m a y e x p l a i n the c l i n i c a l e f f i c a c y of p r o g e s t i n s

otherwise

endocrine

hormone-resistant

therapy

Progestins

are

and

outlined

insulin

culture

is u n d e r c o m p l e x

factors

added

to

the

tumors;

regulation

but

synthesized

exogenous

I n s u l i n is a c o m m o n m e d i u m s u p p 1 e m e n t a n d is growth

of all

by

the

and

cells required

human breast

cell l i n e s i n w h i c h it h a s b e e n t e s t e d ( 4 3 - 4 5 ) .

in

stimulatory

themselves.

serum-free

be

growth

factors

the c o n t i n u o u s

for

proliferation

i n v o l v i n g not o n l y

medium, can

Cell

inhibitory for

that

implications

below.

receptors.

growth

the

Moreover,

cancer insulin

and steroid hormones can reciprocally regulate each others actions (46-49).

We

among

heterologous

the

levels,

an

have

shown

effect

simultaneous

that

in

hormones

that

seems

cell-growth

T47DC0 that

to

be

inhibition

to

first

r o u n d of

control

cell

levels.

little

if a n y g r o w t h

number

of

peak

on

insulin

days

paralleled control

2

(42).

to

3. in

where

insulin

a new

real

increase

in

shows

the

that

during

1 to 3) the

then

first

increase cell

state the

of

settle

down

3 days,

when

4

the and

to a b o u t

8000

there are

in u n t r e a t e d To

insulin

binding

binding

is

receptors,

cells were

of

below

receptors

steroid

hormone hormones

to b i n d a n d

drοtestosterone

specific

specificity incubated or

translocate

pharmacological was

due

(days

compared

to

(42).

the

sufficient

systems.

cells

twice

sites/cell

i n s u 1 i n - b i n d i ng s i t e s / c e l l

determine

classes but

18 , 000

is

established.

increased

insulin

sets, 5-fold

proliferation

levels is

to

a n d not d u e to a c h a n g e in b i n d i n g a f f i n i t y . At s t e a d y s t a t e 5 to 9)

the

cells,

receptor

number

to

In u n t r e a t e d

slow

steady

related

in R 5 0 2 0 - t r e a t e d

sites/cell

are

receptor

increase slightly

during

Thereafter,

Scatchard analysis to a

(Days

is d e m o n s t r a b l e

binding

by a fall

values,

division

In c o n t r a s t ,

progestins insulin

closely

the n u m b e r of i n s u l i n b i n d i n g s i t e s / c e l l the

cells,

regulate

to

analogs, their

levels. R5020;

for

The neither

of

the

increase

5 days with at

concentrations

respective increase estradiol

receptors, in

dexamethasone,

insulin

nor

had any e f f e c t , c o n s i s t e n t with reports

The synthetic glucocorticoid

in

several

dihy-

in o t h e r decreased

682 the n u m b e r of known

variable the

insulin

depending

studies.

corticoid on

insulin binding

to m o d u l a t e

on

RU38

the

cell

486,

a

receptor

by 5 0 % .

are are

type

and

actions

levels when

together with R5020,

Glucocorticoids

levels, and their e f f e c t s

synthetic

and antiprogestiη

insulin

R5020.

sites

receptor

(see

to d i s t i n g u i s h

progestins

and

had

no

effect alone;

receptor

induction

that r e g u l a t i o n of

the

on i n s u l i n

antiestrogen

(ΙΟηΜ),

synthetic antiprogestational

as

experimental

tation or

clinical

486

tools,

inhibitors

--

(ΙμΜ).

studies.

contraceptives

been

However,

available

a candidate

for

antiprogestational clinical

trials

has

and

recently

been

implanbasic RU38

a-(1-propyn1)that

activity

in

has early

of a c t i o n a r e

unclear.

F u r t h e r m o r e , d e v e l o p m e n t of t h i s d r u g u n d e r s c o r e s an o l d

bioassay

problem:

that

progestins

is c o m p l e x

that among

effects

system.

of

circumvented

This

unknown when

therefore

actions

of

this

screening

because

progestational,

properties have

biological

progestational

estrogenized

Its m e c h a n i s m s

and

either

described

antigl u c o c o r t i c o i d

(50,51).

agents,

antiprogestiη,

[17ß-hydroxy-1lß(4-dimethy1 aminopheny1)-17

estra-4,9-dien-3-one ]

promise

a g e n t s - - as a n t i c a n c e r

have

Other

21-hydroxyproges-

nafoxidine

as m i d - c y c l e

none

marker

1,25-dihy-

T h e a n t i p r o g e s t i η , R U 3 8 4 8 6 . D e s p i t e the t h e o r e t i c a l of

by

insulin

in T 4 7 D C 0 .

receptors were

testosterone

in

to the c e l l s

from antiprogestins

that h a d no e f f e c t

(lOnM);

used

antigluco-

i n d u c i b l e p r o t e i n ) m a y be a u s e f u l

d r o x y v i t a m i n D3 ( Ο . Ι μ Μ ) : terone

with

below),

blocked

W e a r e t e s t i n g the p o s s i b i l i t y

hormones

conditions

it w a s a d d e d

it p a r t i a l l y

r e c e p t o r s (or a n y o t h e r

the

steroid

of

the

must

of be

has m a d e

progestins

physiological

superimposed

it d i f f i c u l t

antiprogestational, agents,

T47DC0

been used

are

a

problem

used

for

to c o n t r a s t

interesting

drug

to

and that

the

nm, and drug

to

this w a v e l e n g t h PR

concentrations

in (10

situ. nM)

of

upon

an

distinguish be

screening.

the a g o n i s t

entirely

These

and

cells

antagonist

(52).

can be u s e d Like

anti-

antiestrogenic can

W e f i n d that l i k e R 5 0 2 0 , R U 3 8 4 8 6 a b s o r b s U V at 300

and

requirement

the

[3H]RU38

synthetic

486

approximately

to c o v a l e n t l y bind

photolink

progestin,

two P R s u b u n i t s

low in

683 nuclei

of

T47Deo;

competition for

studies,

unlabeled

[3H]R5020

in s i t u

RU38

glucocorticoid binding

486 has a h i g h a f f i n i t y

n M at 0 - 4 ° C ) a n d transform more state.

Like

in i n t a c t

so

of

is a

nuclear

for P R in v i t r o

cells,

These

biochemical progestins,

To b i o a s s a y

as

we

a 1 hour

asked

RU38

486-pulsed the c e l l s

shown

Thus, conclude with

no

cells

for

have

is

a

it

evidence

cell of

R5020 R5020

alone,

growth

486

When

replenishment, effect

on

cell

the g r o w t h of 1 h o u r

at

least

4 days,

similar

to

that

after of

the

long-acting,

RU38

effects.

were

486

at

This

alone,

number

of

60 or

we

would

properties,

is, h o w e v e r ,

measured

incubated

486

the

marker,

progestiη-1ike is hrs the

not

level

with two

insulin

concentrations

the

hormones

binding

sites

sufficient

cell g r o w t h r a t e , h a d no e f f e c t o n increase,

o n the c o n c e n t r a t i o n

if i n s u l i n

levels

are

properties

used as

of e a c h h o r m o n e .

a biological expected

Thus,

marker,

RU38

of a n a n t a g o n i s t

486 (52).

of

medium

to

these

W h e n the two h o r m o n e s w e r e a d d e d to c e l l s t o g e t h e r ,

of

is

inhibited.

38 486 b l o c k e d 20 to 5 0 % of the R 5 0 2 0 - i n d u c e d

the

insulin

by 5 5 % .

remains

a biological

end-point

RU38

increased

but

as

agonist

T47DC0

translocate PR and alter proteins.

has

antagonist

receptors.

expected,

growth

486

the b i o l o g i c a l

together.

and

inhibition;

long-term for

are pure

RU38

receptor

at a r a t e

486 from

in g r o w t h

controls,

suppressed growth

RU38

PR

R5020.

RU38

containing as

would

is n o t

suppresses

of

2

results.

growth

486 b l o c k s

to h o r m o n e - f r e e

resumed

using

that

the c a s e w h e n insulin

of R U 3 8

cell

B.

binding

the c o m p o u n d

Other synthetic progestins are similarly here

inhibitor

(6 to 8 n M )

both growth

suppresses

combined,

this

Compared

controls. as

pulse

whether

growth. which

are

In

(K500 mg/day

( T a b l e 2), w i t h 789

induration and

to b e e q u a l l y

sub-

using

f r o m 2 to 23+ m o n t h s

was well

have developed

been

those

chosen

reported

patients of

treatment

the d a t a s h o u l d be

17 p a t i e n t s ,

injection the

of

in

the r e s u l t s of 11 c e n t e r s

progestins

in the

studies

treat m e t a s t a t i c

study

compiled

that

of

lesions.

objective positive

ranges

muscular showing

products

months.

et

using high-dose M P A prompted

the

therapy.

progestins

(54-81).

of

versus

have arbitrarily

p a t i e n t s h a v e a c c r u e d , a n d the total rate

sum

all m e a s u r a b l e

suggests

dose"

dose

the

u s e of p r o g e s t i n s

using

We

"high

in

with

side

effects

proven

(90)

than

progestins (90),

antiestrogens,

the

and

has

found are

their

hormones

687 T a b l e 3.

Responses Clinical

to T a m o x i f e n vs. P r o g e s t i n s

Proges t i η

Tamox i f e n Mattsson, Morgan,

in Same

Centers

1980 1982

15/32

(47%)

14/26

(54%) MPA

17/48

(36%)

14/46

(30%) MA

7/27

(26%)

4/28

(14%) MA*

Pannut i,

1982

7/26

(27%)

10/27

(37%) MPA

Johnson,

1983

14/49

(28%)

20/49

(41%) MA

17/58

(29%)

18/45

(40%) MPA*

17/80

(21%)

31/136

(23%) MA*

94/320

(29%)

111/357

Ingle,

1982

Van V e e l e n ,

1983

Al e x i e v a - F i g u s c h ,

1983

TOTAL

*low dose p r o g e s t i n MPA = m e d r o x y p r o g e s t e r o n e MA = m e g e s t r o l a c e t a t e R e f e r e n c e s 86, 94-99

T a b l e 4.

1979

v o n Mai 1 l o t , Forestiere, Bruno,

(25%)

concurrent

(100%)

concurrent

4/23

(17%)

concurrent

14/55

1980 1981

1982

1983

Garcia-Giralt, TOTAL

acetate

R e s p o n s e to C o m b i n a t i o n T a m o x i f e n / P r o g e s t i n R e f e r e n c e s 1 0 3 t o 108

Mouridsen,

Trodella,

(31%)

1984

4/4 8/18

(44%)

concurrent

19/36

(53%)

sequent ial

17/33

(52%)

concurrent

21/35

(60%)

sequent i al

87/204

(43%)

Therapy

688 That the

antiestrogens

treatment

clinical also

trials

from

a

combined

treated

with

studies

of

to

the p r o g e s t i n s ALL

brings

treatment

us

(described negative

further can

there are

still

hormonal

who

agents

data

hormonal

propose

mechanisms

these

to in

periods

(100-102).

Thus may

subsequent

regression

by

tamoxifen

not

treated preclude

combination

of

progestins Our

(T47DQQ),

this

setting.

become or

failed

and

Show

whose

that

tumors

relapsed

as

our

benefit to

other

tumors

report

or

ER that

Similarly,

can be of

whose

in

studies

resistant

studies

progestins

as

are

had

from

are

34%

of

been

previous

these

progestins; other

the

have

latter thereby

treatment

In

PR

pre-treatment of

ER-positive

should conferring

are

addition,

tumors

increased

brief

data,

different

two

tumors.

sensitivity

the

pathways

or

by

Antiestrogens

antiestrogens,

agents,

simultaneous increase

act

Therefore,

positive of

experimental

progestins

conceivable.

properties

with

well

and

estrogen-receptor

antiestrogens to

to

of

progestins

remission.

progestins

due to the e s t r o g e n i c short

show

tumors.

cells

or

Several

clinical,

promote

successful

were

did

role

antiestrogens,

antiestrogens

using

patients tamoxifen

postmenopausal.

this

the

in

to,

had

has

trial.

(83,84,88,90-92).

that

strategies

for

or

but

antiestrogen-resistant

that

dose

each,

therapy

tamoxifen.

tumor

respond

to high

resistant,

on

yet

or

growth

negative.

therapy

Based we

were

for

separate

had been p r e v i o u s l y

and

with

showing

to

357 with

consideration

breast

including

responded

antiestrogen

a

inhibit

fail

estrogen-receptor patients

women

(86,94,95)

below)

for

a n d 29% r e s p o n d i n g

these

therapy,

human

clinical

patients

which

treated

the p a t i e n t s

to

data

progestin

antiestrogen-resistant

cultured

progestins in

of

in

progestins

antiestrogen/progestiη the

of

chemotherapy

subsequent

This

7

useful

from

in t h e same c l i n i c a l

and 320 w e r e

these studies, to

which

results progestins

not o n l y

response

in

tamoχ i f e n

cytotoxic

response

similar

studies

3 )( 8 6 , 9 4 - 9 9 ) .

In many of with

of

may b e e q u a l l y

is suggested

compared with

31% r e s p o n d i n g (Table

cancer

reporting

number

been d i r e c t l y The

and p r o g e s t i n s

of b r e a s t

then

treated levels with tumors promote

synergistic

689 therapeutic

effects.

this

a

model:

breast and

There

total

cancer

of

have been

progestins,

sequentially

(Table 4). remissions

This

level

response

of

for

whose

The

still

a

total

is

as

good

Of

patient found

These in

a

44%

results

an

probably

be

estrogen

should

using to

Only

alternating clinical

the

trials

of

better,

than

not

22%

the

or and 43%. the

with

complete

Though by

combined

warrant rates

further

of

present

the t w o h o r m o n e s one

to

the

two

the p r o g e s t i n

could

a hormone-insensitive has

explored

two

hormones

of

these

(108).

the

antiin

interval

result

in

loss

cell

population

of

repeatedly

value

Clearly,

we 11-tolerated

can

weeks

E x c e s s i v e l y p r o l o n g i n g the

and

with

remissions.

theoretically,

progestin

al.

respectively).

rate

therapy

29%

et

pretreated

response

together, the

leaving study

response

therapy.

precede

one

tamoxifen

11% c o m p l e t e

heavily

combination

administered

the a n t i e s t r o g e n

(102).

of

response

(61% a n d 83%

included

increase

hormonal

of P R a l t o g e t h e r ,

if

support advanced

s t u d y by T r o d e l l a

was

objective

order t o m a x i m a l l y induce PR. between

the

therapy

which

attempt

day c o n v e n t i o n a l

rate was

as,

to

with

simultaneously

objective

note was

population

tamoxifen/progestiη,

study

either

response

or

data

women

a combination

given

chemotherapy and/or hormonal They

clinical

t a m o x i f e n or p r o g e s t i n s w h e n u s e d s i n g l y ;

and 31% respectively. (106)

treated with

response

rates

some

postmenopausal

(103-108)

34%

partial

are

204

more

extensive

hormonal

agents

are

warranted. 2)

The

second strategy

for

u s e of

progestins

is b a s e d

our s t u d i e s w i t h E R n e g a t i v e , P R p o s i t i v e , a n t i e s t r o g e n T47DC0 such

cells, which

cells.

Small

show

that

clinical

progestins

studies

MA, support

the i d e a t h a t a s i m i l a r

have become

resistant

using

therapy.

be c a n d i d a t e s 123

patients

Such

patients

for e n d o c r i n e show

that

22%

respond

to

tamoxifen

(Table

5).

(8%)

if

they

to

but

further

growth

of

of M P A

or

failed

to

tumors tumors

benefit

from

not c o n s i d e r e d

four

trials

progestins

In c o n t r a s t ,

have

doses

t r e a t m e n t , or w h o s e

are usually

therapies,

from tamoxifen (86,95,98,99).

inhibit

high

set of p a t i e n t s , w h o s e

to a n t i e s t r o g e n

h a v e no E R b u t a r e P R p o s i t i v e , m a y a c h i e v e progestin

can

tumors respond

on

resistant

based

to on

after

relapse

rarely

respond

to

progestins

690 Table

5.

Tamoxifen Studies

vs.

Progestin

Cross-Over:

Same

Center

Objective Mattsson,

Ingle,

1980

1982

Van V e e l e n ,

1983

Al e x i e v a - F i g u s c h ,

1983

Tam->fai 1 MPA+fai 1

MPA ΤΑΜ

6/10 0/10

(60%) (0%)

Tam-^fai 1 MA* -*f a i 1

MA* Tarn

2/16 2/18

(12%) (11%)

Tarn -»f a i 1 MPA+fai 1

MPA Tam

5/14 0/14

(36%) (0%)

Tarn MA*

MA* Tam

14/83 12/132

(17%) (9%)

Prog Tam

27/123 14/174

(22%) (8%)

* l o w dose p r o g e s t i n MPA = m e d r o x y p r o g e s t e r o n e MA = megestrol acetate Prog = p r o g e s t i n References 86,95,98,99

Table

ai1 ai1

Tam+fai 1 Prog-+f a i 1

TOTAL

6.

Receptor Content T h e r a p i es

acetate

and

Response

to

Objective

Endocrine

Response

ER+ ER-

312/586 33/264

(53%) (13%)

PR+ PR-

240/352 108/498

(68%) (22%)

ER+ ER+

PR+ PR-

230/323 85/263

(71%) (32%)

ERER-

PR+ PR-

10/29 23/235

(34%) (10%)

Cumulative References

Response

d a t a 14 s e r i e s 1978-1983 1,2,16,78,124-135

691 That breast

progestins

tumor

cells

implications, populations, agents

can

and by

and we

antiestrogens

different

propose

that,

the c o m p l e m e n t a r y be

used

to

inhibit

paths by

has

the

targeting

in

the

of

design

of

clinical

different

therapeutic actions

advantage

growth

important

cell

these

of

two

treatment

protocols. F i n a l l y , an area in

combination

have

been

with

performed

combined with 112)

have

after of

in w h i c h p r o g e s t i n s cytotoxic comparing

tamoxifen.

been

reported,

tamoxifen

addition

combination

reported

randomized response 218

of

and

by

obtained

two

et

non-randomized

(54%)

an

with

two

vs.

randomized

studies

showing

chemotherapy studies

showing

patients

that

al.

in

benefit

a significant

benefit

1973

studies

on

been

then

performed

with

In 6 s t u d i e s ,

progestin/chemotherapy

However,

chemotherapy

the

alone,

treatment

arms.

Nevertheless,

3

studies

with

p r o g e s t i n t h e r a p y , w e r e u n a b l e to s h o w a s t a t i s t i c a l l y between

was

Since

(114-118).

combined

response.

compared

chemotherapy

( 113 ).

have

f r o m 2 7 % to 7 5 %

objective

(114,116,117) difference

(109-

no

in c o n j u n c t i o n w i t h

Stott

rates varying

402

recent

studies

chemotherapy/tamoxifen.

T h e u s e of p r o g e s t i n s first

Similar

chemotherapy

Four and

h a v e a l s o b e e n u s e d is

chemotherapy.

chemo-

significant the

patients

t r e a t e d w i t h the p r o g e s t i n s e e m e d to h a v e a n i n c r e a s e d p e r f o r m a n e e status,

increased

and a protective MPA

has

induced it

is

been by

shown

of

of w e l l

to p r o t e c t

that

B.

patients

adriamycin

with

a progestin,

c h e m o t h e r a p y , a n d be m o r e of

being,

less

pain,

weight

on the b o n e m a r r o w ( 1 1 6 , 1 1 8 - 1 2 0 ) .

vincristine,

possible

presence

sense effect

reduced patients likely

and

against

the

marrow

toxicity

Since

neutropenia

cyclophosphamide

could

gain,

due

receive more

to s t a y o n the p l a n n e d

(121), to

the

intensive schedule

therapy.

Progesterone tumors

receptors

as

markers

of

hormone-dependent

692 E s t r o g e n receptors

(ER) have been used since 1971

(122) as

a guide to t h e r a p y , and they c o r r e c t l y predict a p o s i t i v e r e s p o n s e in 5 0 - 5 5 %

of

patients

(1,2,26,28,124-135)

(123). is

However,

even

improving

predictive

contrast,

receptor-negative

more

accuracy

assessment

useful

by

an

tumors

as

table

additional

rarely

of

tumor 6

shows,

15-20%.

respond

to

In

endocrine

t h e r a p i e s and such p a t i e n t s s h o u l d be c o n s i d e r e d c a n d i d a t e s alternate treatments.

PR

for

Not s u r p r i s i n g l y , few tumors are ER-, PR+

t h o u g h some of these may be falsely n e g a t i v e due to m e t h o d o l o g i c a l r e a s o n s (136).

O t h e r s may have E R that are either s e q u e s t e r e d in

an u n m e a s u r e d c o m p a r t m e n t or n o n f u n c t i o n a l , a n d m a y resemb/le the variant

T47Deo

subline

progestin sensitive. for p r o g e s t i n

that

is

antiestrogen

resistant

but

S u c h tumors s h o u l d be c o n s i d e r e d c a n d i d a t e s

therapy

(see

above).

P r o g e s t e r o n e receptors have also p r o v e n to be m o r e

accurate

than ER in p r e d i c t i n g p r o g n o s i s of limited stage (I a n d II) breast cancer

(3,137-140);

patients with

PR+

tumors

are 3 to 4

times

less likely to d e v e l o p m e t a s t a s e s than those w i t h P R - ones (137). an

independent

p r o g n o s t i c v a r i a b l e , a n d m o r e useful for p r e d i c t i n g

In

more

disease-free

survival Clark

advanced than

grade

either

III

clinical

disease, or

et al (3) r e p o r t e d similar

PR

are

pathological

results

staging

in p a t i e n t s w i t h

II b r e a s t cancer who w e r e u n d e r g o i n g a d j u v a n t therapy. study due

(141)

had

to the small

failed

to detect

number

these

of p a t i e n t s

(138). stage

A n earlier

relationships,

perhaps

evaluated.

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STEROID HORMONES, RECEPTORS AND NEUROTRANSMITTERS

Gary Dohanich, Bruce Nock and Bruce S. McEwen Laboratory of Neuroendocrinology, The Rockefeller University New York, NY

10021

Introduction The brain is characterized by its diversity of chemical signals and modes of communication, just as it is also known for its incredible anatomical complexity.

The two features are,

of course, closely linked, for it is one group of neuronal products which direct nerve cells to form specific contacts and then hold them together during development and another category of diverse cellular products which allows mature neurons to communicate with each other.

It is now recognized

that besides the traditional neurotransmitter substances released from synapses to act upon defined postsynaptic junctions to trigger electrical activity, there are other neuroactive substances with a less precisely-defined mode of action.

These substances, sometimes called "neuromodulators"

show their effects by modifying what other neuroactive chemicals do rather than by acting on their own to excite neurons (1).

Neuromodulators may have other, more-or-less "silent"

effects to influence energy metabolism or the capacity of cells to make, break down or respond to neurotransmitters or other neuromodulators - such modulatory effects may only show themselves under times of stress or prolonged activity. Among the "neuromodulators" are hormones arising from outside of the brain which gain access to the CNS from the cir -

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

702

culation or through the cerebral ventricles.

Protein and

peptide hormones such as prolactin (2) and ACTH (3-5) or TRH (6-8) are reported to alter nerve cell function and influence behavior.

Steroid hormones are also important regu-

lators of brain structure and function and produce effects during early development and in adult life which alter chemical transmission and neuromodulation and influence behavior and affective state.

Steroid hormones gain access to the

brain from the circulation and interact with receptors in certain groups of target neurons; mapping these receptor sites has occupied the efforts of a number of laboratories over the past 16 years (9-13).

Recently, emphasis has shif-

ted from locating receptor sites to how steroids affect brain function (eg. 14).

In these studies the effects of steroid

hormone exposure on neurochemical parameters has revealed a large variety of effects, ranging from direct interactions of certain estrogens with neurotransmitter receptors and metabolizing enzymes to more long-term actions to increase neurotransmitter receptor number or enzyme amount.

In addi-

tion, a new facet has been added by the observation that neural activity, and in particular certain neurotransmitters, may influence how steroid hormones act.

This review will

summarize selected aspects of these studies to provide a picture of the diversity of steroid hormone interactions with neural tissue. Steroid hormone receptors and actions in the brain The brain is responsive to estrogens, androgens, progestins, glucocorticoids and mineralocorticoids (15), and the actions of these hormones in neural tissue follow the general pattern of steroid hormone action throughout the body, which involves intracellular receptors that bind the hormone to the cell nucleus. However, there are three special features of steroid action in brain which deserve emphasis and will be described below.

703 Localization of receptors and effects Steroid receptors are not uniformly distributed throughout the brain but are discretely localized to groups of nerve cells in some brain areas (9-13).

In addition, glial cells

have receptors for at least one class of steroids, namely, glucocorticoids (16).

Each class of steroid receptor has its

own pattern of distribution.

For example, glucocorticoid

receptors are concentrated in hippocampus, septum and amygdala (13), whereas estrogen receptors are found predominantly in hypothalamus, preoptic area and amygdala (9—12). Steroid effects on these receptor-containing neural cells are highly diversified.

In many cases, this is a reflection of

the neurotransmitter phenotype of the cells in question: eg., estrogens regulate cholinergic enzymes in some neurons of the basal forebrain (see below) and tyrosine hydroxylase in some neurons of the arcuate nucleus (17-19) .

There are

many cholinergic and catecholaminergic neurons throughout the nervous system which are not apparently regulated by estrogens, and this is presumably due to the absence of estrogen receptors in these cells.

Sometimes, however, the regulatory

effects are not so clearly delineated according to neurotransmitter phenotype or presence or absence of hormone receptor. For example, estrogens regulate serotonin-1 and alpha-2 adrenergic receptors in some, but not all, estrogen-sensitive cell groupings where these neurotransmitter receptor types are also found (20-22).

And estrogen-sensitive neurons of

hypothalamus, preoptic area and amygdala may also be categorized on the basis of whether they show aromatase activity (conversion of testosterone to estradiol), progestin receptor induction, or both (23). Besides regional differences in hormone response, there are also sex differences in response to hormone which do not

704

coincide with obvious differences in hormone receptor level: eg., estrogenic regulation of cholinergic receptors and enzymes (see below), progestin receptors (24) and serotonin-1 receptors (21).

In such cases as these, there may be addi-

tional regulatory factors besides presence or absence of hormone receptor which determine the response of hormonesensitive cells to hormone (25). Developmental versus activational effects of hormones on neural tissue Receptors for gonadal and adrenal steroids appear early in development.

In the rat, this occurs just prior to or imme-

diately after birth (26).

In neurons, the elaboration of

receptors appears to follow the final cell division and coincides with the beginning of neuronal differentiation

(26).

The effects mediated by these receptors in early development are often qualitatively different from the effects which are mediated by the same receptors in mature neurons.

For

example, glucocorticoids promote the expression of the catecholaminergic phenotype in cells of the autonomic nervous system (27) and promote the development of the serotonergic phenotype in the CNS (28); once these phenotypes are established, they are not so susceptible to hormonal influence. Estrogens, on the other hand, promote neurite outgrowth in groups of neurons of the hypothalamus and preoptic area (29). Such growth is believed to form part of the process of sexual differentiation of the brain, whereby male and female brains become structurally and functionally somewhat different from each other (26).

During this period of morphogenesis and

synaptogenesis, estrogens do not have effects as they do in mature neurons, such as the induction of progestin receptors (30) .

705

Direct versus genomic effects of steroids Besides affecting gene expression via interactions with receptors in the cell nucleus, steroid hormones have direct effects on membrane-mediated processes and enzyme reactions. For example, glucocorticoids facilitate serotonin formation throughout the forebrain via a rapid action at nerve endings (31); and estrogens alter electrical activity of preoptic area and hypothalamus neurons within milliseconds after local application (32).

Moreover, the 2-hydroxylated metabolite

of estradiol interacts with enzymes such as tyrosine hydroxylase and catechol-O-methyl transferase and with receptors for noradrenaline, dopamine and serotonin, albeit at supraphysiological concentrations (33). It is frequently difficult to assess

the contribution which

such direct effects make to overall physiological regulation. On the basis of latency of effect, many steroid actions require minutes to hours (34); furthermore, they are often susceptible to interruption following treatment with RNA or protein synthesis inhibitors (12) leading to the conclusion that genomic actions are involved.

Nevertheless, non-geno-

mic direct effects of steroids may be superimposed upon genomic actions, as is apparently the case for progesterone facilitation of monoamine oxidase (MAO) activity in estrogen-primed hypothalamus (35).

Whereas the ability of pro-

gesterone to increase MAO activity is not blocked by protein synthesis inhibitors, it is dependent on prior estrogen priming in vivo over several days which involves, presumably, a genomic action. In the remainder of the chapter we shall examine in some detail other examples of gonadal hormone-neurotransmitter interactions, which may represent both direct and genomic effects. We shall, however, be looking at them from the standpoint of

706

their relevance to the mechanism of hormone action in regulating sexual behavior and neuroendocrine function related to reproduction.

Gonadal steroids and the cholinergic system of the brain Overview Steroid hormones are known to exert prominent effects on many complex physiological functions.

These actions can range

from a discrete fine-tuning of a particular function to the full activation of a system.

Research on peripheral target

structures indicates that one mechanism by which steroids may influence cellular activity is through the regulation of protein synthesis (36).

Clearly, if central neurons that

concentrate steroid hormones behave in a similar fashion, the consequences for neuronal activity could be profound. Although classical neurotransmitters are not proteins, their synthesis, degradation, and uptake involves enzymes, carriers and receptors, all of which are proteins.

Consequently, one

way that steroids might regulate neurotransmitter activity is by altering the concentrations of proteins associated with these transmitters.

Conceptually, this capacity to regulate

neurotransmitter activity provides a mechanism by which steroids can activate intricate physiological and behavioral responses. In this section, we review evidence pertaining to steroid effects on one neurotransmitter system - acetylcholine. Within this framework, we attempt to implicate the hormonal regulation of the cholinergic system in the mediation of a specific reproductive behavior.

It is now clear that gona-

dal steroids such as estradiol,testosterone, and progesterone

707 have the capability of altering the concentrations and activities of the various neurochemicals involved in cholinergic transmission.

Furthermore, sexually-dimorphic functional

responses to gonadal hormones may arise, in part, from the existence of sex differences in the hormonal regulation of cholinergic neurochemistry.

The exact manner in which these

hormone-induced changes in cholinergic activity might be translated into biological function remains speculative. However, available evidence suggests that certain biological systems which are controlled by steroid hormones have a cholinergic component that is itself regulated by these hormones. One system that is clearly dependent upon the action of gonadal steroids is reproductive behavior.

Lordosis is a posture

assumed by the female rat when mounted by the male during the period of sexual receptivity.

Removal of the ovaries aboli-

shes the capacity for lordosis in female rats while systemic administration of estrogen (estradiol) for several days followed by progesterone restores lordosis in ovariectomized females 4-8 hr after progesterone injection (37). It has been demonstrated that intracerebral administration of agents which elevate cholinergic activity stimulate the occurrence of this behavior in ovariectomized female rats (38-42) . In addition, if ovariectomized females are primed with low doses of estradiol which are behaviorally inactive, the stimulatory effect of cholinergic agonists on lordosis is dramatically enhanced (41) .

This facilitation of lordosis by

cholinergic agonists is prevented if females are pretreated with cholinergic receptor blockers.

Conversely, agents that

antagonize cholinergic activity within the central nervous system have been found to reduce the incidence of lordosis in sexually-receptive female rats (39, 41, 43).

708

The minimum physiological dose of estradiol required to activate lordosis is also capable of inducing cholinergic receptors in the hypothalamus (44) and elevating cholinergic enzyme activity in the basal forebrain (45).

In addition,

both of these cholinergic changes are evident within 24 hr after estradiol treatment, the precise time at which the onset of sexual receptivity occurs (44).

These data indicate

that the regulation of cholinergic neurochemicals by estradiol may be one mechanism by which this gonadal steroid controls sexual behavior in the female rat.

The following sec-

tions review some of the effects of gonadal hormones on cholinergic neurochemistry within the brain.

Muscarinic receptors Cholinergic muscarinic binding is influenced by estrogen in brain regions that mediate female sexual behavior.

Estradiol

increases the number of muscarinic binding sites in the medial basal hypothalamus of ovariectomized female rats (44, 46-49).

In microdissected samples, discrete subregions of

the hypothalamus that heavily concentrate estradiol such as the ventromedial hypothalamus and anterior hypothalamus display similar estrogen-induced increases in muscarinic binding (46).

These increases are dependent upon protein synthesis

since they are not observed when a protein-synthesis inhibitor is administered in conjunction with estradiol (44).

Male

rats, which are capable of only low levels of lordosis behavior, fail to display changes in muscarinic binding in the hypothalamus following estrogen treatment (44, 47-49). Olsen et al have suggested that this regulation of muscarinic binding by estrogen in the medial basal hypothalamus is not relevant physiologically since they have detected significant changes in muscarinic binding over the estrous cycle only in

709

the medial preoptic region (48).

However, available data are

inconsistent in the medial preoptic region of ovariectomized female rats where estrogen has been reported to increase (48), decrease (47), or not alter (49) muscarinic binding.

A varie-

ty of dissection procedures and estradiol regimens might explain these discrepancies.

Reliable changes in muscarinic

binding induced by estrogen have not been observed in other brain regions, including the arcuate nucleus, cerebral cortex, striatum, and amygdala (46, 47, 49). Using a novel tritiated muscarinic antagonist, N-methyl-4piperidyl benzilate, Sokolovsky and coworkers (50) reported that ovariectomy increased muscarinic binding in the median hypothalamus of rats and estradiol replacement returned binding to intact estrous levels.

In an earlier study, these

investigators failed to find differences in total muscarinic binding between males and females at three stages of the estrous cycle (51).

However, a competition analysis of high and

low affinity subtypes of the receptor revealed that the percentage of the high affinity subtype was significantly greater in the preoptic area of proestrous females compared to estrous and diestrous females and males.

Furthermore, when

preoptic homogenates from proestrous females were preincubated with estradiol, the percentage of high affinity sites was substantially reduced (52).

These results suggest that est-

rogen may have important membrane-mediated effects on muscarinic receptor subtypes, in addition to those effects mediated by intracellular receptors. Consequently, the medial basal hypothalamus and the medial preoptic area appear to exhibit alterations in muscarinic receptor density following estrogen treatment. Since both of these brain regions have been implicated in the control of lordosis by a variety of techniques, it is likely that this hormonal regulation of muscarinic receptors contributes

710

to the expression of this behavior.

Cholinergic enzymes Choline acetyltransferase. Anatomically the origin of cholinergic fibers that innervate the preoptic and hypothalamic regions has not been identified.

One possible source of in-

nervation, however, is the vast continuum of cholinergic neurons that populate the basal forebrain areas of the medial septum, diagonal band nuclei, substantia ventral palladium.

innominata, and

Although no distinct anatomical connec-

tion with preoptic and hypothalamic neurons has been discovered to date, cholinergic cells of the basal forebrain are particularly intriguing since their neurochemical activity can be influenced by gonadal steroids. Choline acetyltransferase (CAT) is the major enzyme controlling the synthesis of acetylcholine.

A variety of studies

have detailed alterations in CAT activity in the forebrain region following treatment with steroid hormones.

Work from

this laboratory (45) indicates that estradiol increases the activity of CAT in the horizontal limb of the diagonal band of Broca in female rats.

In minimal treatment paradigms,

ovariectomized females are exposed to pure estradiol from Silastic capsules (1 cm) for 6 to 24 hrs and sacrificed 24 hrs after capsule implantation.

This elevation of CAT acti-

vity in the horizontal limb appears to reflect a protein synthesis-dependent increase in the concentration of CAT (53) Interestingly, a portion of the cholinergic neurons in this region have been reported to concentrate estradiol (54). Although male rats fail to show a similar increase in the horizontal limb, estradiol was found to decrease the activity of CAT in the vertical limb of the diagonal band in males

711

(45).

Several adjacent regions, including the bed nucleus,

medial preoptic area, and anterior hypothalamus failed to exhibit any alteration in CAT activity following estrogen treatment of either sex (45).

Estradiol treatment also did

not affect CAT activity in the medial basal hypothalamus (55). Similar elevations of CAT activity in the horizontal limb of female rats were not observed by Muth et al (56) following estrogen treatment; however, difference in tissue dissection and estrogen regimens may account for this discrepancy. These investigators did observe a significant reduction in CAT activity in the vertical limb of the diagonal band in castrate males following testosterone propionate treatment similar to that observed by this laboratory in the male vertical limb following estradiol administration (45).

In intact cycling female rats, the activity of CAT has been reported to be lower in the anterior portion of the hypothalamus during estrus compared to other stages of the cycle (57).

A similar, though insignificant, reduction has been

observed during estrus compared to diestrus in the preoptic suprachiasmatic area of intact female rats (58).

At present,

the relationship between these lower levels of hypothalamic CAT activity during estrus and increases in basal forebrain CAT activity following estradiol treatment of ovariectomized females is not understood. Cholinergic neurons located in the horizontal limb are known to project to various telencephalic structures (59, 60) and two areas that receive such projections, the hippocampus and amygdala, also display small but significant increases in CAT activity after estrogen treatment of female rats (55). Since these areas probably do not contain cholinergic cell bodies, it is possible that the alterations in CAT activity arise from afferent projections from the cholinergic neurons

712

of the horizontal limb.

Another area that receives projec-

tions from the horizontal limb, the cerebral cortex (59, 60), does not appear to display changes in CAT activity over the estrous cycle of female rats (58).

However, James and Kanun-

go (61) have reported age-dependent increases in CAT activity in the cerebral cortex of male rats within four hours after administration of testosterone or estradiol.

Males

sacrificed more than 12 hours after hormone treatment failed to exhibit increases in cortical CAT activity.

This rapid

cortical effect appears to be quite different from the results obtained in the basal forebrain where alterations in CAT activity are observed at least 24 hrs after the initiation of hormone treatment (45). Acetylcholinesterase. Steroid hormones also have been found to affect the activity of the major degradative enzyme of acetylcholine, acetylcholinesterase regions.

(AChE), in several brain

Work from this laboratory has demonstrated that

estradiol increases the activity of AChE in the horizontal limb of the diagonal band in ovariectomized female rats, an effect that parallels the actions of estrogen on CAT activity (45).

Estrogen did not alter AChE activity in the

vertical limb of the diagonal band, the bed nucleus of the stria terminales, the medial preoptic areas, or the anterior hypothalamus.

Nor did estrogen affect AChE activity in any

brain area examined in castrated male rats. In the cycling female rat, Libertun et al (58) reported significantly lower activity of AChE at estrus compared to diestrus in preoptic-suprachiasmatic area.

No stage differen-

ces were detected in the cortex or arcuate-mammillary region. Changes in AChE activity induced by gonadal steroids have been observed in the cerebral cortex of male rats (61, 62),

713

a region that is devoid of cholinergic cell bodies but which receives cholinergic projections from basal forebrain neurons. Generally, gonadectomy was found to decrease the activity of AChE in male cortex while testosterone and estradiol increased enzyme activity 4 hrs after administration.

Estradiol

was also found to increase AChE activity in the cortex and cerebellum of ovariectomized females 4 hrs after treatment (62).

These increases may have been mediated by induction

of intracellular protein synthesis since actinomycin D, a protein synthesis inhibitor, prevented the elevations in AChE activity in both cortex and cerebellum when administered in conjunction with estradiol (62). Using a similar hormone regimen, Iramain et al reported that estradiol increases AChE activity in the amygdala of castrated male rats and in the amygdala and adenohypophysis of ovariectomized females 4 hrs after injection (63, 64) while progesterone alone increased AChE activity in the amygdala, cerebral cortex, and mesencephalon

of ovariectomized female

rats 4 hrs after treatment (65). Several investigators have noted various sex differences in baseline activity and distribution of AChE in certain brain regions.

In this laboratory, the baseline activity of AChE

was found to be significantly higher in the horizontal limb of long-term ovariectomized female rats when compared to long-term castrated males (45).

AChE activity has also been

reported to be significantly higher in the preoptic-suprachiasmatic area of intact female rats compared to intact males (58).

In the gerbil, the distribution of cells that

stain histochemically for AChE in the preoptic-anterior hypothalamic region is clearly different in males and females (66).

Neurons in this preoptic-anterior hypothalamic region

do not stain darkly enough to be classified as cholinergic cells, rather they may represent a population of cells that

714

receive cholinergic innervation.

Female gerbils display a

rounder and more diffuse distribution and appear to possess a lower level of activity in this area compared to males as determined by densitometry.

Gonadectomy reduced the activi-

ty of AChE in both sexes while treatment with testosterone stimulated the activity of the enzyme. While gonadal hormones induce a variety of effects on CAT and AChE activities in a number of brain regions, the functional significance of these changes is unclear at present. Estrogen does not appear to alter the activities of these two enzymes in the preoptic area or medial basal hypothalamus (45,55 ), two regions where muscarinic receptor density changes following estradiol treatment.

On the other hand,

both enzymes display variations over the estrous cycle in the preoptic and anterior hypothalamic regions (57, 58).

In ad-

dition, the existence of reliable sex differences in the activity, distribution, and hormone inducibility of these enzymes (45, 58, 66) may have important implications for the regulation of sexually-dimorphic functions, such as reproductive behavior.

Acetylcholine The effect of gonadal steroids on the actual release of acetylcholine would be of particular value to the understanding of how hormones regulate cholinergic neural activity.

How-

ever, the effects of steroids on the concentration of acetylcholine have not been well documented, partly due to the difficulties inherent in direct measurement of this transmitter. Furthermore, the turnover of acetylcholine which may represent a useful index of cholinergic activity, has proven to be even more difficult to measure.

Using a radioenzymatic

715

technique, Muth, Crowley and Jacobowitz (56) found that castration of male rats elevated the level of acetylcholine in three discrete brain areas; the rostral diagonal band, medial preoptic nucleus, and ventral tegmental area.

These increa-

ses were partially prevented by treatment of castrates with testosterone propionate.

In female rats, treatment with

estradiol benzoate in combination with progesterone reduced the concentration of acetylcholine in periventricular nucleus and ventral tegmental area compared to ovariectomized control females.

There were no changes observed in acetylcholine

level in either sex in a number of other brain areas that concentrate testosterone and estrogen.

The significance of

these results remains to be determined.

Functional implications Although the precise nature of the changes in cholinergic neurochemistry induced by hormones is controversial, the major conclusion that emerges from these experiments is that steroid hormones, acting as neuromodulators, are indeed capable of regulating cholinergic events within the central nervous system.

Perhaps, the variety of observations made by

investigators reflects the dynamic quality of this regulation, which may be unusually sensitive to the subtle effects of time and environment.

In support of this suggestion, a wide

circadian variation in muscarinic binding in rat forebrain has been observed (67).

Additionally, significant strain and

species differences in cholinergic neurochemistry have been demonstrated by a number of studies (68, 69).

And, in a more

specific instance, the activity of the membrane-bound uptake system for choline, a precursor for acetylcholine, has been shown to be influenced by the simple act of handling the animals prior to sacrifice (70).

716

Evidence of a cholinergic component in hormone-dependent functions other than reproductive behavior, such as gonadotropin release (71-74) and scent marking (75), indicates that this particular hormone-dependent interaction may have important implications for the regulation of certain complex systems of physiology and behavior.

Clearly, the ability of

hormones to alter the level of neurotransmission within the brain is a critical feature of central hormone action.

Modulation of steroid action by neurotransmitters Overview Behavioral, environmental and age related factors can influence steroid-dependent processes.

Often, changes in

steroid secretion mediate the effects of these factors.

How-

ever, responses to steroid hormones are not "hard-wired"; and not all changes in steroid-dependent processes are accompanied by changes in circulating steroid levels.

For example,

time of day and season of year can have important effects on the sensitivity of reproductive processes to steroid hormones (76-85).

Shifts in responsiveness to steroids also occur

during early development, puberty and old age (86-91). At present, only a few modulators of steroid action are known. One modulator of progestin action that has been extensively studied is estrogen.

Exposure to estrogen increases the num-

ber of progestin receptors in some central and peripheral tissues (92, 93) and, thereby, plays a major role in regulating the sensitivity of those tissues to progestins.

Some

nucleotides can influence the binding of steroids to receptors and, therefore, might also modulate steroid action (94-98).

In this section, we review recent evidence for ano-

717

ther class of steroid modulator, neurotransmitters. An obvious site for the modulation of steroid action by neurotransmitters is the brain, where steroid target cells receive many neural afferents of diverse origin.

Surprising-

ly, however, much of the research in this area has been conducted with peripheral organs such as pineal, pituitary and uterus. (99-102).

This work has been discussed in detail elsewhere Here, we focus on research concerning the regula-

tion of steroid action by neurotransmitters in the brain. It should be kept in mind that the idea that neurotransmitters can affect steroid action in brain is relatively new and, therefore, there is not as yet incontrovertible evidence for any given neurotransmitter modulating the action of any particular steroid.

However, taken together with findings

with peripheral organs the existing evidence strongly supports the concept that neurotransmitters can modulate target cell sensitivity to steroid hormones.

Dopaminergic regulation of estrogen action The hypothalamus is an important site of steroid action in brain. Interestingly, a number of cell bodies that concentrate radioactive steroid in hypothalamus appear to be surrounded by catecholamine terminals (103-104). One of the first indications that this catecholamine input might influence the sensitivity of the steroid concentrating cells to steroids was provided by experiments with the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA). Marks et al (105) found that infusion of 6-OHDA into a lateral cerebral ventricle of rats caused a decrease in the concentration of a "soluble estrogen binding protein", presumably cytosol estrogen receptors. Later, Thompson et al (106) found a similar effect with the catecholamine synthesis inhibitor α-methy1-p-tyrosine.

718

Acute treatment of female rats with AMPT decreased receptor-mediated 3H estradiol uptake in anterior and basal hypothalamus (by 35-40%).

Estradiol uptake in dorsal hypothala-

mus and in cerebral cortex and the level of radioactivity in plasma were not affected by AMPT. Experiments with dopamine receptor agonists and antagonists indicate that the effects of 6-OHDA and of AMPT on hypothalamic estrogen receptors might be attributable tc changes in dopamine function.

Acute treatment with the dopamine agonist

apomorphine or bromocriptine increased (by 60-90%) receptormediated 3H estradiol uptake in the nuclear fraction of ante^ rior and basal hypothalamus of female rats.

Estradiol uptake

in cerebral cortex and plasma levels of radioactivity were not affected by the agonists (107, 108).

Some caution should

be exercised with regard to dopamine agonists.

Some dopamine

agonists bind to estrogen receptors (but not progestin or androgen receptors) with a relatively high affinity.

For

example, bromocriptine competitively inhibits 3H estradiol binding to hypothalamic estrogen receptors in vitro with a Ki of about 25 uM.

Also, some agonists,including bromocrip-

tine, might induce estrogen-inducible proteins such as progestin receptors through the estrogen receptor system although this has been tested only in gonadally intact immature rats thus far (109).

Thus, it is conceivable that dopamine

agonists might affect estradiol uptake in hypothalamus through an interaction with estrogen receptors rather than through dopamine receptors.

However, pretreatment with the

dopamine antagonist perphenazine blocked the agonist-induced increase in estradiol uptake in hypothalamus (107, 108). Phenothiazines such as perphenazine do not bind to estrogen receptors, at least up to a drug to 3H estradiol ratio of 1:100,000 (110, 111).

Thus, it seems likely that the ago-

nist-induced increase in estradiol uptake in hypothalamus and

719

the blockade of that effect by perphenazine is attributable to effects on dopamine rather than estrogen receptors. Although the above findings indicate that dopamine might influence estrogen action in hypothalamus, two additional findings should be mentioned.

First, although dopamine ago-

nists dramatically increase 3H estradiol uptake in hypothalamus of female rats, they do not affect estradiol uptake in male rats (107, 108).

Thus, there might be a strong sex dif-

ference with regard to dopamine modulation of estrogen action.

Secondly, whereas Thompson et al (106) found that AMPT

decreased receptor-mediated estradiol uptake in female rat hypothalamus, Carrillo et al (101) found no effect of this drug on hypothalamic estrogen receptors.

One potentially

important methodological difference between the two studies is that in the experiment of Thompson et al (106) AMPT was injected just two hours before 3H estradiol administration (a design similar to that used in experiments with agonists and antagonists) but in the experiment of Carrillo et al (101) the rats were injected for two days with AMPT and estrogen receptors were measured on day three.

It seems likely

that with processes as dynamic as those related to neurotransmission numerous factors, including sex and time course of drug treatment, might interact to determine the effects of neurotransmitter on steroid action.

Noradrenergic regulation of progestin action Interest in the question of whether noradrenergic transmission affects steroid action in brain grew from work with the lordosis response of female guinea pigs.

Lordosis in guinea

pigs, as in rats, is strictly dependent on the synergistic

720 action of ovarian estrogen and progestin (112-114).

However,

despite treatment with an optimal hormone regimen ovariectomized guinea pigs do not display lordosis when norepinephrine is depleted in brain or when αi-noradrenergic receptors are blocked (115-118).

Stimulation of αΐ-receptors with Cloni-

dine potentiates steroid-induced lordosis responding (115, 117, 119). The hypothalamus is thought to be the primary site where estradiol and progesterone act to induce lordosis in guinea pigs; small amounts of crystalline hormone implanted into this area induces lordosis but implants into other areas do not (120, 121).

To determine whether the noradrenergic

innervation of the hypothalamus might affect lordosis responding by modulating the sensitivity of hypothalamic cells to steroid hormones, steroid receptors were assayed after drug treatments that inhibit or potentiate lordosis. In initial experiments (116, 118), cytosol progestin receptors were measured 0.5 - 12 hours after drug injection. The animals were primed with estradiol 24-34 hrs prior to drug injection to approximate as closely as possible experiments with lordosis. Under these conditions, blockade of αχ-receptors (with phenoxybenzamine or prazosin) or inhibition of norepinephrine synthesis (with the dopamine-ß-hydroxylase inhibitor U-14, 624) decreased by about 30% the concentration of cytosol progestin receptors in hypothalamus. Drug treatment had no effect on progestin receptors in other brain areas, including preoptic area, midbrain, and cerebral cortex. Progestin receptor concentration was significantly decreased in hypothalamus by 4 hrs and remained depressed for at least 12 hrs after α^-receptor blockade. Direct stimulation of a^-receptors by injection of Clonidine reversed the effects of norepinephrine synthesis inhibition and restored cytosol progestin receptor concentration in hypothalamus to control levels.

721

Interestingly, recent experiments indicate that it might not be the total number of progestin receptors that noradrenergic transmission affects in hypothalamus.

Whereas interference

with noradrenergic transmission decreases the number of cytosol receptors, the number of nuclear progestin receptors in hypothalamus is increased (122).

This increase in nuc-

lear receptors does not appear to be attributable to a drug-induced release of adrenal progesterone.

First, no

drug-induced increase in plasma progesterone was detected by radioimmunoassay (116, 122) and, secondly, the effect on progestin receptors was limited to hypothalamus (116, 118, 122).

Adrenal progesterone, on the other hand, would be

expected to decrease the number of cytosol progestin receptors throughout the brain.

Also, the drugs themselves do

not bind to progestin receptors (116, 122), therefore, it seems unlikely that the shift in the distribution of the receptors is due to a direct interaction of the drugs with the receptors.

Rather, it seems that noradrenergic function

in some way influences where hypothalamic progestin receptors partition upon cell disruption.

Interference with noradre-

nergic transmission decreases the number of receptors in cytosol and increases the number of receptors found in nuclear preparations.

There are two ways this effect of nor-

adrenergic transmission might be described, depending on whether unoccupied receptors normally reside in cytoplasm or nucleus of target cells. In the past, it has been considered almost dogma that most, if not all, unoccupied steroid receptors reside in the cytoplasm of steroid target cells.

According to this model,

the receptors measured in cytosol are cytoplasmic receptors. When interpreted within this framework, the finding that interference with noradrenergic transmission decreases the

722 number of receptors in cytosol and increases the number in nuclear preparations suggests that noradrenergic transmission affects the distribution of unoccupied receptors within hypothalamic cells.

Depressed noradrenergic function favors a

shift in the distribution of the receptors out of the cytoplasm and into the nuclear compartment. Recently, however, the idea that unoccupied receptors reside in the cytoplasm has been challenged.

Based on new evidence,

it has been argued that virtually all unoccupied steroid receptors are in the cell nucleus (123, 124).

In the absence

of steroid, according to this model, the loose association of the receptor with nuclear elements causes the receptor to partition into the cytosol when the cell is disrupted. When interpreted within this framework, the finding that interference with noradrenergic transmission decreases the number of receptors in cytosol and increases the number in nuclear preparations suggests that noradrenergic transmission affects the strength of the association of unoccupied receptors with nuclear elements.

A reduction in noradrenergic

transmission favors a stronger association between the unoccupied receptor and nuclear elements; therefore, fewer receptors are extracted into cytosol and more receptors are seen in nuclear preparations. At present, both interpretations of the drug effects on unoccupied progestin receptors are viable and until additional information is available concerning the location of unoccupied receptors within target cells, it will be difficult to choose between these alternatives.

Also, at present we can

say little concerning differences in the functional consequences of these alternatives.

However, regardless of whe -

ther noradrenergic transmission affects the distribution of progestin receptors or their strength of association with nuclear elements it remains clear that some aspect of pro-

723 gestin receptor dynamics in hypothalamic cells is sensitive to noradrenergic function.

Other neurotransmitters and steroids Although most research on neurotransmitter modulation of steroid action in brain have been concerned with dopamine effects on estrogen action and noradrenergic effects on progestin action, there have been some experiments with other neurotransmitters and other steroids.

For example, Clark

et al (125) recently found that α^-noradrenergic receptor blockade with prazosin decreases the concentration of nuclear estrogen receptors in hypothalamus and preoptic area of estradiol treated female guinea pigs suggesting that noradrenergic transmission might modulate estrogen action in those areas. The sensitivity of brain regions to adrenal steroids might also be influenced by neural input.

For example, Feldman and

associates have postulated an important role for extrahypothalamic sites in modulating hypothalamic sensitivity to glucocorticoids in rat (126-130) .

Complete or posterior hypo-

thalamic differentiation, dorsal hippocampectomy, or dorsal fornix section decreased the inhibitory effect of dexamethasone on the adrenal secretory response to ether stress.

On

the other hand, electrolytic lesions of the mammillary peduncle or the medial forebrain bundle or bilateral infusion of 6-OHDA into the medial forebrain bundle enhanced the inhibitory effect of dexamethasone (126-130).

Various other

CNS operations did not affect the feedback effect of dexathasone in rats (126-130).

Experiments with species other

than rats also indicate a possible role for extrahypothalamic sites in modulating hypothalamic sensitivity to glucocorticoids.

Stith et al found that electrical stimulation

724

of the dorsal hippocampus, but not amygdala, increased the uptake and nuclear binding of the glucocorticoid 3H hydrocortisone in hypothalamus of male and female cats (131).

Also,

infusion of 6-OHDA into the 3rd ventricle of adrenalectomized dogs has been shown to decrease the concentration of glucocorticoid receptors in hypothalamus with no effect in hippocampus (126) . There is much that is not yet known about the modulation of steroid action by neurotransmitters.

We know almost nothing

concerning the mechanism(s) of neurotransmitter effects on steroid action although there has been speculation in this area (100,106,133).

We also know little about the functional

consequences of neurotransmitter-induced changes in sensitivity to steroids.

There are, however, numerous known phenomena

that might be relatable to the operation of such a mechanism. For example, neurotransmitter-induced changes in sensitivity to steroids might underlie circadian and seasonal changes in behavioral and endocrine responsiveness to steroids and, in addition, shifts in sensitivity to steroids that occur during early development, puberty and old age (76-91,134,135).

From

the finding that neurotransmitters can influence the action of steroids in postsynaptic cells, it also is not a large conceptual jump to envisioning this as an important mechanism by which environmental, behavioral and perhaps emotional events can rapidly and selectively influence steroid-dependent processes. Conclusions In this chapter we have presented a view of steroid hormones and brain function from the perspective of the interaction of various "neuroactive" substances (sometimes called, collectively, "neuromodulators") on brain function, including

725

behavior and neuroendocrine responses.

The picture which

is beginning to emerge is of multiple interactions - of steroids upon neuromodulatory processes and neurotransmitters, and of neurotransmitters upon responsiveness to steroid hormones.

These interactions are often highly specialized and

localized within restricted regions of the brain.

Their

occurrence is also frequently dependent on the stage of development, with permanent effects occurring during early development and qualitatively different, reversible effects occurring in later life. Many of the effects can be related to intracellular steroid receptors, which are produced by neurons in restricted brain regions.

These receptors have characteristics which make

them similar, if not identical, to receptors in other parts of the body.

One of the challenges for future research is to

understand the features of cellular differentiation which explain the diverse effects of steroids, acting via apparently identical receptors, on different tissues and different brain regions.

Another important question for future re-

search is to elucidate the mechanisms by which neurotransmitter activation can modify steroid receptor number or cellular uptake and cell nuclear binding of steroid hormones.

Acknowledgments Research in the author's laboratory is supported by NIH grant NS 07080, by fellowships HD 06258 (Gary Dohanich) and NS 06966 (to Bruce Nock) and by an institutional grant RF 81062 from the Rockefeller Foundation for research in reproductive biology.

We wish to thank Ms. Inna Perlin for editorial

assistance.

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104.

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118.

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(1981).

THE STEROID RECEPTOR OF ACHLYA

AMBISEXUALIS

Robert M. Riehl, David O. Toft D e p a r t m e n t of Cell B i o l o g y , M a y o Rochester, Minnesota, USA

Clinic,

Introduction T h e m e c h a n i s m of a c t i o n of s t e r o i d h o r m o n e s despite

the a c c u m u l a t i o n of s t u d i e s o n the

involved

remains

in the m o l e c u l a r e v e n t s a s s o c i a t e d

steroid-induced

responses.

Information

unclear

individual

steps

with

r e l e v a n t to the

s t r u c t u r e , s t a b i l i z a t i o n and t r a n s l o c a t i o n of

cytosolic

r e c e p t o r s and some of the b i o c h e m i c a l e v e n t s a s s o c i a t e d

with

nuclear steroid-receptor

c o m p l e x e s has b e e n o b t a i n e d

from a

n u m b e r of m o d e l s y s t e m s .

T h e s e c l a s s i c a l m o d e l s are

mainly

c o m p o s e d of t a r g e t t i s s u e s a n d / o r c e l l s from w h i c h r e p r e s e n t o n l y a small of k n o w n s t e r o i d r e s p o n s i v e animal

kingdoms

(1-3).

systems may provide defining

vertebrates

f r a c t i o n of the total s y s t e m s among

population

the p l a n t

Studies utilizing other

and

nonclassical

new i n f o r m a t i o n or insight u s e f u l

the m e c h a n i s m of a c t i o n of s t e r o i d h o r m o n e s

g e n e r a l and b r o a d e n o u r v i e w of the b i o l o g i c a l s t e r o i d a l c o m p o u n d s as i n t e r c e l l u l a r belonging

possess steroid binding proteins a m b i s e x u a l is, is u n d e r study s u b j e c t of this

role of

communication

In this r e g a r d , r e c e n t s t u d i e s have s h o w n that eukaryotic microorganisms

molecules.

some

to the k i n g d o m

(4-8).

in in

Fungi

One of t h e s e ,

Achlya

in our l a b o r a t o r y and is the

chapter.

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

734 Background A brief description of the relevant aspects of the biology of Achlya will be presented here and the reader is urged to consult the more extensive reviews to be found in the list of references (3,9,10).

Notably, Achlya is unique in its

ability to synthesize and respond to steroid hormones.

It is

the only microbe known in which sexual reproduction is controlled by steroid hormones. Reproduction in Achlya is accomplished by either asexual or sexual modes of sporulation.

The asexual mode is thought to

be independent of the steroid hormones and occurs predominantly in response to depletion of exogenous nutrient supplies.

Germination and culture of the asexual spores

(zoospores) is a common laboratory technique for obtaining Achlya cultures in which the process of sexual reproduction can be studied.

Spore germination is evidenced by the

outgrowth of a small tube of protoplasm encased by the plasmalemma and cell wall.

The germ tube continues to

elongate and form apical branches that also grow and branch to ultimately form a meshwork of interconnected tubes called hyphae (somatic cell type) which are collectively known as a mycelium.

Sexual reproduction occurs when male and female

mycelia are in close physical proximity.

Female hyphae

synthesize and release the steroid antheridiol (Figure 1) into the environment from which it is detected by male hyphae that respond by the growth and differentiation of the male sex organs known as antheridial branches and by the production and release of a second steroid, oogoniol. Oogoniol acts upon the female hyphae inducing the formation of the egg-bearing structures or oogonia.

Chemotropic growth

of the male antheridial branches toward the oogonia along a concentration gradient of antheridiol results in contact of the two sex organs followed by syngamy and production of oospores.

The induction of antheridial branches can also be

735 observed by the addition of exogenous synthetic (and chemical analogs) to the culture medium.

antheridiol Within two

hours following exposure to antherdiol, male hyphae produce thin, contorted subapical branches that are readily observable with ordinary light microscopy

(Figure

The following characteristics confer exceptional

2).

advantages

to the Achlya steroid hormone system compared to the classical vertebrate systems:

1) The somatic cells

(hyphae)

of Achlya are a tubular coenocytium, consequently the entire mycelium

is composed of a single "cell" type and

the "target tissue".

represents

2) One facet of the hormone response is

a rapid and readily observable morphological

differentiation.

3) Male mycelia do not synthesize antheridiol;

therefore,

when in axenic culture, there is absence of previous to the hormone.

4) Achlya possess a relatively

exposure

simple

genetic endowment having three sets of chromosomes and a genome size that is only a few times larger than that of Ε. coli .

5) Cultivation of mycelia

in the laboratory can be

accomplished with media of simple composition laboratory glassware and on the bench

in ordinary

top.

The role of steroid hormones in the process of sexual reproduction in Achlya has been well studied

(3,9,10).

However, the mechanism of action of antheridiol strain has received

the majority of experimental

in the male attention

because the chemical synthesis and availability of oogoniol has only been of recent occurrence pure antheridiol has been available

(11).

Since

chemically

(12), some of the

biochemical actions of this steroid have been studied

(10).

The development of antheridial branches has been shown to be dependent on continued RNA and protein synthesis

(13,14).

The synthesis of RNA (14,15) and levels of specific activities of RNA polymerase II (16) and cellulase

(13,17)

are increased by antheridiol.

indirect

studies, it was suggested

From the results of

that chemorecognition of

736 antheridiol could be mediated through a cytosolic receptor in a similar manner to the steroid hormone receptor systems of vertebrates (18). In our studies, we have used a tritium-labeled analogue of antheridiol, 7-deoxy-7-dihydro-antheridiol biologically active.

(7dA), that is

Our objectives were to use the

tritium-labeled ligand to detect the binding protein (7) and characterize the biological significance of binding by analysis of the saturability, specificity and affinity. Following identification of the receptor, the physicochemical properties of the macromolecule could be determined

(8) and

compared with those of steroid receptors in higher organisms.

Methods Chemical Nonradioactive antheridiol and its congeners were supplied by Trevor C. McMorris, Department of Chemistry, University of California-San Diego.

The radioactive analog of antheridiol,

[1,2- 3 H]-7-deoxy-7-dihydro-antheridiol

([ 3 H]-7dA,

specific activity = 40 Ci/mmol) was prepared as described elsewhere (Meyer et al., in preparation). structures of antheridiol and

The chemical

3

[ n]-7dA are shown in

Figure 1. Cell culture

Achlya ambisexualis Raper strains E87 (male) and 734 (female) were cultured as previously described

(7).

Asexual spores

(19) were germinated in PYG medium (20) for 18-24 hrs at 29°C and subsequently innoculated into 2 liters of Ml medium (14)

737 HO

Antheridiol HO

[ 1 , 2 - 3 H ]-7-deoxy-7-dihydro-Antheridiol Figure 1. Chemical Structures of Antheridiol and the Tritium-labeled Analog Employed for Binding Studies.

for growth in aerated suspension culture at 24°C for various time periods.

Mycelia were routinely harvested

for study

after 2-3 days of incubation except where described d i f ferently.

Preparation of fungal cytosol

The techniques for mycelial harvest and disruption are described

in detail elsewhere

(8).

Because preliminary

738 s t u d i e s r e v e a l e d that the a n t h e r i d i o l unstable

in v i t r o , c o n s i d e r a b l e

to o p t i m i z e employed

binding protein

experimentation was

the c o n d i t i o n s and c o m p o s i t i o n of

for h o m o g e n i z a t i o n

optimum binding capacity.

was

performed

buffers

and p r e p a r a t i o n of c y t o s o l P r e s e r v a t i o n of c y t o s o l i c

binding

a c t i v i t y was h i g h l y d e p e n d e n t on the c o n c e n t r a t i o n s of molybdate (8).

and m e r c a p t o e t h a n o l

in the h o m o g e n i z a t i o n

of 25 m M K 2 H P 0 4 , 25 m M

10 m M 4 - m o r p h o l i n e e t h a n e s u l f o n i c

using

KH2P04,

a c i d , 25 m M N a 2 M o 0 4

10 m M 2 - m e r c a p t o e t h a n o l , pH 7.5 at

and

0°C.

The m y c e l i a w e r e h a r v e s t e d by f i l t r a t i o n , m i n c e d

with

s c i s s o r s and a d d e d to PM3 b u f f e r at a ratio of 1:4 wet to v o l u m e . vessels were

immersed

in an i c e / m e t h a n o l

homogenate was filtered

through Miracloth

the f i l t r a t e c e n t r i f u g e d at 2 5 0 , 0 0 0 x g

slush.

resultant cytosol

The

for 1 - 2 hrs at (0.45

a d j u s t e d to pH 7.0 at 0 ° C .

mycelia were frozen

0°C.

μ) and

f l u o r i d e , pH 7.5 at 0 ° C .

e i t h e r by u l t r a f i l t r a t i o n (Millipore

For some

phenylmethylanalytical

a c t i v i t y was

using an i m m e r s i b l e

Corp.) with a molecular weight

concentrated probe

limit of 1 0 , 0 0 0

by p r e c i p i t a t i o n w i t h a m m o n i u m s u l f a t e at 95% of (8).

The a m m o n i u m s u l f a t e p e l l e t was r e s u s p e n d e d

c o n s i s t i n g of 10 m M N a 2 M o 0 4 propanesulfonic studies.

in

of

2 - m e r c a p t o e t h a n o l , 50 m M

a c i d , a n d 0.5 mM

the c y t o s o l i c b i n d i n g

the

to a p o w d e r

a b l e n d e r cup and t h a w e d to 0°C in Τ b u f f e r c o n s i s t i n g 4-morpholinepropanesulfonic

and

Alternatively,

in l i q u i d n i t r o g e n , g r o u n d

100 m M KCl, 25 m M N a 2 M o 0 4 , 10 m M

ten

while

(Calbiochem)

The s u p e r n a t a n t was c l a r i f i e d by f i l t r a t i o n

sulfonyl

weight

D i s r u p t i o n of h y p h a e w a s a c c o m p l i s h e d by four

s e c o n d b u r s t s of a p o l y t r o n at the m a x i m u m s e t t i n g

procedures,

sodium

buffer

Except where noted, most studies were performed

PM3 b u f f e r c o n s i s t i n g

with

and 10 m M

a c i d , pH 7.0 at 0°C

or

saturation in a

buffer

4-morpholine-

(MoM b u f f e r ) for

further

739 Analytical

procedures

Radioligand binding assay. All steroid stock solutions were stored at -20°C in redistilled ethanol and added to 12 χ 75 mm disposable culture tubes immediately prior to addition of sample.

The final concentration of ethanol

volume was no greater than 1%.

in the assay

Incubation was routinely

performed at 0°C in an ice/water slush for the time periods and at the steroid concentrations described the figures and tables.

in the legends to

Unbound steroid was adsorbed

to

dextran-coated charcoal over a 4-minute period after the addition of a 200 μΐ aliquot of a dextran-coated suspension

(5% Norit-A, 0.5% Dextran T-70, 0.01% N a N 3 ) for

each ml of incubation volume and pelleted by at 2000xg for 4 m i n . steroid was measured

The amount of bound

centrifugation

tritium-labeled

in aliquots of the supernatant by liquid

scintillation spectrophotometry

in Beckman Ready

cocktail at an efficiency of 33% determined by standardization. amount of

charcoal

Solv-HP

internal

Nonspecific binding was defined as the

[ 3 H ] - 7 d A bound

in the presence of a fifty-fold

excess of unlabeled antheridiol.

Specific binding was

calculated by subtracting the amount of nonspecific from the total amount of unlabeled

3

[ H ] - 7 d A bound

binding

in the absence of

antheridiol.

Determination of the steroid specificity of [ 3 H ] - 7 d A binding. The amount of

[ 3 H ] - 7 d A bound

in the presence of

increasing concentrations of nonradioactive steroids was determined

to assess their relative binding affinities

the receptor. resuspended

Ammonium sulfate-precipitated

in MoM buffer pH 7.0 at 0°C and 1 ml aliquots

containing 280-320 yg protein were incubated of

for

receptor was

in the presence

[ 3 H ] - 7 d A at a final concentration of 2.5 χ 1 0 - 9

Nonradioactive steroids were added at final ranging from 1 χ 1 0 - 1 1 of specifically bound

to 1 χ 10~ 5 M.

M.

concentrations

The amount

[ 3 H ] - 7 d A in the presence of each

740

F i g u r e 2. S t e r o i d - i n d u c e d r e s p o n s e of A c h l y a a m b i s e x u a l i s male. P h o t o g r a p h s (45x) w e r e t a k e n b e f o r e (left) and 1.5 h a f t e r (right) e x p o s u r e to 1.2 nM [^H]-7dA. c o n c e n t r a t i o n of c o m p e t i t o r i n c u b a t i o n at 0 ° C .

Relative

was m e a s u r e d a f t e r

1.5 h of

binding affinities

(RBA)

c a l c u l a t e d by d e t e r m i n a t i o n s of the c o n c e n t r a t i o n competitor

that r e d u c e d s p e c i f i c

[3H]-7dA binding

the a m o u n t b o u n d

in the a b s e n c e of c o m p e t i t o r

according

equation

to the C

RBA = c

Q-5

0.5

7 d A

to 50% of

(Cg 5)

.

competitor

Sucrose density gradient sedimentation. gradients,

were

of

the c y t o s o l was c o n c e n t r a t e d

s i x - f o l d by u l t r a f i l t r a t i o n

Prior to layering approximately

using an i m m e r s i b l e

( M i l l i p o r e Corp.) w i t h a m o l e c u l a r w e i g h t

[3H]-7dA was then a d d e d to the c y t o s o l at a concentration

probe

limit of

10,000.

final

of 4 nM in the a b s e n c e and p r e s e n c e of 200 nM

on

741

antheridiol. or

S u c r o s e was d i s s o l v e d

in PM3 b u f f e r at pH

in the s a m e b u f f e r w i t h o u t N a j M o C ^ but c o n t a i n i n g

KCl and l i n e a r 5-20% w / w g r a d i e n t s concentrated cytosol proteins were 150,000xg

formed.

Aliquots

(0.3 m l = 227 pg p r o t e i n )

and

7.0

1 Μ of

reference

l a y e r e d o n 4.5 ml g r a d i e n t s and c e n t r i f u g e d

for 16 hrs at 2 ° C .

F r a c t i o n s were c o l l e c t e d

the tube b o t t o m w i t h a n ISCO f r a c t i o n c o l l e c t o r and r a d i o a c t i v i t y d e t e r m i n e d by l i q u i d

at

from

the

scintillation

spectrophotometry.

Results The morphological exogenous

r e s p o n s e of an A c h l y a m a l e m y c e l i u m

3

[ H ] - 7 d A is i l l u s t r a t e d

in Figure 2.

The

p h o t o g r a p h at the left was t a k e n p r i o r to e x p o s u r e hormone. extensive Additional

to 1.2 χ 1 0 ~ 9 Μ

lateral b r a n c h

[ 3 H ] - 7 d A and shows

formation

steroid.

s t u d i e s have s h o w n this r a d i o l a b e l e d a n a l o g u e a c t i v e at c o n c e n t r a t i o n s

10-12 μ .

it is not as p o t e n t as the

While

(22), these r e s u l t s

a very suitable

i n d i c a t e that

indicated

natural [3H]7dA should

in A c h l y a c y t o s o l was v e r y low and v a r i a b l e . a g e n t such as

have b e e n s h o w n to be i m p o r t a n t s t a b i l i z i n g steroid receptors

for the A c h l y a s y s t e m as w e l l .

mercaptoethanol

agents

for

Total e x c l u s i o n of

sites.

in the b u f f e r s o l u t i o n s

true

sodium

from the m e t h o d d e s c r i b e d

p r e p a r a t i o n of the f u n g a l c y t o s o l

resulted

This requirement

is i l l u s t r a t e d

we

activity.

in g e n e r a l , and this was found to be

m o l y b d a t e and m e r c a p t o e t h a n o l binding

activity

Therefore,

to o p t i m i z e b i n d i n g

S o d i u m m o l y b d a t e and a r e d u c i n g

be

analysis.

that s t e r o i d b i n d i n g

t e s t e d a v a r i e t y of c o n d i t i o n s

to

as low as 2 χ

l i g a n d for r e c e p t o r d e t e c t i o n and

initial s t u d i e s

detectable

the

i n d u c e d by the

be b i o l o g i c a l l y

Our

to

The p h o t o g r a p h at the r i g h t w a s taken a f t e r 2 h o u r s

of e x p o s u r e

steroid

to

in an a b s e n c e for

in F i g u r e

Na2Mo04 3 which

for of

742

F i g u r e 3. E f f e c t of S o d i u m M o l y b d a t e o n S p e c i f i c B i n d i n g of [^H]-7dA. The h o m o g e n a t e (· ·) w a s p r e p a r e d as d e s c r i b e d in E x p e r i m e n t a l P r o c e d u r e s and a d j u s t e d to the final c o n c e n t r a t i o n s of s o d i u m m o l y b d a t e i n d i c a t e d on the a b s c i s s a . A l i q u o t s of e a c h c y t o s o l (1 m l = 595 g p r o t e i n ) w e r e i n c u b a t e d for 1.5 h at 0°C in the p r e s e n c e of 2 χ Μ [-^H]-7dA. S p e c i f i c b i n d i n g is s h o w n as a p e r c e n t a g e of the m a x i m u m s p e c i f i c b i n d i n g m e a s u r e d in the p r e s e n c e of the o p t i m u m c o n c e n t r a t i o n of s o d i u m m o l y b d a t e . The m e a n v a l u e s (N = 2) for the m a x i m u m level of s p e c i f i c b i n d i n g in the c y t o s o l was 1 5 , 3 0 3 d p m / m l . depicts

the r e s u l t s of a d j u s t i n g

r a n g e of Na2MoC>4 c o n c e n t r a t i o n s the c y t o s o l . to an i n c r e a s e 25 m M Na2MoC>4. concentration

Specific binding

the h o m o g e n a t e

increased

in Na 2 MoC>4 c o n c e n t r a t i o n Further resulted

to c o n t a i n a

p r i o r to p r e p a r a t i o n

increases

in d i r e c t

in the Na2MoC>4

in d e c r e a s e s

in s p e c i f i c b i n d i n g .

of 5 m M , 10 m M a n d 20 m M m e r c a p t o e t h a n o l l e v e l s of c y t o s o l i c b i n d i n g

98% of the m a x i m u m The total a m o u n t of

relation

to an o p t i m u m of

c o n t r a s t to the d r a m a t i c e f f e c t of Na 2 MoC>4, the increased

of

level, respectively

In

addition

resulted

in

that w e r e 9 3 % , 100% (data not

shown).

[3H]-7dA b o u n d at a c o n s t a n t

protein

and

c o n c e n t r a t i o n was d e p e n d e n t on the pH of the b u f f e r

solution

b u t n o n s p e c i f i c b i n d i n g was i n d e p e n d e n t of the p H .

Maximum

l e v e l s of s p e c i f i c b i n d i n g were m e a s u r e d at pH

7.0.

743 Cytosolic specific binding expressed as a percentage of the maximum specific binding measured at pH 7.0, was 41%, 78%, 97% and 66%, at pH 6.0, 6.5, 7.5 and 8.0, respectively.

Figure 4A shows the results of incubating cytosol for 1 h at 0°C with a range of

[ 3 H ] - 7 d A concentrations

(0.1-4.5 nM) .

The level of nonspecific binding under these conditions was approximately

11% of the total

[ 3 H ] - 7 d A concentration

ranged for 26-66% of the total amount bound.

and

Saturation of

the specific binding sites was achieved at approximately 3 nM with a plateau of specific binding at 1200 fmoles/mg protein.

Scatchard

approximately

(23) analysis of the

specific binding data in Figure 4A is shown in Figure 4B.

ui m oc < TJ

ο m < •o

0

1

2

3

4

[ 3 H]7dA Concentration (nM)

0.1

0.2

0.3

[ 3 H ]7dA BOUND (nM)

Figure 4. Amount of [ 3 H ] - 7 d A Bound as a Function of the [ 3 H ] - 7 d A Concentration in A. ambisexualis E87 Cytosol. Bound steroid was determined as described in Materials and Methods. (A) The amount of [ 3 H ] - 7 d A bound in the absence (o o) and presence (Δ Δ ) of a 50-fold excess of unlabeled antheridiol and the amount specifically bound (· ·) calculated as the difference between the two. (B) Scatchard analysis of the specific binding data.

744 The m e a n e q u i l i b r i u m d i s s o c i a t i o n e x p e r i m e n t s w a s 0.73 nM +

b i n d i n g c a p a c i t i e s of 1 1 0 0 - 2 0 0 0 Specific binding

[^H]-7dA w a s d e t e c t e d not r e s p o n d

fmoles/mg

indicating

A. ambisexualis.

to a n t h e r i d i o l ,

these r e s u l t s

site has "target tissue"

c h r o m a t o g r a p h y of

site

is

of

from c e l l s of Since f e m a l e s i n d i c a t e that

specificity.

[3H]-7dA extracted

Fraction Number

treatment

that the binding

in c y t o s o l s p r e p a r e d

maximum

protein.

In a d d i t i o n , no s p e c i f i c b i n d i n g

the female s t r a i n 734 of binding

from 4 s e p a r a t e

in the c y t o s o l w a s d e s t r o y e d by

w i t h p r o n a s e or t r y p s i n proteinaceous.

constant

.17 S . E . w i t h a range of

from m a l e

Thin

do

the

layer

cytosol

Fraction Number

F i g u r e 5. S u c r o s e g r a d i e n t a n a l y s i s of [^H]-7dA B i n d i n g in Fungal Cytosol. Panel A: S e d i m e n t a t i o n p r o f i l e Γη low i o n i c strength gradients containing molybdate. Panel B: S e d i m e n t a t i o n p r o f i l e in h i g h ionic s t r e n g t h g r a d i e n t s without molybdate. S y m b o l s for b o t h P a n e l s A and B: (· ·) p r o f i l e in the p r e s e n c e of [^H]-7dA a l o n e , (o o) p r o f i l e of [^H]-7dA in the p r e s e n c e of a 5 0 - f o l d e x c e s s of unlabeled antheridiol. Arrows denote fractions containing the peak level of r a d i o a c t i v i t y from [ l ^ C ] - o v a l b u m i n (3.6S) a n d from [ 1 4 C ] - a l d o l a s e ( 7 . 9 S ) . Sedimentation was f r o m left to r i g h t .

745 a f t e r 1 to 6 h o u r s of i n c u b a t i o n at 0 - 2 ° C or a f t e r 1 h o u r 2 3 ° C r e v e a l e d that no m e t a b o l i s m of

at

3

[ H]-7dA occurs

iη v i t r o . The s e d i m e n t a t i o n p r o f i l e of density gradients

[3H]-7dA binding

is s h o w n in F i g u r e 5.

gradients containing a p p e a r e d as a peak

sodium molybdate, specific

in the 8S r e g i o n

a b s e n c e of s o d i u m m o l y b d a t e

and

(Panel A ) .

Additional experiments

in the low salt g r a d i e n t .

test s a m p l e s marked

binding In the

in the 3.6S

region

(data not shown)

that a n 8S form is o b s e r v e d w h e n m o l y b d a t e b u t not

salt

in the p r e s e n c e of 1 Μ K C l ,

s p e c i f i c b i n d i n g d i s t r i b u t e d as a peak (Panel B) .

in s u c r o s e

In low

revealed

is in the

sample,

It w a s not p o s s i b l e

to

in the total a b s e n c e of m o l y b d a t e due to the

i n s t a b i l i t y of the b i n d i n g c o m p o n e n t .

We h a v e

also

f o u n d that the b o u n d s t e r o i d s e d i m e n t s at 3.6S in high gradients

(1 Μ KCl)

in the p r e s e n c e of 10 m M

salt

sodium

molybdate. The a m o u n t of

[3H]-7dA specifically bound

in the

of v a r i o u s c o n c e n t r a t i o n s of n o n r a d i o a c t i v e in Figure

6.

steroids

binding

for the b i n d i n g

7dA = 1 . 0

(by

(natural p h e r o m o n e )

22R,23S-antheridiol

= 0.1 a n d 7 d A - 3 - a c e t a t e = 0 . 0 3 .

i n h i b i t i o n of

[ H ] - 7 d A b i n d i n g by o t h e r

(e.g. C o r t i s o l a n d 7 d A - 3 - a c e t a t e ) , The r e a s o n

Some

at

steroids

at c o n c e n t r a t i o n s w h e r e

competition occurred, were consistently

found to e n h a n c e

for this is u n k n o w n .

is that these s t e r o i d s m a y

c o n c e n t r a t i o n of

A

3

c o n c e n t r a t i o n s g r e a t e r t h a n 1 χ 10~6 m .

possibility

in

= 7.5;

s t e r o i d s and s t e r o i d h o r m o n e s was d e t e c t e d o n l y

[3H]-7dA.

site.

definition);

22S,23R-antheridiol

b i n d i n g of

and

a f f i n i t i e s , c a l c u l a t e d as d e s c r i b e d

M e t h o d s w e r e as f o l l o w s :

significant

is s h o w n

Of the c o m p o u n d s t e s t e d , o n l y a n t h e r i d i o l

its a n a l o g s e x h i b i t e d high a f f i n i t y The r e l a t i v e

presence

[ 3 H ] - 7 d A by r e d u c i n g

increase

the

no

the

One

effective

the a s s o c i a t i o n

of

746 this h y d r o p h o b i c c o m p o u n d w i t h v a r i o u s c o m p o n e n t s of assay

the

system.

When eluted

from D E A E - S e p h a d e x

chromatography

l i n e a r c o n c e n t r a t i o n g r a d i e n t of K C l , b o u n d as a s i n g l e peak at a p p r o x i m a t e l y of c y t o s o l i c p r o t e i n s

columns with a

[3H]-7dA

0.24 Μ K C l .

The

elutes

majority

80%) do not bind t i g h t l y to D E A E

a n d flow t h r o u g h the c o l u m n or are r e m o v e d by w a s h i n g

with

120

10-11

10-10

10-9

10"8

ΙΟ"7

10"6

10-5

STEROID CONCENTRATION (M) F i g u r e 6. S p e c i f i c i t y of [3n]-7dA B i n d i n g . A l i q u o t s of the ( N H 4 ) 2 S O 4 - p r e c i p i t a t e d r e c e p t o r in MoM b u f f e r were i n c u b a t e d in d u p l i c a t e for 1.5 h at 0°C in the p r e s e n c e of [ 3 H ] - 7 d A at a final c o n c e n t r a t i o n of 2.5 χ Ι Ο - 9 Μ and i n c r e a s i n g c o n c e n t r a t i o n s of the i n d i c a t e d s t e r o i d s . The a m o u n t of [-^H]-7dA s p e c i f i c a l l y b o u n d in the a b s e n c e of any c o m p e t i t o r (total s p e c i f i c = 1 5 , 5 0 0 d p m / m l ) was d e f i n e d as 100%. N o n s p e c i f i c b i n d i n g was d e t e r m i n e d as d e s c r i b e d in Experimental Procedures. Each p o i n t r e p r e s e n t s the m e a n v a l u e from two s e p a r a t e e x p e r i m e n t s . Symbols: A =f (22S,23R)-antheridiol; R , S - A = (22R,23S)- antheridiol; 7dA = 7-deoxy-7-dihydro-antheridiol; 7dA-3-acetate = 7-deoxy-7-dihydro-3-ß-aceto-antheridiol; Cort = Cortisol; Choi = c h o l e s t e r o l ; Test = t e s t o s t e r o n e ; Prog = p r o g e s t e r o n e ; Est = e s t r a d i o l ; and Preg = p r e g n e n o l o n e .

747 MoM b u f f e r .

The a m o u n t of

mg of p r o t e i n

[ 3 H ] - 7 d A specifically bound

is e n r i c h e d a p p r o x i m a t e l y

7-fold after

from D E A E c o l u m n s w i t h a d r a m a t i c d e c r e a s e nonspecific binding

(data not

in the level of

shown).

Gel f i l t r a t i o n c h r o m a t o g r a p h y of the a m m o n i u m precipitated

per

elution

sulfate

r e c e p t o r on c o l u m n s of S e p h a c r y l S - 3 0 0 was

p e r f o r m e d and e a c h f r a c t i o n of the e l u a t e w a s a s s a y e d

for

[ 3 H ] - 7 d A b i n d i n g and p r o t e i n c o n t e n t .

four

separate analyses were similar.

The r e s u l t s of

S p e c i f i c binding

of

[ 3 H ] - 7 d A was g r e a t e s t at an e l u t i o n v o l u m e c o r r e s p o n d i n g a K A V v a l u e of 0 . 2 5 8 . linear regression

to

This K A V v a l u e was fit to the

line of the s t a n d a r d curve r e s u l t i n g

e s t i m a t i o n of a S t o k e s r a d i u s

for the b i n d i n g p r o t e i n .

Stokes radius coupled with a sedimentation coefficient

in a n The of

8 . 3 S was used to c a l c u l a t e an a p p a r e n t m o l e c u l a r w e i g h t of 1 9 2 , 0 0 0 by the m e t h o d of S i e g a l and M o n t y

Table

1.

(24).

P h y s i c o c h e m i c a l P r o p e r t i e s of Achlya Steroid Receptor

Sedimentation Coefficient

A summary

of

the

3.6S

(High salt w i t h o u t

Na2MoC>4)

8.3S

(Low salt w i t h N a 2

Mo04)

Stokes Radius

56.6 Ä

Molecular Weight

192,000

Frictional Coefficient

1.5

Axial Ratio

8.9

S e d i m e n t a t i o n c o e f f i c i e n t s w e r e d e t e r m i n e d by c e n t r i f u g a t i o n in s u c r o s e g r a d i e n t s (5-20% w / w ) p r e p a r e d in PM3 b u f f e r as d e s c r i b e d in M e t h o d s . The S t o k e s r a d i u s of the m o l y b d a t e - s t a b i l i z e d (8.3S) f o r m was d e t e r m i n e d by S e p h a c r y l S - 3 0 0 c o l u m n c h r o m a t o g r a p h y in b u f f e r c o m p o s e d of 100 m M K C l , 10 m M N a 2 M o 0 4 , 20 m M 3 - [ N - m o r p h o l i n o ] p r o p a n e s u l f o n i c a c i d , pH 7.0 at 4 ° C .

748 the m o l e c u l a r p r o p e r t i e s of the A c h l y a s t e r o i d r e c e p t o r g i v e n in T a b l e A very

i n t e r e s t i n g a s p e c t of the A c h l y a s t e r o i d

system

is the r e g u l a t i o n of c y t o s o l i c r e c e p t o r

c a n be a c h i e v e d by a l t e r a t i o n s growth medium. Achlya

is

1. receptor levels

in the c o m p o s i t i o n of

A c o m m o n m e t h o d w h i c h we use for

in s u s p e n s i o n c u l t u r e

involves

that the

growing

the g e r m i n a t i o n

of

a s e x u a l s p o r e s in PYG, a rich m e d i u m , for one day f o l l o w e d dilution usually

into a m i n i m a l m e d i u m three d a y s .

(Ml m e d i u m )

for a p e r i o d of

This p r o v i d e s ample a m o u n t s of

mycelia

(about 18 g p e r 2 - l i t e r flask) w i t h g o o d b i n d i n g a c t i v i t y hormone

responsiveness,

illustrates

as d e m o n s t r a t e d a b o v e .

the r e s u l t s of e x p e r i m e n t s four d a y s of g r o w t h

d a y of g e r m i n a t i o n

in P Y G ) .

transferred

Figure 7 24-hour

in Ml m e d i u m

Mycelia containing

l e v e l s of r e c e p t o r a f t e r three d a y s to an e n r i c h e d m e d i u m

maximum

in Ml m e d i u m

(PYG) and the

l e v e l s w e r e f o u n d to d r a m a t i c a l l y d e c r e a s e .

(after one were

receptor

In c o n t r a s t

this d o w n - r e g u l a t i o n of r e c e p t o r , m y c e l i a c u l t u r e d enriched medium possess relatively

and

in w h i c h the m y c e l i a l

l e v e l s of c y t o s o l i c r e c e p t o r w e r e m e a s u r e d at intervals during

by

low levels of

to

in an

receptor

that can be i n c r e a s e d by t r a n s f e r and i n c u b a t i o n in a nutrient-free

salt s o l u t i o n

receptor levels response

to h o r m o n e

We have found that the

(Figure

2) is m o r e o b v i o u s

in a m i n i m a l m e d i u m than in an e n r i c h e d

However, attempts

the

r e c e p t o r m a y be i n v o l v e d .

in

cultures

medium.

in a d d i t i o n

response to

The a b i l i t y to m a n i p u l a t e

levels should prove valuable regulation.

factors

with

visual

to a c t u a l l y c o r r e l a t e b i n d i n g and

h a v e not been d o n e and s e v e r a l

s y n t h e s i s and

Thus,

in A c h l y a show an inverse c o r r e l a t i o n

the s u p p l y of n u t i e n t s . grown

(data not s h o w n ) .

in f u t u r e s t u d i e s on

receptor

receptor

749

α>

5

Ο) \ «ο 0)

ο Ε α

4 o n o t h e r s t e r o i d h o r m o n e documented

receptor systems

effect

in A c h l y a .

r e q u i r e d to p r e s e r v e b i n d i n g In o t h e r s y s t e m s , Na2MoC>4

of

is well

( 2 5 - 2 7 ) , the p r e s e n c e of N a 2 M o 0 4 has not

s h o w n to be a b s o l u t e l y as o b s e r v e d

binding

A l t h o u g h the s t a b i l i z i n g

been activity

serves

750 to s t a b i l i z e

the s t e r o i d r e c e p t o r

in a n o n t r a n s f o r m e d

state

that d o e s not b i n d to n u c l e i but r e t a i n s the c a p a c i t y to hormone.

This nontransformed

state

by a s e d i m e n t a t i o n c o e f f i c i e n t that of the n u c l e a r b i n d i n g

is c o m m o n l y

that is larger

(8 to 10S)

form of the r e c e p t o r

A n u m b e r of p r o p o s a l s r e g a r d i n g

(3 to

i n h i b i t i o n of p h o s p h a t a s e activity

to the r e c e p t o r

than

5S).

the m e c h a n i s m of a c t i o n

Na2Moc>4 o n s t e r o i d h o r m o n e r e c e p t o r s have been m a d e include either direct coupling

bind

characterized

of

that

molecule,

a c t i v i t y or i n h i b i t i o n of

protease

(26,27).

The m e c h a n i s m of a c t i o n of Na2MoO,j in A c h l y a is u n k n o w n but m a y r e l a t e ,

in p a r t , to i n h i b i t i o n of p r o t e a s e

The A c h l y a b i n d i n g p r o t e i n enzymes

(7) and b i n d i n g

is v e r y s e n s i t i v e

activity

to

is d e s t r o y e d by

w i t h p r o n a s e e v e n at low t e m p e r a t u r e s .

activity.

proteolytic treatment

S i n c e this

organism

g r o w s r e a d i l y using c o m p l e x p r o t e i n as a m a i n n u t r i e n t , l i k e l y to c o n t a i n an a r r a y of p r o t e a s e a c t i v i t i e s . of the p r o t e a s e

inhibitors:

(0.5 m M ) , l e u p e p t i n (10 yg/ml)

i^-macroglobulin

buffers without molybdate

not p r e v e n t the loss of c y t o s o l i c b i n d i n g shown).

Clearly,

further research

e f f e c t of m o l y b d a t e w e l l as in o t h e r

activity

that m o l y b d a t e

i n t e r a c t s w i t h the

complex.

W i t h the a v i a n p r o g e s t e r o n e

molybdate

is not d i m i n i s h e d

protein

system

not the as

systems.

It is also p o s s i b l e

that

did

(data

is r e q u i r e d to d e f i n e

in the A c h l y a s t e r o i d b i n d i n g

p r o t e i n d i r e c t l y and forms a s t a b l e or slowly

indicating

Inclusion

phenylmethylsulfonylfluoride

(10 pg/ml) or

in h o m o g e n i z a t i o n

it is

binding

dissociating

r e c e p t o r , the a c t i o n of

after extensive

purification

it i n t e r a c t s d i r e c t l y w i t h the

receptor

(28).

In a d d i t i o n to the e f f e c t s of s o d i u m m o l y b d a t e , the p r o p e r t i e s of the A c h l y a s t e r o i d b i n d i n g p r o t e i n remarkably

s i m i l a r to the m o l e c u l a r p r o p e r t i e s of

molecular

are the

751 well-characterized organisms

(25,27).

s t e r o i d h o r m o n e r e c e p t o r s of

higher

It is an a c i d i c p r o t e i n w i t h a m o l e c u l a r

w e i g h t of a b o u t 192,000 and it does not a p p e a r to be since

it has a f r i c t i o n a l

c o e f f i c i e n t of 1.5 (Table

with other steroid receptors,

globular 1).

As

the 8S form in c y t o s o l can be

c o n v e r t e d to a 4S form by h i g h salt

treatment.

A l t h o u g h the A c h l y a s t e r o i d b i n d i n g p r o t e i n and o t h e r

steroid

r e c e p t o r p r o t e i n s are s i m i l a r , the A c h l y a r e c e p t o r

is v e r y

s p e c i f i c for fungal s t e r o i d

binding

a f f i n i t i e s of a n t h e r i d i o l

ligands.

and its c o n g e n e r s are

consistent with their relative morphogenesis a f f i n i t y of in v i t r o

The r e l a t i v e

in the A c h l y a m a l e E87

[3H]-7dA binding

generally

a b i l i t y to induce (22).

(Kd = 0.7 nM)

is c o n s i s t e n t w i t h o u r and o t h e r

sexual

Also,

the

measured (22)

observations

that the m a l e E87 r e s p o n d s to a 7dA c o n c e n t r a t i o n of 0.24 a n d the r e s p o n s e a b o v e this

i n c r e a s e s w i t h i n a 1 0 - to 20-fold

concentration.

As with other steroidal expression

nM

range

s y s t e m s , the m o d u l a t i o n of

is an e a r l y e v e n t

gene

in a n t h e r i d i o l a c t i o n

(10);

h o w e v e r , the e x i s t e n c e of n u c l e a r r e c e p t o r forms has not yet b e e n t e s t e d in A c h l y a . The p r e s e n t s t u d i e s are of proposals

regarding

molecules. observed

interest

in light of

the e v o l u t i o n of c e l l u l a r

Vertebrate

recent

communication

h o r m o n e s and t h e i r r e c e p t o r s have

in m i c r o o r g a n i s m s

suggesting

that these

been

forms of

c e l l u l a r c o m m u n i c a t i o n m i g h t have a r i s e n v e r y e a r l y in the e v o l u t i o n of e u k a r y o t e s and have b e e n h i g h l y c o n s e r v e d

(29).

To our k n o w l e d g e , A c h l y a

is the only fungal o r g a n i s m , and

only primitive eukaryote

b e l o w the

r e c e p t o r has b e e n d e s c r i b e d .

insects, where a

However, recent

by F e l d m a n and c o w o r k e r s have d e m o n s t r a t e d estrogen binding proteins

steroid

investigations

the p r e s e n c e

in S a c c h a r o m y c e s c e r e v i s i a e

and Paracoccidioides brasiliensis

(6).

the

They have

also

of

(5),

752 i d e n t i f i e d a p r o t e i n that b i n d s g l u c o c o r t i c o i d s albicans

(4).

U n l i k e the a n t h e r i d i o l

p r o t e i n s do not show a strong r e s e m b l a n c e receptors

in h i g h e r o r g a n i s m s .

of m u c h i n t e r e s t

evolutionary predecessors higher

However,

to

binding

steroid

they are

in that they c o u l d be i n v o l v e d

p e r c e p t i o n of e n v i r o n m e n t a l

in C a n d i d a

r e c e p t o r , these

s i g n a l s or they m a y

certainly

in the represent

to s t e r o i d a l c o n t r o l s y s t e m s

in

organisms.

B e c a u s e of its n o v e l t y , A c h l y a p r o v i d e s some opportunities antheridiol

receptor

understanding systems.

for i n v e s t i g a t i o n .

is of c l e a r s i g n i f i c a n c e

the e v o l u t i o n a r y

unusual

The c h a r a c t e r i z a t i o n

organism offers unique advantages

s i m p l i c i t y of

for

the

to

a s p e c t s of s t e r o i d a l

F u r t h e r m o r e , the r e l a t i v e

of

control

this

laboratory

i n v e s t i g a t i o n s on the m e c h a n i s m of s t e r o i d h o r m o n e

action.

References 1.

Geuns, J.M.C.:

2.

Callard, I.P., Klotz, K.L.: 314-321 ( 1 9 7 3 ) .

T r e n d s B i o c h e m . S e i . 7, 7-9

(1982).

3.

O'Day, D.H., Horgen, P.A.: S e x u a l I n t e r a c t i o n s in E u k a r y o t i c M i c r o b e s , A c a d e m i c P r e s s , N e w York 1 9 8 1 .

4.

L o o s e , D . S . , F e l d m a n , D.: ( 1982) .

5.

F e l d m a n , D., Do, V . , B u r s h e l l , Α . , S t a t h i s , P., D.S.: S c i e n c e 218, 2 9 7 - 2 9 8 ( 1 9 8 2 ) .

6.

Loose, D.S., Stover, E.P., Restrepo, A, Stevens, D.A., F e l d m a n , D.: P r o c . N a t l . A c a d . Sei. U . S . A . 80, 7 6 5 9 - 7 6 6 3 ( 1983) .

7.

Riehl, R.M., Toft, D.O., Meyer, M.D., Carlson, G.L., McMorris, T.C.: E x p . Cell R e s . 153, 5 4 4 - 5 4 9 ( 1 9 8 4 ) .

Gen. Comp. Endocrinol.

J . B i o l . C h e m . 257,

J . B i o l . C h e m . in the

21,

4925-4930 Loose,

8.

Riehl, R.M., Toft, D.O.:

9.

Griffin, D.H.: in F u n g a l P h y s i o l o g y , p p . 2 8 6 - 2 9 1 , a n d S o n s , N e w York ( 1 9 8 1 ) .

press. Wiley

753 10.

Timberlake, W.E., Orr, W.C.: in Biological Regulation and Development, Vol. 3B, Hormone Action (Goldberger, R.F. and Yamamoto, K.R., eds.) pp. 255-283, Plenum Press, New York (1984).

11.

McMorris, T.C., Le, P.H., Preus, M.W., Schow, S.R., Weihe, G.R.: J. Org. Chem. 48, 3370-3372 (1983).

12.

McMorris, T.C., Sheshadri, R., Arunachalam, T.: Chem. 39, 669-676 (1974).

13.

Kane, B.E., Jr., Reiskind, J.B., Mullins, J.T.: Science 180, 1192-1193 ( 1973) . Timberlake, W.E.: Dev. Biol. 51, 202-214 (1976).

14. 15. 16.

J. Org.

Silver, J.C., Horgen, P.A.: Nature (London) 249, 252-254 (1974). Horgen, P.A., Iwanochko, M., Bettiol, M.F.: Arch. Microbiol. 134, 314-319 (1983).

17.

Thomas, D. des S., Mullins, J.T.: ( 1967) .

18.

Horgen, P.A.: Biochem. Biophys. Res. Commun. 75, 1022-1028 ( 1977) . Griffin, D.H., Breuker, C.: J. Bacteriol. 98, 689-696 (1969) .

19. 20. 21.

Science 156, 84-85

Cantino, E.C., Lovett, J.S.: Physiol. Plant. 13, 450-458 (1960) . Barksdale, A.W.: Mycologia 62, 411-420 (1970).

22.

Barksdale, A.W., McMorris, T.C., Seshadri, R., Arunachalam, T., Edwards, J.Α., Sundeen, J., Green, D.M.: J. Gen. Microbiol. 82, 295-299 (1974).

23.

Scatchard, G.: ( 1949) .

24.

Siegel, Μ., Monty, K.J.: 346-362 (1966).

25.

Niu, E.-M., Neal, R.M., Pierce, V.K., Sherman, M.R.: Steroid Biochem. 15, 1-10 (1981).

26.

Housley, P.R., Grippo, J.F., Dahmer, M.K., Pratt, W.B.: in Biochemical Actions of Hormones, (Litwack, G., ed.) Vol. 11, pp. 347-376, Academic Press, Orlando (1984).

27.

Sherman, M.R., Tuazon, F.B., Stevens, Y.-W., Niu, E.-M.: in Steroid Hormone Receptors: Structure and Function, (Eriksson, H., Gustafsson, J.-Α., eds.) pp. 3-21, Elsevier, Amsterdam (1983).

28.

Puri, R.K., Grandics, P., Dougherty, J.J., Toft, D.O.: J. Biol. Chem. 257, 10831-10837 (1982). LeRoith, D., Shiloach, J., Berelowitz, M., Frohman, L.A., Liotta, A.S., Krieger, D.T., Roth, J.: Federation Proc. 42, 2602-2607 (1983).

29.

Ann. Ν. Y. Acad. Sei. 51, 660-672 Biochim. Biophys. Acta 112, J.

THE RECEPTOR FOR 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN:

SIMILARITIES AND

DISSIMILARITIES WITH STEROID HORMONE RECEPTORS

Lorenz Poellinger, Johan Lund, Mikael Gillner and Jan- Äke Gustafsson

Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital F69, 141 86 Huddinge, Sweden.

Introduction The receptor protein for 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD), a

putative gene regulatory protein involved in the induction of specific forms of cytochrome Ρ-Ί50, seems to share many characteristics with steroid hormone receptors. The scope of the present review is to overview and compare the present knowledge of the TCDD receptor to that of the steroid hormone receptors. Topics for this comparison include current models of gene regulation, physicochemical characteristics of the receptor proteins, ligand binding specificities and receptor interaction with DNA.

Background Steroid hormones seem to regulate the expression of specific genes by their interaction via specific intracellular recognition sites, i. e. soluble receptor proteins. In analogy to this endocrine model of gene regulation, several lines of evidence indicate that intracellular, soluble receptor proteins are essential mediators of the biological responses produced by certain xenobiotics, i.e. halogenated or non-halogenated polycyclic aromatic hydrocarbons

(1, 2). TCDD is one of the most potent

agonists known today, both for these receptor-mediated biological responses and for receptor binding itself (2, 3). The chlorinated dibenzodioxins are formed as unwanted contaminants during the synthesis of halogenated phenols. TCDD may accidentally be formed during the manufacture of 2,4,5-trichlorophenol which is, in turn, used to synthesize the

Molecular Mechanism of Steroid Hormone Action © 1985 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

756 herbicide 2,H,5-trichlorophenoxyacetic acid (2,4,5-T) and similar compounds (if). Furthermore, it appears that various polychlorinated

dibenzo-

dioxins, including TCDD, are formed as unwanted products during incomplete combustion of organic chlorinated compounds (5). TCDD also seems to be a remarkably persistent and metabolically inert compound (10) having a half-life of more than 10 years in soil (11) and, furthermore, exhibiting high biological availability after ingestion of TCDD-contaminated soil by rats and guinea pigs (12). However, some metabolic alteration or biological degradation of TCDD has recently been reported to occur _in vivo in animals (10).

Investigations with laboratory animals have shown that TCDD and structurally related compounds, e.g. chlorinated or brominated dibenzo-p-dioxins, dibenzofurans or biphenyls, produce a similar characteristic pattern of toxic responses (2, 6, 7). TCDD was shown to be the most toxic compound of these congeners thus far examined and, furthermore, appears to be one of the most potent toxic and teratogenic synthetic low-molecular compounds known (2, if, 8, 9). Thus TCDD serves as a prototype for these toxic, structurally related halogenated aromatic hydrocarbons. Several toxic lesions produced by halogenated aromatic hydrocarbons in the mouse, such as thymic involution, cleft palate formation in fetuses, teratogenesis, hepatic porphyria and epidermal hyperplasia and hyperkeratosis appear to be mediated by the TCDD receptor (12, 14, 15). Furthermore, the binding affinities of a large number of these compounds for the TCDD receptor correspond well with the rank order of their toxic potencies (6). Many of these quite characteristic lesions are reported to be relatively species-specific and to occur in a limited number of species (if, 16). In most species, however, administration of a single lethal dose of TCDD leads to the same pattern of a prolonged "wasting" syndrome including loss of adipose tissue and general debilitation of the animal leading to death within a few weeks (2, 17). This extreme acute lethality of TCDD suggests that TCDD is probably affecting some fundamental biological process within mammalian cells. A very striking and consistent feature of TCDD-toxicity is a depletion of lymphoid tissues in general and the thymus in particular (18), an effect which closely resembles the effect of glucocorticoids on the same tissues. It has also been reported that the induction of tyrosine

757 aminotransferase by dexamethasone can be inhibited by 3~methylcholanthrene and benzo(a)pyrene (19) - two well established ligands of the TCDD receptor ( 2 0 - 2 Ό . However, glucocorticoids do not compete with [3H]TCDD for TCDD receptor binding sites (20, 25-27), nor does TCDD compete for the glucocorticoid receptor binding sites (17, 23). Furthermore, adrenalectomized

rats respond to TCDD with thymic involution (28) and no endocrine

manipulation tested appears to have any effect on [3H]TCDD receptor binding in the rat liver

(29).

Several endocrine effects have been described following exposure to halogenated aromatic hydrocarbons, e.g. lowered plasma concentrations of progesterone (30, 31), corticosteroids

(32) and thyroxine (33), distur-

bances of the estrous cycle (30, 31)) and impaired reproduction

(30, 35).

One particularly striking and often studied biochemical effect produced by TCDD and related compounds is their potency to induce specific isozymes of cytochrome P-i450 and the associated monooxygenase activity aryl hydrocarbon hydroxylase (AHH) (1, 3 6 )

together with several non-monooxygenase

activities, e.g. UDP-glucuronosyltransferase, DT-diaphorase and ornithine decarboxylase (1). For chlorinated dibenzo-j?-dioxin congeners, there exists a good correlation between their binding affinities for the TCDD receptor and their potencies to induce AHH activity in vivo (20). There also seems to exist a

good correlation between the potency of these

compounds to induce AHH activity and their toxic potency (2, 37). TCDD induced keratinization of a mouse teratoma cell line has been used as one in vitro model of toxicity (37). However, many TCDD receptor containing tissues Jji vivo and cells _in vitro respond to TCDD with the induction of AHH activity but display no evidence of a toxic response (16, 3 8 ) .

A

genetic model, where the TCDD receptor appears essential but not sufficient for the expression of toxicity has been proposed (15). In this model, two genetic loci are conceived: the Ah and hr loci, which determine the presence of the TCDD receptor and the expression of receptor mediated toxicity, respectively

(15).

758 Gene Regulation by Steroid Hormones and TCDD - Level of Regulation

The synthesis of a wide variety of proteins has been demonstrated to be under hormonal control (39). For several of the genes encoding these proteins, the hormonal control of the observed alteration in gene expression has been attributed to the level of transcription, more specifically to an increased rate of transcription. This concept is, to a large extent, based on experiments where molecular cDNA probes were isolated from hormonally induced specific mRNAs and the radioactive cDNAs then used to quantitate specific mRNA levels, especially the kinetics of the induction process (40-42). The rapid hormonal induction of mRNA and the insensitivity of this response to inhibitors of protein synthesis have been taken as indications of a primary hormonal effect. A quantitative and temporal relationship between hormone-receptor complex formation, nuclear translocation and RNA induction have been taken as further support for this concept (40, 43). Also, sequences in the 5' flanking regions of hormonally regulated genes have been demonstrated to be required for the hormone responsiveness of these genes (44-55). As to the molecular mechanisms responsible for the hormone-receptor induced alteration in the transcription of certain genes, little is known. However, recent data from experiments where a glucocorticoid response element from murine mammary tumor virus (MMTV) has been inserted near originally hormone insensitive genes thereby rendering these genes hormonally controlled following transfection into glucocorticoid receptor containing cells (56, 57), may shed new light on this process and will be discussed in some detail later.

It should be pointed out that an increased rate of transcription after hormone administration is not the only mechanism proposed for the hormonal regulation of gene expression. Effects of steroids on the stability of mRNA molecules (40, 58, 59) as well as on protein-processing (60) have been proposed as additional mechanisms by which steroids may modulate gene expression. Several studies have demonstrated that administration of TCDD or TCDD-like inducers leads to a large increase in translatable mRNA's encoding the TCDD induced cytochrome P-450 isozyme(s) (61-63). It has also been shown

759 by _in vitro nuclear transcription studies that this process reflects an increased rate of transcription of the specific cytochrome P-450 gene(s) (6H-66) rather than an effect on mRNA stability - another plausible level of regulation of gene expression (67; c.f. above). Nevertheless, the latter mechanism cannot be completely ruled out in view of the limited amount of data available. In cultured cells, it has been shown that treatment with TCDD results in a large increase in the rate of transcription of specific cytochrome P-450 encoding RNA within 30 min (66). A quantitative and temporal relationship between the nuclear accumulation of the TCDD-receptor complex and the increase in synthesis of mRNA encoding a specific form of cytochrome P-M50 has also been demonstrated (68). Full accumulation of mRNA also occured in the presence of cycloheximide (69). Using variant hepatoma cells with specific cytochrome P—i)50 induction-responsive as well as induction-resistant phenotypes, it has also been shown that the presence of a functional TCDD receptor constitutes an essential component in mediating selective stimulation of the rate of cytochrome P-450 transcription (66, 69). The rapidity of this induction is similar to that observed with glucocorticoid activation of transcription of mammary tumour virus (70). No specific data are available on the effect of TCDD on cytochrome P-450 processing.

Current Models for the Mechanism of Action of Soluble Receptor Proteins

It is today widely accepted that specific intracellular recognition sites are involved in gene regulation by steroid hormones and that all steroid hormones act via a similar scheme (71. 72). This model was first proposed by Gorski et al. (73) and Jensen et al. ( 7 Ό

and involves a) the uptake of

the hormone into the cell; b ) the subsequent noncovalent binding of the steroid hormone to specific, saturable and soluble receptor proteins; c) an increased affinity of the hormone-receptor complex for the cell nucleus induced by the receptoi—ligand interaction itself. In this model, the nucleus is believed to be the primary site of action and, furthermore, cytoplasmic receptors are postulated to exist either occupied or non-occupied by a steroid, whereas nuclear receptors are postulated to exist only when occupied by a hormone. Recently, however, it has become clear that

760 several issues in this model, are somewhat controversial. Among such issues are e.g. a) the question concerning the intracellular distribution of steroid receptors in target tissues in the presence or absence of hormone (75-79); and b) the nature of the putative nuclear "acceptor" site of occupied steroid receptors (72, 79). Recent reports have indicated that, in fact, unoccupied steroid receptors may exist in a state of equilibrium between the cytoplasmic and nuclear compartment and that this equilibrium may be shifted in favor of the cytoplasm by manipulations such as tissue homogenization and subcellular fractionation (75, 76, 80, 81). Two independent studies using either immunohistochemical techniques (i.e. immunocytochemical staining using a monoclonal antibody against the estrogen receptor) (77) or cytochalasin Β induced enucleation to obtain cytoplast and nucleoplast fractions from an estrogen responsive cell line (78) supported the notion that unoccupied steroid receptors predominantly reside within the cell nucleus. Based on these studies it was suggested that the classical "two-step" model for steroid hormone action requires some modification in so far as the steroid induced increased affinity of steroid receptor for DNA should be regarded as an intranuclear event, rather than as a step whereby cytoplasmic receptors are rendered capable of interacting with nuclear structures leading to a cytoplasmic - nuclear translocation.

It has been proposed by several authors that the unoccupied steroid receptor form with negligible affinity for nuclei, DNA or other polyanionic resins may represent an oligomer of several steroid or nonsteroid binding entities. Not until after dissociation of this oligomer into a monomeric steroid binding form (for physico-chemical characteristics, cf. below), does the receptor exhibit a high affinity for nuclei or polyanions (82-81). This model, however, is based on observations _in vitro and it remains to be proven whether it is valid for the situation in whole cells.

The nature of putative intranuclear "acceptor" sites for steroid hormone receptors has been the subject of intensive research efforts over the past decade (72, 85). Recently, however, reports that steroid receptors bind to specific DNA sequences and thereby enhance transcription of specific clusters of regulated genes (50, 52, 51, 86-98) have provided an attrac-

761 tive model to explain the extreme selectivity in protein induction by steroid hormones. This mechanistic model is particularly interesting in view of its analogy to the regulation of procaryotic genes by specific proteins, e.g. the lac repressor or the catabolite gene activator protein of Escherichia coli. Furthermore, certain features of procaryotic gene regulatory proteins - i.e. requirement of the ligand for DNA-binding and separateness of ligand- and DNA-binding sites - are shared by steroid hormone receptors (cf. below).

In analogy to the "two-step" model proposed for the mechanism of action of steroid hormones, an accumulation of TCDD-receptor complexes in the cell nucleus following occupation of cytosolic receptor sites has been reported by several authors (25-27, 99, 100). Furthermore, it has been suggested that transcription of TCDD-regulated genes requires nuclear localization of the TCDD-receptor complex (66, 68, 69). As is the case with the model for the mechanism of action of steroid hormones (cf. above), questions have been raised whether unoccupied TCDD receptors solely reside in the target cell cytoplasm as proposed in the "two-step" model of action (1, 2) or whether both unoccupied and occupied TCDD receptors reside within the nuclear compartment but in two affinity states for nuclear "acceptor" sites (101). In similarity to experiments carried out with genes regulated by steroid hormones, the identification of cis-acting regulatory elements within or near the specific cytochrome P-M50 gene(s) should help to elucidate the nature of possible intranuclear target sites for occupied TCDD receptors.

Physicochemical Properties of Eucaryotic Soluble Receptor Proteins Hydrodynamic Properties and Molecular Weights. A comparison of available data on soluble receptors concerning even such basic characteristics as size and hydrodynamic properties is severely hampered by the great differences in experimental conditions used by different investigators. In addition, although a model for the structure (s) of different soluble receptor forms extracted from mammalian tissues is beginning to evolve (102), virtually nothing is known about the

762 native form(s) of soluble receptors. Taking these limitations into consideration, it becomes necessary to define the basis for the selection of data for the comparison of receptors. In Table I we have selected data from the literature regarding the monomeric forms of soluble receptor proteins, preferentially from studies on purified preparations. Reports where inadequate precautions against proteolysis were taken, leading to registration of anomalously small receptor sizes, have been avoided.

As shown in Table I, the labeled glucocorticoid receptor, either in pure form or in crude cytosolic preparations, sediments as a 3.4 -3.7 S entity in hypertonic sucrose gradients (103, 104). During gel filtration, the glucocorticoid receptor displays a Stoke's radius of 5.6-6.0 nm (103. 104). This would imply an apparent molecular weight in the range of 85,000 to 96,000, a frictional ratio of 1.74-1.90 and an axial ratio of 15-20. These data appear to be confirmed by SDS-PAGE of either purified receptor preparations (103, 105, 106) or photoaffinity-labeled receptor in crude cytosol (107-109), where the molecular weights estimated range between 87,000 and 94,000.

In the case of the estrogen receptor, the monomeric form of the receptor sediments as a 4.2 S entity in hypertonic sucrose gradients (110, 111). If the incubation with ligand is performed at 20~37°C it appears that the -4 S estrogen receptor forms a homodimer that sediments as a -5 S entity (110). The Stoke's radius estimated for the monomeric form of the estrogen receptor ranges from 4.4 to 6.4 nm (110, 111) giving apparent molecular weights of 76,000 to 114,000. From these data it is possible to calculate frictional ratios of 1.45-1.84 and axial ratios of 8-20. By SDS-PAGE of the purified estrogen receptor its molecular weight was estimated to 70,000

(111) .

Available evidence indicates that the progesterone receptor in chick oviduct (112-114) exists in the form of two subunits, A and B, whereas the progesterone receptor in rabbit uterus seems to exist as a hormone binding unit of one and the same size, not excluding, however, the possible existence of two subunits of equal size. The A subunit from chick oviduct sediments as a 3 . 6 S entity and has a Stoke's radius of 4.6 nm whereas the

763 Β subunit sediments as a 4.2 S entity and has a Stoke's radius of 6.3 nm (112). From these data, apparent molecular weights of 71,000 and 114,000 were calculated for the A and Β subunits, respectively (112). It is also possible to calculate frictional and axial ratios of 1.54 and 10 for the A subunit and 1.81 and 15-20 for the Β subunit. Due to the general availabilty of a photoaffinity label for the progesterone receptor, R 5020, extensive data is available on molecular weights as determined by SDS-PAGE of the photoaffinity labelled receptor. These studies indicate that the A subunit has a molecular weight of 79,000-80,000 and the Β subunit a molecular weight of 108,000-109,000 (113, 114). The rabbit progesterone receptor seems to have a molecular weight of 70,000 as determined by SDS-PAGE of a purified uterine receptor preparation (115).

The androgen receptor purified from rat ventral prostate sediments at 4.5 S in hypertonic sucrose gradients and displays a Stoke's radius of 4.8 nm (116). These data suggest an apparent molecular weight of 83,000, a frictional ratio of 1.41 and an axial ratio of 6-8. SDS-PAGE of the purified androgen receptor indicated a molecular weight of 86,000 (116). The same value was obtained when using a photoaffinity label for the androgen receptor in the rat ventral prostate (117).

The vitamin D receptor, purified from chick intestine (118), has been reported to sediment as a 3.3 S entity in hypertonic sucrose gradients and a molecular weight of 68,000 was calculated from gel filtration experiments (118). From these data one may calculate a Stoke's radius of 4.9 nm, a frictional ratio of 1.67 and an axial ratio of 12-15. SDS-PAGE of the purified vitamin D receptor yielded a molecular weight of 55,000 (118). Recently, higher molecular weights of 91,000 to 1 1 0,000 (1 19-121 ) have been reported for the vitamin D receptor, a fact that the authors attribute to their precautions against proteolysis.

The data summarized above indicate that the monomeric forms of steroid hormone receptors, during conditions minimizing proteolysis, exist as components with sedimentation coefficients of 3-3 to 5.0 S, Stoke's radii of 4.4 to 6.4, frictional ratios of 1.41 to 1.90, axial ratios of 6 to 20 and molecular weights in the range of 60,000 to 120,000. The frictional

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Table I also presents comparative data on the physicochemical characteristics of the TCDD receptor.

Data from our own laboratory indicate

that the TCDD receptor in the rat sediments as a 4.4-4.5 S entity in hypertonic sucrose gradients, that it has a Stoke's radius of 6.1-6.6 nm, a frictional ratio of 1.73-1.79, an axial ratio of 12-15 and an apparent molecular weight of 111,000-136,000 (23, 123). The TCDD-receptor in the mouse has been estimated to sediment in the 5.7 to 7.5 S range in hypertonic sucrose gradients (21, 25, 68). Furthermore, Stoke's radii ranging from 6.0 to 7.5 nm have been described (ibid), and the apparent molecular weight has been estimated to be between 196,000 and 245,000 (21, 68). From these data a frictional ratio of 1.40-1.67 and an axial ratio of 6-15 may be calculated. However, in a recent study using identical experimental conditions for the rat and mouse TCDD receptor we were unable to detect any physico-chemical differences between the two receptors (23).

In summary, the hydrodynamic properties and molecular weights described for the different steroid hormone receptors show both similarities and dissimilarities; it is clear that the corresponding parameters estimated for

the TCDD-receptor fall within this range of variation and that the

TCDD receptor does not display any particular hydrodynamic property distinguishing it from the steroid hormone receptors.

Charge

DEAE-cellulose chromatography and isoelectric focusing are methods often utilized to assess differences in charge of proteins. Isoelectric focusing may be carried out in density gradients in columns, or more conveniently in flat-bed Polyacrylamide gels (IFPAG). IFPAG-analysis requires considerably shorter separation times (2 h) than isoelectric focusing in

767 columns, thus receptor p r o t e o l y s i s and ligand d i s s o c i a t i o n may be minimized. I n the f o l l o w i n g we have t h e r e f o r e , when p o s s i b l e , quoted data obtained from IFPAG.

IFPAG-analyses have revealed apparent i s o l e c t r i c p o i n t s ( p l ) f o r the g l u c o c o r t i c o i d receptor of 5 to 6 and 6.1-6.2 from human lymphocyte cytosol

(124) and r a t l i v e r cytosol

(125), r e s p e c t i v e l y . Similar

pi-values

( 5 . 9 - 6 . 4 ) have been reported f o r the estrogen receptor both in human mammary tumor cytosol

(126) and in r a t l i v e r cytosol

(125). These data (pi

- 6 . 4 ) were confirmed by i s o e l e c t r i c focusing in columns of a pure receptor preparation from c a l f

uterus ( 1 2 8 ) . A pi of 6.2 has been reported f o r the

progestin receptor from r a t uterus as well as human breast carcinoma cytosol

(129).

When the androgen receptor from r a t p r o s t a t i c cytosol was analyzed by i s o e l e c t r i c focusing in sucrose density gradients the non-DNA-binding form of the non-activated ( c . f . below) receptor focused at a pH of 5.8, whereas the DNA-binding form of the receptor focused at a pH of 6.5 ( 1 3 0 ) . Chromat o f o c u s i n g , a r e c e n t l y developed method f o r protein separation based on d i f f e r e n c e s in charge, has been used t o estimate the pi of the vitamin D receptor t o 6.0-6.2 ( 1 3 1 ) .

In the case of the TCDD receptor the apparent pi as determined by IFPAG i s 6.0 in human lymphocyte c y t o s o l (132) and 6.2 in r a t l i v e r c y t o s o l

(133)·

At pH 7.2, the TCDD receptor from r a t l i v e r cytosol was reported t o e l u t e from DEAE-cellulose at 0.2-0.3 Μ NaCl ( 2 3 ) . DEAE- c e l l u l o s e chromatography at pH 7.4 has also been e x p l o i t e d t o separate the DNA-binding form

of the

r a t l i v e r g l u c o c o r t i c o i d r e c e p t o r , e l u t i n g at 0.06 Μ potassium phosphate, from the non-DNA-binding form, e l u t i n g at 0.24 Μ at a pH of 7.4 ( 1 3 4 ) .

In summary, s t e r o i d hormone receptor proteins seem t o have i s o e l e c t r i c points around 6. The same i s the case with the n a t i v e TCDD-receptor. Limited

p r o t e o l y s i s (135) i s o f t e n used t o improve the r e s o l u t i o n of

soluble receptor preparations during IFPAG a n a l y s i s . This treatment a f f e c t s the apparent pi of some receptor proteins ( 1 3 5 ) . I f

subjected t o

l i m i t e d p r o t e o l y s i s , the apparent pi i s increased f o r the estrogen

768 receptor of mammary tumours (126), is unaffected for the estrogen receptor from rat (127), and is decreased for the glucocorticoid (125) and TCDD receptor (133) from rat liver. It may be speculated that these differences are due to the loss of peptides with differing charges from different receptors. Both the TCDD and glucocorticoid receptors elute from DEAEcellulose around a salt concentration of 0.2 Μ (23, 131).

Ligand Binding Specificity

Most of the information on the specificity of binding of steroid hormones to their receptor proteins has been derived from competitive binding experiments. In such experiments the affinity of an unlabeled competitor is estimated from its ability to displace a radiolabeled ligand for the receptor. The capacity of an agonist to elicit a biological response is not always directly correlated to its receptor affinity,

since distribu-

tion factors, such as absorption, metabolism and plasma protein binding, determine the concentration of unmetabolized agonist available for receptor binding (136). It is thought that long-term retention of the agonist at the receptor sites, due to a low dissociation rate, is required for a sustained biological response (136)

From studies of substituent effects on the binding of adrenocortical hormones binding to the glucocorticoid receptor, it appears as the 3 20-oxo groups, and the 11-

and 21-hydroxyl

and

groups are essential for

binding (137-139). Schmit and Rousseau (110) have studied fifteen glucocorticoid receptor ligands and concluded that the basic structure of ring A for high receptor affinity is 1 α,2ß-half-chair. Furthermore, it was concluded that the Β and C rings are semirigid chairs, and that their conformation is not much affected by substituents. On the other hand, the receptor binding affinity may be affected by substituents in these two rings (112). The shape of the D-ring is critically dependent on the nature of the substituents. A 17-hydroxyl group reduces whereas a 17-methyl group increases the affinity for the receptor (111). Furthermore, these sub-

769 s t i t u e n t s influence the o r i e n t a t i o n of the pregnane side ohain. However, the p a r t i c u l a r conformation of the s i d e chain required f o r high a f f i n i t y binding has not yet been established

(142).

In a study of the thermodynamics of binding of 29 c o r t i c o s t e r o i d s to the r a t hepatoma c e l l g l u c o c o r t i c o i d r e c e p t o r , Wolff et a l . (143) found that the changes in both enthalpy and entropy of binding decreased as the temperature was increased, which i n d i c a t e s that the s t e r o i d receptor binding mainly i s of hydrophobic nature.

A phenolic r i n g seems t o be a common f e a t u r e of both estrogens and a n t i estrogens, e . g . d i e t h y l s t i l b e s t r o l and tamoxifen, r e s p e c t i v e l y . HShnel et a l . (144) have suggested that the phenolic 3-hydroxyl group of

estrogens

i s of greater importance f o r binding than the 17-hydroxyl group, although both are required f o r h i g h - a f f i n i t y binding of the estrogen r e c e p t o r . Certain t r i p h e n y l e t h y l e n e d e r i v a t i v e s hydroxylated in two of the rings have higher a f f i n i t y f o r the estrogen receptor than e s t r a d i o l , but i t

is

not e n t i r e l y c l e a r how the hydroxyl groups of these ligands can be superimposed on the hydroxyl groups of e s t r a d i o l t o depict common binding f o r these hydroxyl groups on the estrogen receptor

sites

(145).

Duax et a l . (146) have found that the progestin receptor i s able t o bind a number of s t e r o i d s with s i g n i f i c a n t structural v a r i a t i o n s in the B-, C-and D-rings. The only s t r u c t u r a l f e a t u r e that was common t o a l l s t e r o i d s with a f f i n i t y f o r the progestin receptor was the 3 - o x ° -

configuration a l -

though not a l l s t e r o i d s with t h i s structure bind t o the receptor with a high a f f i n i t y . Several of the S-oxo-A^-steroids with high a f f i n i t y f o r the progestin receptor a l s o had the 1 β, 2a-inverted half chair conformation (146). These f i n d i n g s l e d Duax e t . a l . to propose that high binding a f f i n i t y f o r the progestin receptor i s due t o a t i g h t binding of

the

inverted Α - r i n g t o the binding s i t e .

A r e l a t i v e f l a t n e s s of the Α - r i n g , as in 5a-reduced or

Δ^-structures,

seems t o be required f o r high binding a f f i n i t y f o r the androgen receptor (147). A 17$-hydroxyl group a l s o seems necessary f o r high a f f i n i t y b i n ding, whereas structures with a 17a-hydroxy group are unable t o bind t o

770 the receptor (117). A 3~oxo group increases binding affinity, but is not necessary (1 -48, 119). 19-Nortestosterone binds with higher affinity than testosterone, a 17a-methyl group does not decrease binding affinity, and a 7a-methyl group further increases the binding affinity (147). Introduction of a 3,9,11-triene structure resulted in an increased binding affinity indicating that binding increases with increasing flatness of the steroid nucleus (150).

A problem in studies of the mineralocorticoid receptor has been the possible coexistence of mineralocorticoid and glucocorticoid receptors in mineralocorticoid target cells, since most ligands binding to one of these receptors also bind to the other receptor, albeit not with the same affinity (151). Because of this problem we have chosen not to discuss the properties of the mineralocorticoid receptor in this review. Fortunately, the

problem of insufficient specificity of mineralocorticoid receptor

ligands has recently been diminished by the synthesis of specific glucocorticoids, such as RU 26988, which do not interact with the mineralocorticoid receptor or corticosteroid-binding globulin and which may therefore be used to block the glucocorticoid receptor binding sites (152).

The Β ring of the steroid nucleus is opened in the vitamin D secosteroids. In these, the 1a- and 25ß-hydroxyl groups are reported to be the most important substituents for high affinity binding to the vitamin D receptor (153, 154). The binding is also reported to be sensitive to modifications in the A ring and the side chain (154).

The TCDD receptor binds TCDD with a high binding affinity (Kd = 0.3 χ 10~9 Μ in mouse liver cytosol) (20). The TCDD receptor preferentially binds compounds known to induce microsomal AHH. These compounds are polycyclic aromatic hydrocarbons, e. g. 6-naphthoflavone, 3-methylcholanthrene, benzo(a)pyrene, and chlorinated hydrocarbons, e.g. dioxins, dibenzofurans, and biphenyls (2). From studies of the binding of halogenated dibenzodioxins to the TCDD receptor it has become clear that at least 3 and preferentially 4 halogens in positions 2, 3. 7 and 8 are required for high binding affinity. The dioxin nucleus is not essential for binding since some other molecules, e. g. anthracene and biphenylene substituted with 4

Figure 1. Plots of molecules with the van der Waals radii of the atoms included. TCDD (2,3,7,8-tetrachloro-p-dibenzodioxin), top (A), and side view (C), and budesonide (11g,21-dihydroxy-16a,17a[(22R)-propylmethylenedioxyJ-pregna-1,4-diene~3,20-dione) top (B) and side view CD).

772 chlorines at similar positions as in TCDD, also bind to the receptor with high affinity (6). Biphenyls chlorinated at similar positions as in TCDD binds to the TCDD receptor with a lower affinity than the corresponding dioxins (155). Planarity, or the ability to attain planarity, may therefore be a prerequisite for binding.

It has been noted that these chlori-

nated ligands fit into a 3 χ 10 Ä rectangle where the centers of the chlorine atoms are situated in its corners. As pointed out (2), this concept does not account for the binding of unhalogenated ligands, like ß-naphthoflavone, to the receptor. We have instead attempted to visualize the true three-dimensional space occupied by some receptor ligands. Therefore the molecular structures of these compounds were studied with a vector display driven by a VAX 11/750 computer running an interactive program using crystallographic data as inputs. All potent receptor ligands studied could be fitted into a rectangle of 6.8 χ 13.7 Ä, when the van der Waals radii of their atoms were included (156).

The affinity of TCDD for the TCDD receptor corresponds to that of steroid hormones for their receptors. To compare a high affinity ligand for the glucocorticoid receptor with TCDD, we have used the method described above to plot the (22R)-epimer of Budesonide (157), and TCDD (158) in two projections (Fig 1). Budesonide can be regarded as an analogue of triamcinolone acetonide, where the 9a-fluorine is replaced by a hydrogen, and the two methyl groups of the acetal are replaced by a hydrogen and a propyl group. It is evident that Budesonide fits quite well into the 6.8 χ 13.7 X rectangle. On the other hand, it can be seen that the overall thickness of the Budesonide molecule is considerably greater than that of TCDD (3.1* X). The reason for this difference is that Budesonide is, due to its partially saturated structure, like other steroids, pleated, whereas TCDD due to its aromatic structure is planar. Since all known ligands of the TCDD-receptor are very lipophilic, it is probable that the main driving force for binding is of hydrophobic nature, as in the case of the glucocorticoid receptor

(113).

It is interesting that the requirement for binding of chlorinated ligands to the TCDD-receptor is such that the chlorine atoms should be separated by certain distances, whereas the intervening structure is of less impor-

773 t a n c e , as long as i t i s planar or may a t t a i n a planar conformation.

This

s t r u c t u r a l requirement resembles t h a t f o r l i g a n d binding t o t h e e s t r o g e n r e c e p t o r where hydroxyl groups s e p a r a t e d by c e r t a i n d i s t a n c e s seem t o be r e q u i r e d f o r b i n d i n g , whereas t h e i n t e r v e n i n g s t r u c t u r e appears l e s s c r i t i c a l . Furthermore i t i s i n t e r e s t i n g t h a t p l a n a r i t y of ligands f a v o r s TCDD r e c e p t o r binding as i s a l s o t h e case with androgen r e c e p t o r b i n d i n g . S t e r o i d r e c e p t o r s e x h i b i t some c r o s s - s p e c i f i c i t y with regard t o l i g a n d b i n d i n g , e s p e c i a l l y in case of t h e g l u c o c o r t i c o i d and m i n e r a l o c o r t i c o i d r e c e p t o r s . On t h e o t h e r hand, no s t e r o i d has yet been shown t o bind t o t h e TCDD-receptor.

DNA-Binding P r o p e r t i e s of Soluble Receptor P r o t e i n s The i n t e r a c t i o n of s t e r o i d hormones and t h e i r r e c e p t o r p r o t e i n s with the c e l l nucleus of t a r g e t t i s s u e s i s a presumed e s s e n t i a l s t e p in t h e mechanism by which t h e s e hormones modulate n u c l e a r e v e n t s such as gene e x p r e s s i o n (72). Nuclear s t e r o i d r e c e p t o r s may o f t e n be e x t r a c t e d from nuclei with 0 . 3 - 0 . 6 Μ KCl or NaCl (73, 159, 160). The c e n t r a l i s s u e about t h e accumulation of s o l u b l e r e c e p t o r s in t h e nucleus i s what d e f i n e s a p u t a t i v e n u c l e a r a c c e p t o r and/or b i o l o g i c a l e f f e c t o r s i t e f o r t h e s e p r o t e i n s . Among t h e p o t e n t i a l s i t e s , DNA has been suggested t o be of prime importance (72, 161). The "nuclear t r a n s f e r " phenomenon of s t e r o i d hormone r e c e p t o r s - in i t s e l f a c o n t r o v e r s i a l i s s u e , c f . above - i s u s u a l l y mimicked _in v i t r o by t h e a d d i t i o n of hormone t o c y t o s o l , followed by " r e c e p t o r a c t i v a t i o n " . This i s a poorly understood process in which t h e r e c e p t o r i s rendered competent t o bind t o i s o l a t e d n u c l e i or v a r i o u s n u c l e a r t a r g e t elements f o l l o w i n g c e r t a i n m a n i p u l a t i o n s , such as prolonged s t o r a g e , d i l u t i o n , gel permeation chromatography, warming, exposure t o s a l t s or p r e c i p i t a t i o n by ammonium s u l f a t e (71), 160, 162-165). This process may involve a conformational change of t h e s t e r o i d - r e c e p t o r complex, r e s u l t i n g in t h e exposure of p o s i t i v e l y charged r e g i o n s on t h e s u r f a c e of t h e molecule (166). A f t e r " a c t i v a t i o n " , t h e s t e r o i d - r e c e p t o r complex d i s p l a y s an increased a f f i n i t y f o r both n a t u r a l and s y n t h e t i c polyanions i n c l u d i n g chromatin (128,

774 167-169), nuoleosomes (170), purified DNA (166, 171-173), DNA-cellulose (171-177), RNA (178-180, phospho-cellulose (177, 181), heparin-agarose (111, 131), ATP-Sepharose (182, 183), carboxy-metyl(CM)-Sephadex (166, 184), sulfopropyl(SP)-Sephadex (166) and glass beads (166, 185). However, the overall K d of the steroid receptor interaction with bulk DNA sequences has been estimated to only -10-11 Μ (186). Clearly, the DNA-binding property of steroid receptors seems to be physiologically significant. Yamamoto et al. (187) examined the differences in binding to DNA-cellulose of the glucocorticoid receptor from two mutant phenotypes of glucocorticoid-resistant S 49 mouse lymphoma cells in which the receptor appeared to be normal with respect to hormone-binding. One class of cells exhibited decreased nuclear binding (nt~) whereas the other showed increased nuclear binding (nt*) of the mutant receptor complexes. The different nuclear binding properties of these receptors correlated well with their respective affinities for DNA-cellulose. A mutation affecting the polynucleotide binding domain of the receptor resulting in a glucocorticoid-resistant phenotype was suggested. The polynucleotidebinding domain was thus shown to be distinct and separated from the hormone-binding domain. All studies on receptors associated with the nti-phenotype have revealed a molecular weight of the mutant glucocorticoid receptor of approximately 40,000 rather than 94,000 M r characteristic of the wild-type receptor (109, 188, 189). Interestingly, α-chymotrypsin or trypsin cleavage of the 94,000 M r rat liver glucocorticoid receptor leads to the formation of a 39,000 M r fragment that retains both steroid- and DNA-binding activities (106, 190); like the nt* receptor, this fragment binds with increased affinity to bulk DNA sequences (109, 187, 189). Accordingly, there seems to exist a third, non-steroid- and non-DNA-binding domain of the glucocorticoid receptor important for its biological activity (189, 191-193). Polyclonal antibodies raised against highly purified glucocorticoid receptor (194) have been used to detect and characterize this domain, which was shown to carry the main antigenic determinants (191). Further-

775 more, 40,000 M r mutants of the glucocorticoid receptor as well as 40,000 M r proteolytic fragments of wild-type glucocorticoid receptors lack this major immunoreactive region (194-196).

Limited proteolysis has also been used for the characterization of the progestin (198-203). estrogen (200, 204-208), androgen (209) and putative mineralocorticoid (200) receptors. Based on these findings and on studies performed on the glucocorticoid receptor, it has been proposed that all steroid hormone receptors may have the same basic structure (193): three protein domains separated by protease-sensitive regions.

It has been demonstrated that most classes of steroid hormone receptors (glucocorticoid, estrogen, progesterone and androgen receptors) have to undergo the losely defined process called "activation"

(cf. above) in

order to acquire affinity for polyanionic resins such as DNA-cellulose (160, 162, 165). Several molecular mechanisms leading to steroid receptor "activation" have been proposed: a) dimerization of receptor subunits (110, 176); b) limited proteolysis of "unactivated" receptor (204-206); c) conformational changes of the receptor molecule (138, 166, 210, 211); and d) the dissociation of receptor oligomers into constitutive subunits (82-84, 211). There has been some controversy regarding the existence of an "activation" process for vitamin D

receptors, in part due to reports

that both unoccupied (120, 212) as well as occupied (118, 212-214) vitamin D receptors readily interact with DNA-cellulose. Recently, however, it has been shown, by means of "mixing" experiments, that unoccupied vitamin D receptors exhibit a lower affinity for DNA than occupied receptors (120). The ionic strengths required to dissociate steroid hormone receptors from DNA-cellulose closely resemble those required to extract them from nuclear binding sites (i.e. -0.1-0.5 Μ KCl or NaCl).

The concept that DNA may be an important component of genomic binding sites for steroid-receptor complexes is further strengthened by recent studies demonstrating selective binding of glucocorticoid and progesterone receptors to specific cloned portions of hormonally regulated genes (52, 54, 86-98). Several types of experiments have demonstrated that at least some of the cloned DNA-fragments that interact selectively with steroid

776 receptors _in vitro (89, 96) are capable of mediating hormonal responsiveness in vivo (46, 47, 50, 89, 97) leading to the formulation of the concept that these specific binding regions - subsequent to interaction with the receptor protein - might function as hormone- and receptor-dependent transcriptional enhancers (57).

It has been reported that less than

10?

of protein-bound

[3H]TCDD

as-

sociates with DNA-cellulose without any preceding manipulations known to lead to "activation" of steroid receptors for

30

min at

25~37°C

ceptor complexes

>50?

(123).

However, upon incubation

and/or gel permeation chromatography of TCDD-reof the specific

[3H]TCDD

binding in rat liver

cytosol was retained on DNA-cellulose (R. Hannah, J. Lund, L. Poellinger, M. Gillner, J.-Ä. Gustafsson, unpublished). DNA-binding of the TCDD-receptor required the presence of ligand, and limited proteolysis of the receptor by trypsin or α-chymotrypsin abolished the DNA-binding

(123;

Hannah et al., unpublished), in analogy to studies performed on the glucocorticoid receptor

(190).

The DNA-binding form of the TCDD receptor

has, however, not yet been characterized. Furthermore, there is no consensus as to the nature of genomic target sites for the TCDD-receptor complexes (2, 68), and it remains to be established if the nuclear form of the TCDD receptor (of. above) interacts with cloned portions of genes regulated by TCDD. There is also a paucity of data concerning the nature of the primary biochemical response to the "nuclear transfer" phenomenon of TCDD receptor-complexes. Clearly, further studies are needed in this field before any conclusions can be drawn about analogies between steroid receptors and the TCDD receptor as DNA-binding and gene-regulatory proteins .

Concluding Remarks

Apparently the steroid hormone receptors and the TCDD receptor share certain characteristics with regard to size, charge and DNA-binding. It is also thought that these soluble receptor proteins regulate the expression of certain genes in a similar fashion. However, to evaluate common features with regard to this receptor-mediated regulation of gene ex-

777 p r e s s i o n , f u r t h e r work i s c l e a r l y needed on t h e i n t e r a c t i o n of b o t h t h e TCDD r e c e p t o r and s t e r o i d hormone r e c e p t o r s w i t h r e g u l a t o r y e l e m e n t s of c o n t r o l l e d g e n e s . Such s t u d i e s have been performed w i t h t h e g l u c o c o r t i c o i d and p r o g e s t e r o n e r e c e p t o r s (52,

86-98).

I n view of t h e many s i m i l a r i t i e s between t h e t h e TCDD r e c e p t o r and t h e s t e r o i d hormone r e c e p t o r s , i t i s t e m p t i n g t o s p e c u l a t e t h a t t h e s t r u c t u r a l genes f o r a l l t h e s e r e c e p t o r p r o t e i n s have e v o l v e d from a common a n c e s t r a l gene by gene d u p l i c a t i o n and s u b s e q u e n t d i v e r g e n t e v o l u t i o n . Based on DNA sequence homology such a c o n c e p t has been proposed f o r c e r t a i n s u b u n i t s of an o l i g o m e r i c s t e r o i d b i n d i n g p r o t e i n , t h e p r o s t a t i c s t e r o i d

binding

p r o t e i n (PSP) ( 2 1 8 ) . C e r t a i n s u b u n i t s of PSP have been shown t o have amino a c i d homologies w i t h a n o t h e r s t e r o i d b i n d i n g p r o t e i n , r a b b i t

uteroglobin

(219) and a common a n c e s t o r f o r t h e s e p r o t e i n s has been s u g g e s t e d

I n o r d e r t o t e s t t h e v a l i d i t y of s p e c u l a t i o n s r e g a r d i n g

(219).

evolutionary

r e l a t i o n s h i p s between s o l u b l e r e c e p t o r p r o t e i n s , t h e a v a i l a b i l i t y of a cDNA-probe c o r r e s p o n d i n g t o a p o r t i o n of t h e r a t g l u c o c o r t i c o i d r e c e p t o r coding s e q u e n c e s h o u l d p r o v e e x t r e m e l y h e l p f u l ( 2 2 0 ) . Using t h i s cDNA probe i t should be p o s s i b e t o o b t a i n t h e gene f o r t h e g l u c o c o r t i c o i d r e c e p t o r and p e r h a p s a l s o , by d e c r e a s i n g t h e h y b r i d i z a t i o n s t r i n g e n c y , o b t a i n the genes f o r o t h e r s o l u b l e r e c e p t o r

to

proteins.

Acknowledgements

T h i s work was s u p p o r t e d by g r a n t s from t h e Swedish Cancer S o c i e t y and t h e Swedish C o u n c i l f o r P l a n n i n g and C o o r d i n a t i o n of R e s e a r c h . Dr. C. Camb i l l a u (CRMC2-CNRS Campus Luminy - Case 91 3, 1 3288 M a r s e i l l e ,

Cedex,

F r a n c e ) i s g r a t e f u l l y acknowledged f o r p e r f o r m i n g t h e computer s u p p o r t e d p l o t t i n g of t h e m o l e c u l e s i n F i g .

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786 150. Delettr70%) of specific binding applied to the column.

Recoveries on the 30 cm TSK3000 SW columns were

consistently 87 to 93%.

Column calibration with marker

proteins suggested this component to be a very large species of >80°A.

0

20

40

60

80

0

20

40

60

βΟ

FRACTION NUMBER

Fig. 4 Separation of progestin receptor isoforms from human uterus using HPSEC. Labeled cytosolic proteins were applied to and eluted from a 30 cm TSK3000 SW column as described (29). Progestin receptors labeled with [3H]C>RG-2058 and incubated in the presence (•) and absence (•) of excess ORG2058 are presented in A. The receptor isoform pattern eluting under identical conditions using t^H]R5020 as ligand in the presence (Δ) and absence (A) of excess unlabeled steroid is presented in B. The HPSEC system was pre-calibrated with a series of pure proteins, thyroglobulin (TG), ferritin (FE), bovine serum albumin (SA) and cytochrome c (CC).

A small but distinct and reproducible secondary isoform (Fig. 4B) was demonstrable between fractions 50 and 60 with R5020, (ca. 50 A), but only represented 10 to 15% of the specifically bound radioactivity.

No free steroid radioactivity appeared

in these separations, indicating that dextran coated-charcoal clearance of the unbound ligand was complete and that no

802 discernible column-induced dissociation of steroid occurred. [3H]ORG-2058 also appeared bound specifically by two isoforms [3H]R5020

with virtually identical ratios as observed for (29).

However, with ORG-2058 as ligand, the primary receptor

isoform was observed between fractions 40 and 47 representative of a protein of ca. 70 A.

(Fig. 4A)

The smaller,

secondary receptor peak appeared between fractions 73 and 83 with O R G - 2 0 5 8 . 2058

The nonspecific binding was minimal with ORG-

when compared with that of

R5020,

similar to the density

gradient sedimentation data reported elsewhere

(29).

Clinical application of the HPSEC mode for the analysis of estrogen receptors in human tumor samples is rapidly evolving. However, quantification is essential since the levels of estrogen receptor have prognostic value in the determination of hormonal response 2,3,24,27.

At the same time, our

laboratory (e.g. 3,16) and others (24,25) have proposed that the distribution of receptor isoforms has clinical significance also.

Clearly, if HPSEC analysis proves to

provide quantitative and qualitative information on hormone receptors in human tumors, it will find wide application in the clinical laboratory due to its speed.

In this regard,

(21,28) suggested that an automated system employing high performance ion-exchange chromatography (HPIEC) could be highly useful in the determination of the profile of receptor isoforms.

The relationship of receptor isoform profiles

("fractionated receptors") to ultimate responsiveness has yet to be established (3). HIGH PERFORMANCE CHROMATOFOCUSING (HPCF) As described earlier (18,29) all chromatography was performed in cold-room at 0-4°C.

Buffers were filtered under vacuum

through Millipore 0.45 μπι HAWP filters before use.

Free

steroid or the ligand-labeled cytosols were applied to SynChropak AX-300 or AX-500 (250 χ 4.1 mm I.D.) anion-exchange

803 columns valve

( S y n C h r o m ) w i t h a n A l t e x M o d e l 210 s a m p l e

(Beckman).

112 p u m p s .

injection

Elution was carried out using Altex

T h e a b s o r p t i o n p r o f i l e of the e l u a t e w a s

a t 280 nm w i t h a H i t a c h i

100-40

w i t h a n i n - l i n e flow cell

spectrophotometer

(Beckman).

Model monitored

equipped

pH w a s m e a s u r e d

in-line

with a Pharmacia pH monitor. Two different column equilibration and elution programs u s e d d e p e n d i n g u p o n the i n i t i a l b u f f e r c o n d i t i o n s of receptor preparations.

The columns were

e q u i l i b r a t e d to the s t a r t i n g pH u p p e r limit)

initially

( s l i g h t l y a b o v e the

using a common cationic buffer.

high performance chromatofocusing

were

the

on AX-300

desired

I n the c a s e of and AX-500

c o l u m n s , w e h a v e u s e d 25 m M T r i s - H C l c o n t a i n i n g 1 m M d i t h i o t h r e i t o l a n d 20% a t 0°C

(v/v) g l y c e r o l a d j u s t e d to pH 8.1 - 8.3

(18) or a s i m i l a r p h o s p h a t e b u f f e r

chromatofocusing molybdate-stabilized

(29).

receptor

m M s o d i u m m o l y b d a t e w a s i n c l u d e d in the c o l u m n buffer.

For components,

C y t o s o l s p r e p a r e d in h o m o g e n i z a t i o n b u f f e r

were

e l u t e d w i t h a 30:70 m i x t u r e of P o l y b u f f e r s 96 a n d 74.

This

p o l y a m p h o l y t e s o l u t i o n w a s d i l u t e d 1 0 - to 2 0 - f o l d w i t h glycerol,

f i l t e r e d w i t h a 0.45 μπι f i l t e r

(Millipore)

a d j u s t e d to b e t w e e n p H 4.0 a n d 5.0 a t 0 - 4 ° C . experiments,

column equilibration buffer at 1-2 The

For

20%

and

most

1.0 m l f r a c t i o n s w e r e c o l l e c t e d a t 1.0

C o l u m n s w e r e r e g e n e r a t e d to t h e i r s t a r t i n g pH

(8.3)

ml/min. with

ml/min.

[125i]iodoestradiol-17 ß-labeled receptor complexes,

s p e c i f i c b i n d i n g c o m p o n e n t s , a n d f r e e s t e r o i d in e a c h were detected radiometrically

in a M i c r o m e d i c s 4/600

c o u n t e r or w i t h the new B e c k m a n M o d e l 170 detector.

[3H]R5020

and

non-

fraction gamma

flow-through

t 3 H ] O R G - 2 0 5 8 w e r e m e a s u r e d in M o d e l

3801 l i q u i d s c i n t i l l a t i o n c o u n t e r

(Beckman).

S o m e t i m e s the pH

of a l t e r n a t e f r a c t i o n s w a s d e t e r m i n e d a t 0 ° C u s i n g a Model

10

equilibration

Beckman

3500 pH m e t e r w i t h a c o m b i n a t i o n g l a s s e l e c t r o d e .

c o u n t i n g e f f i c i e n c y of !25;ι;0(}ίηθ a v e r a g e d 65%

in the

The

804 conventional counter, as judged by reference to independent determinations of disintegratins per minute using a Beckman 4000 two-channel gamma counter.

Tritium counting efficiency

was 35-42%. Chromatofocusing is a technique which separates proteins on the basis of their surface charge properties and uses a column which is essentially a weak ion-exchanger.

The enhanced

ability to resolve ionic species which differ in charge by as little as 0.1 pH unit provide a unique method, especially in the HPLC mode to separate and charaterize proteins. Our first experiments with estrogen receptor indicated that the AX-300 column formed a stable pH gradient with the polybuffers, and that a number of cytosolic proteins were well resolved within the gradient (18).

We were concerned that the

initial (i.e. "loading") peak may be composed of unresolved species, namely, that certain proteins would be excluded based upon size and charge properties.

However, HPCF of labeled

estrogen receptors from human uterus (Fig. 5) showed that isoforms were not eluted in the loading peak but were recovered in the gradient when the A-500 column was employed. Similar results were observed when estrogen receptors in lactating mammary gland of the rat were separated (Fig. 6).

805

Fig. 5. Separation of estrogen receptor isoforms from human uterus in the presence or absence of molybdate by HPCF. A. Cytosol was incubated with [125χ]iodoestradiol-178 in the presence or absence (·) of excess diethylstilbestrol. Activity was eluted from the AX-500 column with a mixture of Polybuffers 96 and 74 adjusted to pH 4.5. B. Cytosol was prepared in buffer containing 10 mM molybdate and incubated with [125j]iodoestradiol-17ß in the presence or absence (·) of excess diethylstilbestrol. The primary eluent was a mixture of Polybuffers 96 and 74 containing 10 mM sodi um molybdate and adjusted to pH 5.0. The secondary eluent (arrow) was Polybuffer 74 (no molybdate) adjusted to pH 3.5. Taken from (18).

806 Fig. 6. Separation of receptor isoforms by HPLC chromatofocusing on AX-300. Cytosol was prepared from 14-day lactating mammary glands and incubated with [125i]iodoestradiol-17ß as described earlier. For elution, a 30:70 mixture of Polybuffers 96 and 74, diluted 1:10 with 20% glycerol and adjusted to pH 5.0, was used. Taken from (18).

HPCF of progestin receptors was accomplished primarily on the AX-500 column as described previously

(18).

The AX-500 column

in the HPCF mode exhibited problems found with the HPIEC system, namley that radioactivity appeared just after the void volume and prior to application of the pH gradient (Fig. 7). This peak was in the exact location in which free steroid eluted in the HPLC mode (29).

Even in the presence of 10 mM molybdate, the AX-500 column appeared to strip labeled-steroid from the receptor.

However,

a second peak appeared at a pH of 5.6 - 6.1 which contained specifically bound steroid

(Fig. 7).

The results presented

with [3H]R5020 in Figure 7 were virtually identical to those observed when [ 3 H]ORG-2058 was used as ligand (29).

Thus,

based on pH, the progestin receptor isoform focused at a pi value regardless of the ligand used in contrast to the HPSEC profiles

(29).

The origin and significance of progestin receptor polymorphism remains obscure.

Although certain components may represent

distinct physiological species, proteolytic cleavage may occur with a labile receptor.

Dougherty et al. (30) identified two

8S forms of the progesterone receptor from chick oviduct. Interestingly each form contained a 90,000 molecular weight component which did not associate with progestin and either a 75,000 or 110,000 molecular weight steroid binding

species.

Various combinations of these components could give rise to considerable size heterogeneity as we observed with receptors from human uterus

(29).

807

Fig. 7. HPCF of progestin receptor isoforms in human uterus. Cytosol obtained from human uterus was incubated with [^H]R5020 in the presence (0) and absence (·) of excess unlabeled R5020. Following removal of the free steroid, the incubate was injected and eluted on an AX-500 column in a chromatofocusing mode as described earlier (29).

One advantage of HPCF is that stabilizing agents such as sodium molybdate may be included in the buffer systems. Molybdate has several useful properties, including the preservation (stabilization) of larger forms of the receptor and the ability of block receptor activation (e.g. 31).

Thus,

molybdate is a valuable tool in correlating the interrelationships between receptor isoform structure and biologic function. HIGH PERFORMANCE ION EXCHANGE CHROMATOGRAPHY (HPIEC) This is one of the most effective HPLC modes for the study of receptor isoforms (21,29).

The resolution of isoforms and

their high recovery provide an excellent means of characterizing receptors for structure/function relationships.

808 Briefly, a portion

(150-200

ul) of i n c u b a t e c l e a r e d of

l i g a n d is a p p l i e d to a n A l t e x

322

(Beckman, Berkeley,

Chromatograph equipped with either AX-300, AX-500 (SynChrom)

anion exchange columns.

phosphate

(30 m i n ) of the c o l u m n w i t h b u f f e r

f o l l o w e d by e l u t i o n w i t h a l i n e a r g r a d i e n t of (21,29).

Subsequently

r e t u r n e d to s t a r t i n g c o n d i t i o n s . n m

absorbing

spectrophotometer c o u n t e d for

filter.

was

filtered

T h e level

the e l u a t e t h r o u g h a H i t a c h i

e q u i p p e d w i t h a low v o l u m e flow

from

100-40

cell.

1 ml f r a c t i o n s of the e l u a t e w e r e c o l l e c t e d

[16α - 1 2 5 χ ] i o d o e s t r a d i o l - 1 7 ß

using a

M o d e l 170 F l o w - t h r o u g h D e t e c t o r . radioactivity

Beckman

[^HjLigands were measured

in c o l u m n f r a c t i o n s w i t h a M o d e l

liquid scintillation counter

(Beckman).

The

c o n c e n t r a t i o n w a s d e t e r m i n e d by m e a s u r i n g fractions with an in-line detector

from

the c o n d u c t i v i t y

(21), l a c t a t i n g m a m m a r y

g l a n d of the r a t a n d

c a r c i n o m a of the r a b b i t

(19).

is s h o w n in F i g u r e 8.

and AX-1000

separation

from h u m a n b r e a s t

i s o f o r m e l u t e d a t 190

p h o s p h a t e w h i l e o t h e r s w e r e o b s e r v e d a t 52 m M a n d 100 phosphate

(21).

cancer

endometrial

A representative

The m a j o r

of

Bio-Rad.

c o l u m n s in the H P I E C m o d e u s i n g h u m a n u t e r u s a n d b r e a s t

isoforms

by

3801

phosphate

W e h a v e c o m p a r e d the use of the S y n C h r o p a k A X - 5 0 0

p r o f i l e of e s t r o g e n r e c e p t o r

and

Micromedics

g a m m a c o u n t e r h a v i n g a 62% c o u n t i n g e f f i c i e n c y or the counting

of

s p e c i e s w e r e d e t e c t e d as they e m e r g e d

the c o l u m n by d i r e c t i n g Approximately

after

the c o l u m n

All buffers were

p r i o r to use w i t h a 0 . 4 5 u m M i l l i p o r e A280

was

potassium

p h o s p h a t e a t pH 7.4 w h i c h a p p r o a c h e d 500 m M 90 m i n gradient initiation

is

Each column was

e q u i l i b r a t e d p r e v i o u s l y w i t h low i o n i c s t r e n g t h A wash

or A X - 1 0 0 0

All chromatography

p e r f o r m e d in the c o l d r o o m a t 0 - 4 ° C . buffer.

unbound CA)

T h e i s o f o r m s e l u t i n g a t 100 a n d 200

cancer mM

mM mM

p h o s p h a t e h a v e b e e n o b s e r v e d in c y t o s o l s f r o m m a m m a r y g l a n d of the r a t a n d h u m a n u t e r u s c y t o s o l of e n d o m e t r i a l

(21) a l s o .

cancer

T h e i s o f o r m s in the

fo the r a b b i t e x h i b i t a 50 m M

s p e c i e s a n d 175 m M c o m p o n e n t w h e n s e p a r a t e d o n H P I E C

(19).

809

Fig. 8. HPIEC separation of ionic forms of the estrogen receptor from human breast cancer tissue on AX-1000. Cytosol was prepared and incubated in the presence (0) or absence (·) of 500-fold excess of inhibitor. Elution was performed at 1.0 ml/min using a gradient of potassium phosphate at pH 7.4. The elution of the labeled ligand alone was previously determined under identical conditions and is marked with an arrow. The recovery of radioactivity from the column was 91% for the aliquot of cytosol incubated in the absence of diethylstibestrol. A tracing of species absorbing at 280 nm is given by the continuous line. Taken from (21). Additionally, the on-line method of analysis using the Model 170 Flow-Through Detector permitted the detection of very low levels of receptor isoforms in small quantities of cytosol (10— 20 μΐ).

Figure 9A and 9B compare the manual and on-line

profiles of 6.9 fmol of total receptor-bound from human uterine tissue separated by HPIEC.

radioactivity Measurements

obtained manually are shown in panel A, with the continuous tracings presented in panel B.

An elution pattern is similar

to that for estrogen receptors in breast tissue emerged (32). Two non-specific binding components that did not intract with the column were also present in uterine cytosol.

However, the

two receptor isoforms were shown to have slightly different surface charge properties as characterized by an altered

810

elution from the phosphate gradient. The first species of receptor eluted at a phosphate concentration of 150-180 mM and was equivalent to 1.4 fmol of bound steroid (22% of total). The second isoform eluted between 225-255 mM phosphate and was equal to 5.0 fmol of receptor (78% of total). A 93% recovery was observed in this representative experiment. The presence of different ionic isoforms of the receptor in breast and uterine tissues revealed by the rapid format with HPIEC allows the comparison required of clinical studies.

Fig. 9. HPIEC separation of micro quantities of ionic isoforms of the estrogen receptor from human uterus. The curves shown are the results of receptors separated in the presence (0) or absence (·) of 200-fold excess of diethylstibestrol. Elution was performed on 10 μΐ of cytosol (10 mg/ml) equivalent to 6.9 fmol receptor at 1.0 ml/min using a gradient of potassium phosphate ( ). (A) 1 ml fractions were collected and radioactivity measured manually, or (B) radioactivity recorded continuously using in-line Model 170 Radioisotope Detector with conductivity flow cell. Total binding is indicated by (·) in A and by (—) in B, and nonspecific binding is indicated by (0) in A and by ( ·) in B. Recovery of radioactivity from the column was 93% for the total bound curve determined by counting a 10 μΐ aliquot before sample injection. Specific binding was 30 fmol receptor/mg cytosol protein determined by multi-point titration analysis. Taken from (32).

811 SUMMARY High performance liquid chromatography of steroid hormone receptors provides rapid analyses, sensitivity and high recoveries of these elusive, labile proteins.

Based upon the

demonstration that receptors exhibit polymorphism, we propose that their level of cellular organization and interrelationships are more complex than considered originally.

Some of the cellular events which may give rise

to receptor heterogeneity include proteolysis, phosphorylation and other post-translational modifications, protein-protein interactions as well as protein-nucleic acid interactions. Some isoforms may be generated due to handling artifacts while others may be the result of authentic physiologic processes. Regardless, HPLC in its various modes should be useful in future studies of receptor characterization.

Although the

biological significance of receptor polymorphism is not well understood currently, there are important biological implications which include the utility of receptor isoform profiles as new markers in the clinical setting. ACKNOWLEDGMENTS Studies from the author's laboratory have been supported in part by USPHS grants CA-19657, CA-34211, CA-32101, and CA-31946 from the National Cancer Institute and by grants from the American Cancer Society (PDT-210) and Phi Beta Psi Sorority.

The important contributions of numerous research

fellows are acknowledged, particularly those of Drs. R.D. Wiehle, G.E. Hoffmann, A. Fuchs, T.W. Hutchens, Ν.A. Shahabi and Α. van der Walt.

The author also expresses his deepest

appreciation to Ms. Dana Gibson for her assistance in the preparation of this typescript.

812

1.

McGuire, W.L., Carbone, P.P. and Vollmer, Ε.P., eds. Estrogen Receptors in Human Breast Cancer. Raven Press, New York (1975).

2.

Anonymous Cancer 46, 2759-2963 (1980).

3.

Wittliff, J.L., Cancer 53, 630-643 (1984).

4.

McGuire, W.L., Raynaud, J.P. and Baulieu, E.E., eds. Progesteroine Receptors in Normal and Neoplastic Tissues. Raven Press, New York (1975).

5.

Creasman, W.T., McCarty, K.S. Sr., Barton, T.K., and McCarty, K.S., Jr.

Obstet. Gynecol. 55, 363 (1980).

6.

Erlich, C.E., Young, P.C., Cleary, R.E. Gynecol. 141, 539-546 (1981).

Am. J. Obstet.

7.

Käuppila, Α., Kujansuu, Ε., Vihko, R. 2166 (1974).

8.

Carlson, J.A. Jr., Allegra, J.C., Day, T.G. and Wittliff, J.L. Am. J. Obstet. Gynecol. 149, 149-153 (1984).

9.

Mörtel, R., Levy, C., Wolf, J.P., Nicolas, J.C., Röbel, P. and Baulieu, E.E. Cancer Res. 41, 1140-1145 (1981).

Cancer 50, 2157-

10. Daxenbichler, G., Grill, H.J., Geir, W. , Wittliff, J.L. and Dapunt, 0. In Steroid Receptors and Hormone Dependent Neoplasia (J.L. Wittliff and 0. Dapunt, eds.) pp. 59-67, Masson Publishing USA, Inc., New York (1980). 11. Boylan, E.S. and Wittliff, J.L. (1973). 12. Gardner, D.G. and Wittliff, J.L. 2096 (1973). 13. Goral, J.E. and Wittliff, J.L. (1975).

Cancer Res. 33, 2903-2908 Biochemistry 12, 3090Biochemistry 14, 2944-2952

14. Wittliff, J.L. I_n Methods in Cancer Research (H. Busch, ed.). Vol. XI, pp. 293-354. Academic Press, New York (1975). 15. Wittliff, J.L., Mehta, R.G. and Kute, T.E. In Progesterone Receptors in Normal and Neoplastic Tissues (W.L. McGuire, E.E. Baulieu and J.P. Raynaud, eds.), pp. 39-57. Raven Press, New York (1977). 16. Wittliff, J.L., Lewko, W.M., Park, D.C., Kute, T.E., Baker, D.T. Jr. and Kane, L.N. Iji Hormones, Receptors and Breast Cancer (W.L. McGuire, ed.) pp. 325-359. Raven Press, New York (1978). 17. Wittliff, J.L., Feldhoff, P.W., Fuchs, A. and Wiehle, R.D. In Physiopathology of Endocrine Diseases and Mechanisms of Hormone Action (R. Soto, A.F. DeNicola and J.A. Blaquier, eds.), pp. 397-411. Alan R. Liss, Inc., New York (1981) .

813 18. H u t c h e n s , T . W . , W i e h l e , R . D . , S h a h a b i , N.A. a n d J.L.

J. Chromatogr.

266, 1 1 5 - 1 2 8

Wittliff,

(1983).

19. S h a h a b i , N . A . , H u t c h e n s , T . W . , W i t t l i f f , J . L . , H a i m o , S.D., Kirk, M.E. and Nisker, J.A. Iji H o r m o n e s a n d C a n c e r 2 (F. B r e s c i a n i , R . J . B . K i n g , M . E . L i p p m a n , M . Namer and J.P. Raynaud eds.), pp. 63-71. Raven Press, New Y o r k (1984). 20. W i e h l e , R . D . , H o f m a n n , G . E . , F u c h s , A . a n d W i t t l i f f , J. C h r o m a t o g r . 307, 39-51 (1984).

J.L.

21. W i e h l e , R . D . a n d W i t t l i f f , J . L .

313-

326

J. C h r o m a t o g r . 2 9 7 ,

(1984).

22. S h e r m a n , M . R . , P i c k e r i n g , L . A . , R o l l w a g e n , F . M . ,

Miller,

L.K. F e d . P r o c . 37, 1 6 7 - 1 7 2 (1978). 23. M c C a r t y , K . S . J r . , C o x , C . , S i l v a , J . S . , W o o d a r d , B . H . , Mossier, J.A., Haagensen, D.E., Barton, T.K., McCarty, K . S . S r . , a n d W e l l s , S.A. J r . C a n c e r 4 6 , 2 8 4 6 - 2 8 5 0 (1980) . 24. C l a r k , G . M . , M c G u i r e , W . L . , H u b a y , C . A . , P e a r s o n , O . H . , Marshall, J.S. N. E n g l . J. M e d . 309, 1 3 4 3 - 1 3 4 7 . 25. P o w e l l , B., G a r o l a , R . E . , C h a m n e s s , G . C . , M c G u i r e , C a n c e r R e s . 39, 1 6 7 8 - 1 6 8 2

W.L.

(1977).

26. K n i g h t , W . A . , L i v i n g s t o n , R . B . , G r e g o r y , E . J . a n d

McGuire,

W.L. C a n c e r Res. 37, 4 6 6 9 - 4 6 7 1 (1977). 27. F i s h e r , B., R e d m o n d , C . , B r o w n , Α . , W i c k e r h a m , D . L . , W o l m a r k , N., A l l e g r a , J . C . , E s c h e r , G., L i p p m a n , M . , S a v l o v , Ε., W i t t l i f f , J . L . a n d F i s h e r , E . R . e t al. J. C l i n . O n e . 1, 2 2 7 - 2 4 1 (1983). 28. W i t t l i f f , J . L . a n d W i e h l e , R . D . In: H o r m o n a l l y S e n s i t i v e T u m o r s , (V.P. H o l l a n d e r , ed.) Academic Press Inc. (1985) in p r e s s . 29. V a n d e r W a l t , A . a n d W i t t l i f f , J . L . J. C h r o m a t o g r . in p r e s s .

(1984)

30. G r e e n e , G . L . , N o l a n , C . X . , E n g l e r , J . P . a n d J e n s e n , P r o c . N a t l . A c a d . S e i . U.S. 77, 5 1 1 5 - 5 1 1 9 . 31. N i s h i g o r i , H. a n d T o f t , D . O . (1980)

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32. B o y l e , D . M . , W i e h l e , R . D . , S h a h a b i , N.A. a n d J.L. J. C h r o m a t o g r . (1985) in p r e s s .

E.V.

77-83

Wittliff,

AUTHOR INDEX

Andreasen, P.A. Auricchio, F.

199 279

Bardin, C.W. Baulieu, E.E. Binart, N. Bodwell, J.E. Brinkman, A.O. Buchou, T.

587 31 31 637 563 31

Callison, S. Castoria, G. Catelli, M.G. Catterall, J.F. Cidlowski, J.A. Clark, J.H.

173 279 31 587 141 399

d'Arville, C. Dickerman, H.W. Dohanich, G. Dougherty, J.J.

659 505 701 299

Faber, L.E.

61

Gase, J.M. Gillner, Μ. Gorski, J. Gustafsson, J.A.

31 755 539 755

Holbrook, N.J. Horwitz, K.B.

637 659

Isohashi, F.

225

Janne, O.A. Joab, I. Jordan, V.C. Junker, K.

587 31 603 199

Kishimoto, S. Kontula, K.K. Kovacic-Milivojevic, B. Kumar, S.A.

249 587 85 505

LaPointe, M.C. Leavitt, W.W. Lieberman, Μ.Ε.

85 437 539

816

Litwack, G. Lund, J.

309 755

Maeda, Y . Markaverich, B.M. Matsumoto, K. McEwen, B.S. Mendel, D.B. Μ ester, J. Migliacchio, A . Moudgil, V.K. Mulder, E. Muldoon, T . G . Munck, A . Murayama, A . Myers, J.E.

61 399 249 701 637 31 279 351 563 377 637 1 61

Nakao, M. Nishizawa, Y . Nock, B. Noma, K . Notides, A . C .

249 249 701 249 173

Pasqualini, J.R. Poellinger, L .

471 755

Radanyi, C. Reker, C.E. Renoir, J.M. Riehl, R . M . Rotondi, A .

31 85 31 733 279

Sakamoto, Y . Sasson, S. Sato, B. Schmidt, T.J. Sedlacek, S.M. Shull, J.D. Shyamala, G. Silva, C.M. Simons, S.S. Sumida, C.

225 173 249 309 659 539 413 141 111 471

Tai, Pink-K. K . T o f t , D.O.

61 733

Vedeckis, W.V.

85

Watson, C.S. Wei, L . L . Welshons, W.V. W i t t l i f f , J.L.

587 659 539 791

SUBJECT INDEX

Acceptors, microsomal Accessory proteins Acetylcholinesterase Achlya Activated receptor binding by ATP-Sepharose Chromatin DNA nuclei oligonucleotides Activation estrogen receptor glucocorticoid receptor progesterone receptor Activation, mechanism Affinity labeling Agonist-antagonist, relationship Allosteric binding mechanism Androgen action Androgen-induced proteins Androgen receptor activation cyproterone acetate binding DNA binding molybdate effects nuclear binding photoaffinity labeling polyribonucleotides binding prostate, rat RNA, binding seminal vesicles, ram Anterior pituitary estrogen receptor prolactin gene transcription protein synthesis Antheridiol Antiandrogens cyproterone acetate Antibodies estrogen receptor glucocorticoid receptor progesterone receptor

382 517 712 733 231,354 226

322,328,505,671,783 235,783 511,515 174-176,261,362,381 85,103,199,225,309,327,362,637,646 34,38,362 48,105,106,353 111,672 644 174,175,188 587,590,591 383 563-569 568-570 580-582 567,568 563,589,593 580-582 384,385,570-582 575 565-570 387,543,544 539-541,544-546,548 547 733 587 567-570 472,614 119 37,51,64,72

818 A nti estrogens binding site biological responses chemotherapy mechanism metabolism structure-activity relationship

610,612,613 471,492,495 603,604,627-629 609-613 606-608 618-623

Antiglucocorticoids Antiprogestin Aqueous two phase partitioning Aurintricarboxylic acid

113-115,682 682 199,200 359,360,365

Brain

acetylcholine acetylcholinesterase choline acetyltransferase cholinergic system dopaminergic regulation gonadal steroids localization of receptors muscarinic receptors noradrenergic regulation Breast cancer

714 712 710 710 717 706 703 708 719 659

Oa2+—calmodulin—Kinase Calpastatin cAMP cAMP-dependent kinase cDNA Choline acetyltransferase Cholinergic system Chymotrypsin Clomiphene Cortisol 21-mesylate Cyproterone a c e t a t e

288 654 389 303 590 710 706 89,91,93 603,604 113-115 567-570

Deoxycorticosterone-agarose Dephosphorylation DNA binding accessory proteins nonspecific probes DNA sequences recognition Dopaminergic regulation

305 230,289,452,453 580-582,671 517 507 509 509,524 717

819 Enclomiphene Endocrine therapy Equilibrium model Estradiol binding in mammary tumor Estradiol e f f e c t s on f e t a l tissues gene transcription histone acetylation protein synthesis receptor synthesis RNA polymerase activity

182,184 690,792,795 61,81 404

Estradiol, mechanism of action Estriol kinetics and responses Estrogen priming responses Estrogen binding endogenous inhibitor low molecular weight stabilizer

293,381,428,431,438 181,183

Estrogen-induced mammary tumors Estrogen receptor activation aggregation factor affinity labeling anterior pituitary, binding factors biological activity, actions breast cancer cooperativity cytosol form dephosphorylation dimer structure dissociation kinetics distribution DNA binding equilibrium binding fetal, new born pig uterus GH 3 hormone binding hydrophobic domain inactivation lactating mouse, rat leydig cell tumor magnesium ions, e f f e c t of mammary gland

487 539,541,544-546,549 485 485 497 485

445,484 481 399,400,402,407 255 404 174-176,256 269 379 543,544 8-26

264,471,663 792 175,180,188 543,544 289 177-179 183,185-187 189,378,539,550,552,553,555,560 174

180 472,473,477,478 551 284 23,28 280,450 404,420,427 252 25 391,413,422,423

820

microsomal monoclonal antibodies nuclear form nuclear localization nuclear receptor nuclear translocation nuclear type II sites oligonucleotide binding ontogeny phosphoamino analysis phosphorylation processing proteolized forms purification regulatory factor T47 D T47 D c o cells transformation translocation tumor biopsy type II binding sites unoccupied vero-receptor forms virgin mice Estrogen sulfates

378.380 472,473,800 390,543,544 539,550,552,553,560 437,543,544 21,25,28 401 511,515 478 291 279,285-288,293 438 7,22,23,178 176 439,450-453

Flutamide

596

Gene cloning Gene expression Gene regulation GH 3 cells estrogen receptor distribution prolactin synthesis Glucocorticoid receptor activation affinity labeling antibodies AtT-20 cells degradation dexamethasone mesylate DNA binding domain gene cloning hormone binding site, domain kinase activity kinetics leukocytes mero receptor

590 383,539-541,587 758 557,558 539,550,552,553,555,560 556

662 662,680

380.381 21

795 399,447 262,448,539,550 5 427 491,493

102,199,225,309,637,646 111

119 86

637 115,367 89,91,99,123 107 89,91,92,114 336 637,644 637 87,94,638

821

molecular weight molybdate-stabilized mutants nt mutants phosphorylation physiologic significance proteolysis proteolytic map proteolytic pathway purification pyrophosphate, e f f e c t s on rat thymus ribonuclease sensitivity RNA interaction structure subunit dissociation subunits sulfhydryl group association translocation inhibitor variants Glucose oxidation Gonadal steroids

117,119,129,132 142,639 95 95,205,212 132,309,367-371 141 86,91,639 92 92,93 134,308,318,324 238-240,646 637,638 96,98,99 97,99 637 86,88 145,150 166,648 225,226,229 132,133,205 415 706

Heterogenous receptor forms High performance chromatofocusing ion exchange liquid chromatography size exclusion Histone acetylation Hormone binding domain Hormone-dependent tumors

144 802 807 791 797,798 485 89,91,114,134 691

Immunoaffinity chromatography Immunoblotting Inhibitors of ATP binding Insulin receptors

66,76 43 359 681

Keoxifen

604

Leukocytes, human LHRH Lithium Low molecular weight inhibitor

637 386,389 214 260

Mammary gland estrogen action estrogen receptor progesterone receptor responsiveness

404,414 422,423 416 404,414,418

822

Mammary tumors Mammary tumor virus, mouse MER 25 Microsomal acceptors Minicolumn analysis Molybdate effects mRNA, testosterone-induced Muscarinic receptors

404,623-627 125-128 603,604 382 641 50,142,299,300,305,360,365,424,742 597 708

Nafoxidine Neuromodulators Neurotransmitters Non-steroid binding subunits Noradrenergic regulation Nuclear acceptor sites Nucleotides

603,604 701 701,716,723 147 719 438,577-579 199,230,351

oligonucleotide binding o-phenanthroline Oxytocin receptor

512 359,360,365 459,460,464,465

Phosphatase inhibitors Phosphatases Phosphocellulose binding Phosphorylation of estrogen receptor glucocorticoid receptor progesterone receptor Photoaffinity labeling Polyribonucleotides PPi effects Pregnancy Progesterone action Progesterone binding globulin Progesterone receptor activated form antibodies, against breast cancer cross-linking DNA binding fetal tissue immunoaffinity chromatography mammary gland monoclonal antibodies non-activated form nuclear processing ontogeny

101 101,289,304,452,453 564,565 279,285,293 132,309,367,369,370 299 66,79,111,574-577,672 646 464 439,447,498 475 34,38,69 37,55 659,671,684 49 671 475,477,479 66,76 416 37,55,61,64,72 34,38,45,46,299 666 478,483

823 phosphoamino acids phosphorylation photoaffinity labeling physiological significance purification uterus receptor form, 8S regulation by progestins steroid affinity chromatography stimulation by estradiol subcellular distribution subunits T47Do0 cells transformation Progesterone withdrawl Progestin action therapy treatment Prolactin messenger RNA synthesis Prolactin-estrogen interplay Prolactin gene transcription Proteases Protein synthesis Proteolysis Purification of estrogen receptor glucocorticoid receptor progesterone receptor Pyridoxal 5'-phosphate R 5020 RU 38486 Receptor activation adenine nucleotides, by allosteric mechanism ATP, by hormonal requirement KCl, by mechanism of nucleotides, by Receptor binding factors Receptor kinase activity Receptor recycling Receptor regulation

302 299 66,79,672 141 36-44,63,69,300,301,305 61,801 299 665 300,301,305 484,496 51 38,61,77,78,145 666

35,48 455-460 660,678,719 684,685 659,684 540 540 419 539-541 3,6,7,16,23,86,101 485 91,143,205 176 134,309,318 36-44,69,300,301,305 231,359,360,365 674,676 682,684 199,209 173,353 263 257 256,353 199,351 97,104-106 304,306,336,371 292 386,391,437,439,451

824

Receptor stabilization by molybdate Receptor subunits Ribonuclease Ribonucleic acid Rifamycin AF/103 RNA polymerase

299 38,61,77,78,145 96,99 97,100,332 359,360,365 485

Steroid affinity chromatography Steroid receptor achyla antheridiol brain, in the DNA binding fungi interaction with nucleotides isoforms kinetics mechanism of action modification non-hormone binding subunits oligonucleotide binding physicochemical properties regulation T47D C0 cells

300,301,305,318 733-754 736,737 702 123-128,236,505,671,783 733 351 791,794,806,809,810 644 759 164 55,80 512 761 386 660

Tamoxifen Testosterone, responses to Tetrachlorodibenzo-p-dioxin receptor Tfm/y mouse Translocation modulators ATP-stimulated low molecular weight Triamcinolone acetonide Trioxifene Trypsin Tungstate Tyrosine aminotransferase induction Tyrosyl receptor kinase

493,495,603-635,687,690 591 755 595 225,226 236 225 117,118 604 91,93,94 232 113-115 290

Uterine cytosol inhibitor Uterotrophic effects Uterus androgen receptor estrogen receptor fetal and new born pigs progesterone receptor

402,403,407 482,496 437-465 566,567 471,514 471,472 471

Vanadate Variants glucocorticoid receptors

452 132,133,205,211

w DE

G κ. Fotherby

s. B. Pal (Editors)

Walter de Gruyter Berlin-New York Hormones in Norma! and Abnormal Human Tissues Volume 1 1980.17 cm χ 24 cm. XIV, 658 pages with figures and tables. Hardcover. DM 145,-; approx. US $48.40 ISBN 311 0080311

Volume 2 1981.17 cm χ 24 cm. XII, 552 pages with figures and tables. Hardcover. DM 135,-; approx. US $45.00 ISBN 3110085410

Volume 3 1982.17 cm χ 24 cm. X, 297 pages with figures and tables. Hardcover. DM 150,-; approx. US $50.00 ISBN 3110086166

K. Fotherby S. B. Pal (Editors)

The Role of Drugs and Electrolytes in Hormonogenesis 1984.17 cm χ 24 cm. XII, 360 pages. Numerous illustrations. Hardcover. DM 180,-; approx. US $60.00 ISBN 311008463 5

K. Fotherby S. B. Pal (Editors)

Steroid Converting Enzymes and Diseases 1984.17 cm χ 24 cm. IX, 261 pages. Numerous illustrations. Hardcover. DM 180,-; approx. US $60.00 ISBN 3110095564

Prices are subject to change without notice

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Walter de Gruyter Berlin-New York

DE

G K. Fotherby

Exercise Endocrinology

S. B. Pal (Editors)

1985.17 cm χ 24 cm. XII, 300 pages. Numerous illustrations. Hardcover. DM 230,-; approx. US $76.70 ISBN 311 009557 2 There has been a marked increase in the amount of interest taken in exercise physiology during the past few years. This is partly due to the higher standards demanded in most sports and the most efficient use of strength, energy and stamina required of the sports-person, which can have a significant effect on the final result. This book will be of importance to both "trained" and "untrained" subjects in helping them to understand the physiological changes which take place during exercise.

Μ. K. Agarwal (Editor)

Μ. K. Agarwal (

Editor

)

Hormone Antagonists 1982.17 cm χ 24 cm. IX, 734 pages. Numerous illustrations. Hardcover. DM 180,-; approx. US $60.00 ISBN 311 0086131

Principles of Recepterology 1983.17 cm χ 24 cm. VII, 677 pages. Numerous illustrations. Hardcover. DM 220,-; approx. US $73.30 ISBN 311009558 0

Μ. K. Agarwal M. Yoshida

Immunopharmacology of Endotoxicosis

(Editors)

Proceedings of the 5th International Conference of Immunology. Satellite Workshop. Kyoto, Japan, August 27,1983 1984.17 cm χ 24 cm. XIV, 376 pages. Numerous illustrations. Hardcover. DM 170,-; approx. US $56.70 ISBN 311 009887 3

Μ. K. Agarwal (Editor)

Adrenal Steroid Antagonism Proceedings · Satellite Workshop of the VII. International Congress of Endocrinology. Quebec, Canada, July 7,1984 1984.17 cm χ 24 cm. VIII, 399 pages. With numerous illustrations. Hardcover. DM 175,-; approx. US $58.30 ISBN 3110100908

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