Recent Advances in Steroid Hormone Action 9783110850260, 9783110107623

Table of contents :
Preface
Contents
STEROID HORMONE RECEPTORS: RECENT ADVANCES
Interaction of Steroid Receptors with DNA and Chromatin
A NEW MODEL FOR STEROID REGULATION OF GENE TRANSCRIPTION USING CHROMATIN ACCEPTOR SITES AND REGULATORY GENES AND THEIR PRODUCTS
SPECIFIC ASSOCIATION OF ANDROGEN RECEPTORS AND ESTROGEN RECEPTORS WITH THE NUCLEAR MATRIX: SUMMARY AND PERSPECTIVES
ANTIESTROGEN ACTION: PROPERTIES OF THE ESTROGEN RECEPTOR AND CHROMATIN ACCEPTOR SITES
CHARACTERISTICS OF ESTROGEN RECEPTORS ISOLATED FROM ESTROGEN RESPONSIVE AND UNRESPONSIVE NORMAL MOUSE MAMMARY GLANDS
Cloning of Steroid Receptors and Gene Expression
THE OESTROGEN RECEPTOR: STRUCTURE AND FUNCTION
THE GLUCOCORTICOID RECEPTOR: PURIFICATION, CHARACTERIZATION AND CLONING OF THE cDNA
ROLE OF ANDROGENS IN THE REGULATION OF GENE EXPRESSION IN THE MOUSE AND RAT PROSTATE
REGULATION OF EXPRESSION OF XENOPUS VITELLOGENIN GENES BY ESTROGEN
ESTROGEN RECEPTOR REGULATION OF VITELLOGENIN AND RETINOL BINDING PROTEIN GENE EXPRESSION
Steroid Hormone Receptors: Structure and Modifications
ROLE OF cAMP-DEPENDENT PROTEIN KINASE IN GLUCOCORTICOID RECEPTOR FUNCTION
STEROID HORMONE RECEPTOR PHOSPHORYLATION
THE RAT UTERUS AS A MODEL FOR STEROID RECEPTOR AND POSTRECEPTOR CHANGES DURING AGING
Analysis of Steroid Receptors in Cancer Cells
AUTOCRINE REGULATION OF BREAST CANCER CELL GROWTH BY ESTROGENINDUCED SECRETED PROTEINS AND PEPTIDES
WILD-TYPE AND MUTANT GLUCOCORTICOID RECEPTORS OF MOUSE LYMPHOMA CELLS
ESTROGEN STRUCTURE-RECEPTOR FUNCTION RELATIONSHIPS
Advances in Methodological Approaches
IMMUNOCHEMICAL ANALYSIS OF THE GLUCOCORTICOID RECEPTOR
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF STEROID RECEPTOR PROTEINS: RAPID HIGH RESOLUTION CHARACTERIZATIONS AND THE OPPORTUNITY FOR RESOURCE CONSERVATION
PURIFICATION OF VARIOUS STEROID HORMONE RECEPTORS (GLUCOCORTICOID, PROGESTERONE, MINERALOCORTICOID) BY AFFINITY CHROMATOGRAPHY. DESIGN OF SUITABLE ADSORBENTS AND THEIR APPLICATIONS
Author Index
Subject Index

Citation preview

Recent Advances in Steroid Hormone Action

Recent Advances in Steroid Hormone Action Editor V K. Moudgil

W DE

G Walter de Gruyter • Berlin • New York 1987

Editor Virinder K. Moudgil, Ph. D. Professor of Biological Sciences Chairman, Interdepartmental Biochemistry Oakland University Rochester, Michigan 48063 U.S.A.

Library of Congress Cataloging in Publication Data Recent advances in steroid hormone action / editor, V. K. Moudgil. p. cm. Includes bibliographies and indexes. ISBN 0-89925-313 X (U.S.) 1. Steroid hormones-Receptors. I. Moudgil, V K. (Virinder K.). 1945[DNLM: 1. Gene Expression Regulation. 2. Receptors, Steroid-analysis. 3. Receptors, Steroid—physiology. 4. Sex Hormones—physiology. 5. Steroids-physiology. WK150 R2947] QP572.S7R39 1987 599'.01927~dcl9 DNLM/DLC 87-30300

CIP-Kurztitelaufnahme der Deutschen

Bibliothek

Recent advances in steroid hormone action / ed. V K. Moudgil. - Berlin ; New York : de Gruyter, 1987. ISBN 3-11-010762-7 NE: Moudgil, Virinder K. [Hrsg.]

Copyright © 1987 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 nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. Binding: Luderitz & Bauer GmbH, Berlin. - Printed in Germany.

Preface Involvement of steroid hormones in numerous complex physiological and developmental processes is well known. Influence of steroids on mineral balance, regulation of normal and tumor cell proliferation, sexual differentiation, and regulation of reproductive function has long been recognized. Ever since the initial characterization of estradiol receptor by Toft and Gorski, the literature on receptors for various steroids has grown to enormous proportions. In the volume "Molecular Mechanism of Steroid Hormone Action" published in 1985, a comprehensive account of recent advances in steroid receptor biochemistry was attempted to provide the reader with an overview and state-of-the-art information on various aspects of steroid hormone action. Since the publication of that volume, many reports have appeared that provide a glimpse at the molecular organization of steroid receptors. Applications of new technology have assisted investigators in closely examining facets of steroid receptor structure and function which provide clues and insight into the gene-regulation by these fascinating proteins. In keeping with these advances, the emphasis of this volume is centered on the molecular aspects of steroid receptor structure and function. The volume is compiled of chapters from eminent investigators who have made significant contributions in the area of steroid hormone action. The chapters have been prepared to provide a reader with sufficient background, methodological details, and discussion that is easy to comprehend. The book has been organized into several sections. The purpose of the introductory chapter is to summarize the detailed accounts presented in each contribution and to discuss certain aspects of steroid hormone receptors not covered in the individual chapters. The generally accepted model of steroid hormone action dictates that receptors are regulators of gene expression. This postulation is based on the known interaction of steroid receptors with nuclear components of target cells. An entire section devoted to this topic includes contributions from the laboratories of Drs. Spelsberg, Barrack, Ruhs and Shyamala. A closer examination of structure and function ofsteroid receptors is now possible due to recent advances in the molecular biology of steroid receptors. The developments in the field of cloning of receptors and regulation of gene expression are the theme of the section composed of chapters reviewing the work from the laboratories of Drs. Chambon, Govindan, Parker, Tata and Shapiro. It has long been recognized that steroid hormone receptors are labile entities. In recent years, it has become evident that phosphorylation-dephosphorylation processes may modulate hormone function. Chapters by Drs. Gruol and Bourgeois, and Rao and Fred Fox, provide a glimpse into the modification of receptor structure by phosphorylation. Age-dependent modifications in the function of steroid hormones have been traced to alterations in estradiol receptor by Dr. Chuknyiska. With

VI

the evidence that the progression of certain hormone-dependent cancers can be arrested by endocrine therapy, interest in the characterization of steroid receptors in malignant cells has been accelerated and is the focus of discussion in chapters by Drs. Vignon and Rochefort, Gehring and Brooks and co-workers. The magnitude of success that has been achieved in the molecular biology of steroid receptors owes its origin, at least a good part of it, to new methodological developments in the field, including the areas of immunochemistry, chromatographic high resolution and purification of steroid receptors. Contributions by Drs. Formstecher and Lustenberger, Harrison and Pavlik and their collaborators have illuminated this section with much needed details. I hope the book will serve as a valuable treatise for all interested in the cellular and molecular aspects of steroid-receptor interactions. I am grateful for the support, cooperation and encouragement received from the contributors of this volume. Thanks are due to the staffat Walter de Gruyter for their patience, cooperation and activities in the timely publication of the book. Secretarial assistance of Ms. Rita Perris is greatly appreciated. Parviz, Sapna and Rishi deserve sincere thanks for their support and understanding throughout the duration of this pleasant but challenging undertaking. July 1987

The Editor

Contents Introduction Steroid hormone receptors: Recent advances V K. Moudgil

1

Interaction of Steroid Receptors with DNA and Chromatin A new model for steroid regulation of gene transcription using chromatin acceptor sites and regulatory genes and their products T. C. Spelsberg, M. Horton, K. Fink, A. Goldberger, C. Rories, B. Gosse, K. Rasmussen, J. Hora and B. Littlefield

59

Specific association of androgen receptors and estrogen receptors with the nuclear matrix: Summary and perspectives Evelyn R. Barrack

85

Antiestrogen action: Properties of the estrogen receptor and chromatin acceptor sites M. E Ruh and T. S. Ruh

109

Characteristics of estrogen receptors isolated from estrogen responsive and unresponsive normal mouse mammary glands G. Shyamala

133

Cloning of Steroid Receptors and Gene Expression The oestrogen receptor: Structure and function S. Green, V Kumar, A. Krust and E Chambon

161

The glucocorticoid receptor: Purification, characterization and cloning of the cDNA Manjapra V Govindan

185

Role of androgens in the regulation of gene expression in the mouse and rat prostate M. G. Parker, J. S. Mills, M. Needham, R.White and E Webb

243

VIII

Regulation of expression of Xenopus vitellogenin genes by estrogen J. R.Tata

259

Estrogen receptor regulation of vitellogenin and retinol binding protein gene expression D. J. Shapiro, M. C. Barton, J. Blume, L. Gould, M. J. Keller, D. Lew, D. M. McKearin, D. A. Nielsen and I. J.Weiler

289

Steroid Hormone Receptors: Structure and Modifications Role of cAMP-dependent protein kinase in glucocorticoid receptor function Donald J. Gruol and Suzanne Bourgeois

315

Steroid hormone receptor phosphorylation Kanury V S. Rao and C. Fred Fox

337

The rat uterus as a model for steroid receptor and post-receptor changes during aging R. S. Chuknyiska

367

Analysis of Steroid Receptors in Cancer Cells Autocrine regulation of breast cancer cell growth by estrogen-induced secreted proteins and peptides E Vignon and H. Rochefort

405

Wild-type and mutant glucocorticoid receptors of mouse lymphoma cells U. Gehring

427

Estrogen structure-receptor function relationships S. C. Brooks, N. L.Wappler, J. D. Corombos, L. M. Doherty and J. E Horwitz

443

Advances in Methodological Approaches Immunochemical analysis of the glucocorticoid receptor R.W Harrison, W J. Hendry, M.Turney, E. Kunkel, E.Thompson, R. A. Denton and B. Gametchu

467

IX

High performance liquid chromatography of steroid receptor proteins: Rapid high resolution characterizations and the opportunity for resource conservation Edward J. Pavlik, Katherine Nelson, John R. van Nagell, Jr., Holly H. Gallion, and Richard J. Baranczuk

477

Purification of various steroid hormone receptors (Glucocorticoid, progesterone, mineralocorticoid) by affinity chromatography. Design of suitable adsorbents and their applications R Formstecher and R Lustenberger

499

Author Index

537

Subject Index

539

STEROID HORMONE RECEPTORS: RECENT ADVANCES V i r i n d e r K. M o u d g i l Department

of Biological

Rochester, Michigan 1.

Introduction

In

this

each of

chapter,

this

wishes

a

reader

to be

reading avoid

and

prove sent

a

this

hormone

2. S t r u c t u r e Virtually

all

literature

upon

(SRs)

their

The

lack

was

in v i t r o

steroid

of

partly

to

the

a

is

review

not of

the of

information

does

to

an

who

in-depth purposely written

efforts

possible

that

and

to

pre-

literature

on

references

to

are

steroid

not

covered

receptors

and on

composition

studying

corresponding ligand

ligand.

receptors binding

response

structure of

of

to

Thus

have

re-

itself

may

a

other

hormone

unoccupied

adequately

of

receptor

w h i c h may p r e c e d e

cellular

nonavailability

ap-

one

elsewhere.

structure, the

of

of some

some

chapter

contain

unoccupied The

on

to I

the

structure

to

final

the

duplication it

concentrated

receptor

due

work p r i o r

summarizing

that

have or

of unoccupied on

does

subject

and r e p o r t e d

unexplored. to

it

features

times

contributions.

treatise

has

at

re-

sensitive

techniques.

The p r e s e n c e exposure

her

certain

binding

in

the

Although

properties

prior

salient

developments/systems

studied

alterations

ceptor

a

the

those

and p r o p e r t i e s

modifications (1).

be

action, of

practically

confer

or

comprehensive

physicochemica1

mained

his

the

effort,

with

to an a u t h o r ' s

of

and

receptors

molecules the

redundant

familiar

would

here but are widely

steroid

a

space-effective.

discussions

University,

i t may a p p e a r

references while

complete

steroid or

summarize

While

introduced

as

less

is not

analysis

the use of

by o t h e r s

to

chapter.

summarizing

to

Oakland

48063

I attempt

contributed

peal

Sciences,

of

ligand

has been

b i n d i n g domain o f SRs. of

unoccupied

steroid

known

to p r o t e c t

Earlier receptors

studies to

or

stabilize

demonstrated

elevated

Recent Advances in Steroid Hormone Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

the that

temperature

2 resulted be

in

the

prevented

gand may a l s o DNA

(4).

The shown

boratory

had

chicken

the

(6,

7).

that rat

dependent

on

o f SRs a r e

and

that

in

of

spite

hypothesis result

upon

significant biology

SRs

native

their

the p r e f a c e

to

Hansen

and

Gorski

phase

partitioning

electrostatic estradiol GR

aqueous

then

certain

glycol

mixed

in each p h a s e in

turn

titative

with

amount o f is

properties

The

the the

receptor

of

receptor

assessment

of

of

high

the

of

behavior

of

SRs.

was

That of

voiced

in

aqueous

twoand

rat

been

uterine

successfully

the

process

system

are

weight

phases they for

conformational The

al-

attributes

that and

of

com-

combined with

a

receptor

phase,

which

electrostatic

technique both

are

of

polymers

separate

The a m o u n t

affinity

in

precede

immunochemistry

of

ATPP

the

provide occur

conformational

molecular

When

molecule. the

or

study

the

between

could

liganded

to

preparations, its

any,

(9).

had

in each p h a s e .

function of

result

and

properties

changes

technique

11)

phases of

if

the

series

technique

dextran).

receptor

is a

of

or

the

i n Re f .

two

the

These

ligands.

unexplored,

this

unoccupied

mixtures

in

absolutely (8).

SRs

characterize

This

(reviewed

transformation.

(polyethylene

of

(ER).

and

transformation

remain

employed

properties

site

physico-chemical

in

to

con-

accompany

advances the

structure

(ATPP)

receptor

of

SRs,

(transformed)

conformational

v i t ro

in was that

(GR) w a s

nontransformed

f i r s t volume (10)

by A n d r e a s e n

posed and

the

in

la-

v i t ro

study

binding

either

in

accomplished

of d i f f e r e n c e s ,

recent

of

4S

receptor

conformation

which

receptor

from t h i s

recent

interaction with

that

receptor

and m o l e c u l a r

used

the

1igand-occupied of

terations

in

that

to

with

transformation

from a

steroid

or demonstration

the

structure

emerged

li-

interacting

be

of

could of

unoccupied

could

9S ( u n t r a n s f o r m e d )

suggested

for

of

extent

glucocorticoid of

for

transformation

(PR)

the

picture

occupancy

measure

support

receptor

Presence

P r e v i o u s work

although

i n f l u e n c e d by t h e i r

unoccupied the

the

liver the

observations

A direct

that

3).

receptor

(5).

ligand,

A clearer

(2,

behavior

to be d i f f e r e n t

added

b i n d i n g s i t e s which

1igand

physico-chemical

of

of

steroid

the

a f f i n i t y of

progesterone

demonstrated version

of

demonstrated

absence

lower

cytosol

addition

influence

has been of

loss of

by

allows

unoccupied

quanand

3 liganded (10)

receptors

used

induced which

the change

in

precedes

the

was o b s e r v e d same

PI

that

but

gesting

a

The of

the

also

estrogenic

allowed in

inhibition

support

other

the

changes

have

analysis

and

that

of

1 igandreceptor,

in

of

vitro.

It

ER h a v e

the

is

sug-

but

not

estradiol

causes

a

to

change

independent

in

of

re-

that in

structural

that

from t h o s e

3. S u b c e l l u l a r since in

the

the

literature mones

on

receptor

uterus about

specific proteins

explain

hormone

initial

rat for

the

action,

which

a

to

this

generally

cell

via

to

recently

rat

liver

other

reported GR

at

0°C

receptors

steroid

the

hormone

that

exist

in

of

events model

of

popular

years

the

notion

the a

of

course

proposed to

(15,

steroid bind

has of

mediated

cytoplasm in

was

diffusion

[ H]estradiol

effects

were

accepted model,

passive

the

appear

of

synthesis

two-step

extended

have

characterization

protein

target

in

induces accompany

observations

been

(13)

or

transformation.

(14)

which

have

al .

induced Gorski's

independently

The

may

c a u s e d by r e c e p t o r

twenty

sequence

and

action

from

precede

noted

compared

transition

reports

binding

change

localization

ER

1igand

et

exhi-

unoccupied

conformation

recent

conformation.

Moudgil

between

antiestrogen

receptor

applications

is distinct

that

the

which

4-OH-tamoxi fen was

1 i gand-dependent

receptor

systems.

of

transitions

ligands,

receptor

indicating

the

conformational

properties

in

together,

notion

in

of

Binding

change

1 i gand-dependen t

ing

Gorski

a

coefficient

binding

and a n t i h o r m o n a l

(12).

changes

general

receptor

To

and

conformational

the

partitioning

ER

Taken

conformational

ing

partition in

ER monomer

steroids

estrogens.

Ever

unoccupied

hydrophobic

to hormonal

nontransformed

to

the

nontransformed

differ

Further,

i n d u c e a much s m a l l e r

lab

and

different

less

of

difference

involve by

it

demonstrate

h e a t - 1 r a n s f o r m a t i on

forms

properties.

technique

with

of

Hansen

to

transformation.

bited to

of

unoccupied

ER

makes

t h e ER b o u n d

and

properties

both

properties

ceptor

the

conditions.

an a d v a n t a g e

process

these

receptor

surface

identical to

significantly

that

electrostatic its

under

technique

its

bind-

existed steroid via

specific

target of

in hor-

a

16).

hormone receptor

cells. steroid Accord-

enters in

a the

4 cytoplasm. vivo

The

results

receptor

In

Once

DNA

recent

not

(SRc)

in

years,

to

leading

the

its

to

this

its

the

to

receptor the

translocation

cellular

in

steroidin

SRc

the

interacts

protein

synthesis.

undergone

réévaluation

and

have

challenged,

although

has

seem

in

translocated

influence

model

cytoplasmic

changes

to

nucleus,

assumptions

been

its

unequivocally.

Historically receptor

speaking,

owed

its

autoradiography

the

origin

essentially

in

speed

the

high

Following the

an

hormone

were

in

tion

of

of

all

in

cells

ligands

to

had met

with

at

in

general

terpretation

of

were

When

caused

were

exposed

demonstrated

the

studies

incuba-

radioactive

these

two

observations

decades,

were

of

receptors

Consequently,

nearly

these

uptake

the

cells

Although

for

tissues.

or

of

and work.

isolated

target

autoradiography

temperature

of

earlier

vivo,

a majority

nucleus.

results

the

the

in

cytoplasm.

acceptance

the

of

unoccupied

fractionation

of

receptors

steroid

4°C,

the

the

most

fraction. at

elevated

appear

a

nuclear in

in

slices,

incubated

radioctivity

biochemical

(cytosol)

of

tissue

the

and

cytoplasmic

unoccupied

supernatant

intact

obtained

the

of

employed

administration

radioligand

presence

concept

to

techniques

Accordingly,

to

steroid

conformational

chromatin

and

fundamental

of

some

complex

nucleus. with

binding in

the

not

in-

uni f o r m l y

adop t e d .

Many

factors

tors

in

nucleus were

appear

cellular or

cytoplasm

concentrated

1979; McCormack called

into

in

cells

ing

task

was

who

employed

in

GHg

et

cells

ted

cells.

fer

and

depending

or

were

cell

by

were

the

(19)

could

be

these

subsequent

of

in

tissue

important

experimental 90%

of

the

intact

nucleoplasts

allowed

al.,

localiza-

This

Gorski

nucleoplasts

et

observations challengand

Greene

to

answer

immunocytochemistry that

the

extracts

hormone

intact. of

recepin

(Sheridan

steroid

still

of

appeared

the

buffer

These of

the

centri fugation

ER

whether

different

found

localization

laboratories and

demonstrated

Homogenization

the

with

1980).

enucleation

using

al.

upon

analysis

membranes

undertaken

to

Unoccupied

diluted

and G l a s s e r ,

where

questions

Welshons

contribute

consideration

tion

the

to

compartments.

approaches. unoccupied

of in

appearence

the

dilute of

ER

enucleabuf-

unoccu-

5 pied

receptors

antibodies, lization zation

of

of

the

EE

in

that

associated may

nuclear

sites.

lead

As

changes, of

on

nuclear

on

this

Jensen

(26)

argued

possible

to

has

cytosol

of

localibeen

studies

fraction

receptor

its

loca-

also

above

of

which

is

binding

with

the

the

with

the

prove

SRc to

be

collectively thought

could

correct,

referred

to

simply

dence

or

that

is

as

and

complexing Schrader

stored

its

with

(27)

be

to

necessary

represent

ligand

binding

mation)

are

tended

that

SRc

state

evolving

absence

of

are

intra-

of

evidence much

subcellular

a

and in

(28).

the

regulatory

ear-

it

exclu-

was at

models

localization

nuclear

localization

of

SR

in

(29).

the

con-

elements

revised

accomplished

of

transforof

Although

be

accepted

(26). of

phenomena

model

of

is universally

plasma

entity

to

remains

evi-

without

tenets

Furthermore,

proposing

the

(activation/

origin.

of new

compelling from

since

is

of

compartment

revised

nuclear

support

no

it

portion

result

nonreceptor

proposed

cellular

a

fundamental

alterations

views.

means,

small

hormone

unchanged

SRc

a

is

receptors

opposing

as

nuclear

the

recognized

place concepts

hormone

with

the

of

that

there

the

remain in

out

steroid

that

subsequent

receptors, of

or

suggested

their

overwhelming

question

to

localization

of

unfilled

of

and

gardless of

uptake

steroid

technical

cytoplasm

Also,

transfer

unchallenged

nuclear

rule

of

generated

current

the

pool.

hypothesis

has

the

in

receptor

has

two-step

with

present

demonstrating

membrane

localization

topic

unequivocally

receptor

synthesis

an

and

were

HRc,

nuclear

the

of

association

which

literature

sive

the

postulations

discussion

lier

of

monoclonal

nuclear

event ( s) .

the

total

in

t r a n s f o rma t i on and w e r e

translocation

The

results

nucleus,

these

anti-ER

steroids

population

tighter

grows, not

cells. other

recovered that the

a

Should or

nuclear

nuclear

The

with to

conformational

for

(21-25).

Using

demonstrated

target for

receptor

hormone

activation

of

also

receptors

represent

loosely

the

(20)

a number

the may

fraction.

Greene

reported

homogenates

as

cytosol

and

unoccupied

subsequently suggest

in

King

there

rethis is of

before

the

presence

and

6 4. A c t i v a t i o n / t r a n s f o r m a t i o n Upon

tissue

homogenization,

recovered

in

incubated

with

little

to

ties

the

of

vation the

in

form

matin,

(6,

a

form

exhibits

accumulated

transformation under

cussion

on

of

recent

SRc

of

of

intact

transformation of developments

which

A)

o f RNA

the

RNA

(36-54).

past a

Liao

and

and e s t r o g e n (44, of

45).

be

The

preparations Such

of

SRs

al.

that

were to

in

in

its

transformed or

chro-

ATP-Sepharose,

cells

(31).

has

(35).

roles

and

Although has

been

systems,

the

also

The

receptors will

has

as

been

de-

following

dis-

f o c u s on

some

f o r RNA,

heat-

heat-stable

of

that

that

crude

the

the

SRs

androgen a n d RNA structure factor

cytosol

exogenous

increased

of

protein

RNase-labile

with in

accumulating

function

suggested

demonstrated

changes

been

and

complexes

had e m p l o y e d

their

refered from

nuclei

cell-free

structure

incubated

resulted leading

been

receptor The

acti-

describe

transformation

originally

a

studies

to

also

are

proper-

known a s

4-5S.

evidence

(38)

by

altered

cytosol

processes.

the

may e x i s t

et

initial

treatments

perties

in

coworkers

Hutchens

has

implicate

years,

role

receptors

GR c o u l d

cytosol.

several

plays

in

the

treatments

isolated

target

steroid

temperatures of

kinetics

conditions

and p h o s p h o r y l a t i o n

that

as for

performed

in

by

used

cytosol

receptor

shock p r o t e i n s

During

the

dissociation

physiological

Involvement

been

very

binding

physicochemical

have

sediments

process

studies

of

the

exhibit

be

conditions,

above

phosphocel1ulose,

its

the

from

scribed the

in

about

elevated

are

can

nuclear

collectively

terms

affinity

and

ionic

transformation

that

increased

alteration

knowledge

term

high

The

they

that The

incubation

34). in

receptors

where

31).

at by

receptors

alteration

DNA-cel l u l o s e

shows

33,

(both

The

v i t ro to

7,

hormone

transformation

an

receptor

preparations and

hormone

complexes

(30,

in v i t ro under (32),

receptors

fraction

form

some a l t e r a t i o n s

steroid

same p r o c e s s ) .

8 - 1 OS

sites

hormone

induce

or

as

to

0°C

hormone

steroid

nuclear

receptor

of

the

supernatant

hormone

for

10 nM ATP a t

thought

to

the

the

presence

with

speed

can be a c q u i r e d

incubating the

high

affinity

capacity

of s t e r o i d

in

receptor

ribonuclease.

physicochemical

DNA b i n d i n g

and

proslower

7 sedimentation

rate.

been

(47,

reported

ted by

that

ribonuclease

hydrolyzing

48,

49,

have

51).

RNA

identified

the

9-10S

form,

low

and

the

9-10S low

molecular

hepatic

tor,

RNA

and

(49)

have

inhibits

thought

to

factor,

monomeric

be

a

4S

7.7

important

However,

the

cytoplasmic analysis

receptor

the

which

(57)

stabilizes

of

receptor

itself.

composed

of

Rowley

receptor

a

role

of

the

is

in an

et

al .

complex of

the

the

more

of in-

process

integral

is re-

association

merited

RNA

RNA

of fac-

reportedly

integrity SRs

of

and w h e t h e r

which

nonspecific

nontransformed

as

Accordingly,

structure

structural

putative

(46,

a].

et

DNA.

to

RNA component

the

receptor form

chymotrypsin-sensitive

receptor

the

for

transformation,

a

androgen

possibility

RNA w i t h

of

binding

a hetero-oligomeric

weight

identified

factor

sedimentation

also

sugges-

of

oligomeric

and Tymoczko

weight

its

GR h a s

studies

transformation

(56)

promotes

r i b o n u c l e o p r o t e i n;

ceptor. depth

molecular

GR i s

the

caused

these

nontransformed

and Tymoczko

digestion,

this

contains

its

puri fied

some o f

of

treatment

from

a

GR t o RNase

o f RNA w i t h

Results

Anderson

the

some

Association 48).

of

part

of

SRs. Although

the

remains

question

unsolved,

significance may

be

into

In

nucleus

or

recent

years,

which

integral

(51). fied

initially

as

a

transfer

mechanisms tional However, detect

of

RNA (tRNA)

or

recent

RNA i n p u r i f i e d , tumor

of

cell

line

the gene

may

that

by

the

(52).

was

reflect

expression of

this

via

The

(50,

turn51).

regarding

studies

RNA-

and

RNA may

postulated

cobe

mouse GR

was

identithat

the

post—transcriptional influencing

hormone-induced laboratory

conclusion

transfer

influence

3 6 , 000

u n t r a n s f o r m e d mouse GR (58).

its

transformed

above It

GR may

potential interaction

Vedeckis

a Mr

oligomeric in

from

it

observations

stabilization work

of

SR-RNA

complex,

reported

5.2S

reported

regulating

more

the

or

association

o f gene e x p r e s s i o n

suggested

of

activity

efficiency

been

are

The the

retention,

important

have

The RNA s p e c i e s

particles

of

regulation

some

component

tRNA b i n d i n g

tary

nuclear

interaction

receptor-RNA

functioning.

stabilization

s p e c i f i c RNA o r

workers, an

cellular in

receptor

the

ribonucleoprotein

for

involved

the

over o f

regarding

has

translaproteins. failed

from A t T - 2 0

thus

became

to

pituiinescap-

8 able

that

and to

the

contains

untransformed

no

RNA.

dissociation

of

mouse

Accordingly, the

oligomeric

GR

is

RNA

binding

entirely

proteinaceous

occurs

untransformed

subsequent

GR-complex

into

monome r s .

The

role,

if

any,

transformation,

that

has

raised

the

question

formed

SR,

as

of

receptor

ployed

the

portant

role

in

transformed

from

complex

sedimentation

and

bilized

form

peptide. two

8S

the

A

SRs

Toft

paration

the

the

B

A

and

not

of

peptide and

90

peptides

K

type

bind

PR

plus II

type K)

nature em-

subsequent play

to

an

stability

gene

im-

of

a

expression

of

reported

90

to

PR

in

the

8S,

contained

(Mr of

110

K)

(63).

PR

using

al.

peptide,

binding exist

which

avian that

et

II

that

the

peptide. as

not

did

of

the et

their

highly

complex not

phosphoproteins.

al . by prewith

purified

the

ste-

peptide

Furthermore,

K

containing

bind B

of

employed

in

K

and 90

associated

the

sta90

peptide

Birnbaumer

obtained

which

sedimen-

existence

90K

methods

was

contained

molybdate,

contained

and

8S

a

a major

the

the

(65)

I

a

molybdate-

90 K p e p t i d e

type

as and

Laboratories

contained

demonstrated

type

of

slower

Consequently

which

complex

to

SRc

further

contained K

presence

of

exist

300,000

hetero-oligomeric

I

progesterone

that a

the

SRs

around

native

preparations

and

Dougherty

8S

in

avian

These

reported

non-steroid

were

untrans-

the

may

cytosols,

The

60).

receptor

Subsequently,

roid;

(59,

puri f i c a t i o n

Toft

preparations

in

have

receptor

is

and

receptor

with by

of

still

weight

8-10S.

isolated

79

tissue

transformation

laboratory

the

did

purity

alteration

stabilized

62).

(Mr

attempted and

of

functioning

target

of

form

forms,

receptor

Baulieu

be

had

Toft' s

and

RNA

influenced

association

molecular

block

(61,

receptor

peptide

PR.

can

to

binding

Baulieu

of

coefficient

shown

DNA

(64)

preceding

extraction

protein

ting,

of

of

studies

cells.

their

of

RNA

effective

Proteins

agent

degree

the

Heat-Shock

structure

was

process Recent

polyribonucleotide

of

large

an

the

the

association

the

If

the

receptor

SR-responsive

Upon

or

in

complex.

reported,

studies.

activation,

have

more

whether

preparation

for

Role

of

previously

receptor

B)

RNA m i g h t

become

all Joab

plus three

et

al .

9 (66)

extended

reported

the

hormone tors

these

binding

of

four

lated

that

exists

in

observations

presence

of

the

component

steroid

suggests a wide

in the

range

of

other

systems

peptide

nontransformed

hormones. that

to

90 K Da

receptor

a

chick

Additional

nonhormone

as

when

oviduct

evidence

binding

systems,

they

common,

recep-

has

accumu-

component

tissues

non-

of

and

SRs

species

(67-71) . The

relationship

non-steroid metry

of

with

be p r e s e n t Although widely tion,

that

the

in

the

the

and

These

conditions

Eukaryotic by or

in

di f f e r e n t

molecular different literature

proteins

highly

proteins

and

of

90 K p e p t i d e

K hsp

(hsp

GRc lost

as

cell

a

recently contain on

com-

binding

and

and

as well

a

trans-

nontransformed steroid

is

prepara-

as

90),

Their are

heat

the

mode

in

their the

existence

disunder

known

to

weights

8,000

clues, a major

other

such

led hsp

the

heat-

action

of

presence

is

in

These reported

di f f e r e n t

constitute -

90,000.

including

that

of

organism.

occur

proteins

SRs a s

environmental

has been

shock

indirect

like

to

understood,

90 KDa p e p t i d e , of

respond

specific proteins,

tolerance

molecular

of

of

not

they

t h e r e w e r e many

cation

of

is

thermal

Chicken

abundance

90

re-

it

only

phosphoproteins

Although

conserved.

systems

sizes.

that

intact

organisms

(72).

(hsp)

molecular the

in

65).

of

(71)

is

containing

producing a set

proteins

are

al .

to

(64,

receptor exists

et

es-

(71).

to c o r r e l a t e w i t h

proteins

in

that

are

been

appears

process

nontransformed

suggest

activation

prokaryotic

stress

heat-shock known

and rapidly

the

and

Mendel

proteins

not

demonstrated,

is absent

structures

thermal

in

phosphoprotein

binding

has

the

stoichio-

peptides

directly

forms.

and

the

90 K p e p t i d e

of molybdate

investigators

heteromeric

during

cell-free

absence

binding

90 KDa n o n - s t e r o i d sociate

been

molybdate-stabi1ized

K non-steroid

formation.

the

90 K p e p t i d e

not

8 - 1 OS SR

and

hormone-binding

90 K p e p t i d e

that

subunits

clear,

8-10 S complexes

the of

binding

not

However,

known

reported

shock

is

has

of

stress

the

over

involvement

puri fied

plexes

steroid

transformation

component 90

forming

certainty.

in e x c e s s the

the

90 K p e p t i d e

proteins

tablished

ceptor

between

binding

to

the

four In

the

size

and

the

identifi-

(73-75).

Although

proteins,

increases

in

10 abundance

when

cells

are

stressed

certain cytotoxic agents, protein In o r d e r

to e l u c i d a t e

reported

success

gene

encoding

this

functions because 90

tein

of

of

hsp,

its

is also a m a j o r (72,

in

hsp

90

to

form c o m p l e x e s

which

are

The

hsp

kinases

and

sociation forms bine

4S,

the

cell-free

to d r a w

this

to

90

Catelli segment

known

inactive

to

binding

of

protein

of SRs a n d

SRs be

( 73,

Dis-

DNA

with

recom-

the

8-10S

may,

binding

to

hsp in

regulation

in the p u t a t i v e

in intact

Vs

90

receptors,

established

Processes

77).

tyrosine

complexes

inactive receptors

Phosphorylation-Dephosphorylation

including

attempts

binding

will

pro-

complexes.

active

If

forms

non-DNA

example,

viral

these

their

the

tyrosine

kinase

both

al.

attention

oncogenes,

with

et for

biochemical For

of

protein

from

complexes

interesting

by

cytosolic

significant

viral

form

hsp

inactive

transformation

of

tyrosine associate

equilibrium between active and

C)

to

communication).

DNA

generating

of

of

personal

transformed

role

to

receptors

transform

in

be

separation

(Baulieu,

succeed the

90 m a y

steroid

or

therefore,

90,

cDNA

w i t h a number

products

vi r u s - 1 r a n s f o r m i n g

of

hsp

short

addition

likely

Rous Sarcoma role

of

a

In

is

ubiquitous

or

76).

cloning

protein.

temperature

r e l a t i o n w i t h SRs or o t h e r p r o t e i n s .

is k n o w n

kinases

elevated

characteristics

(75)

hsp

it

in u n s t r e s s e d c e l l s

by

the of

cells.

Transformation

of R e c e p t o r s Several

mechanisms

transformation mechanisms and

of

include

dissociation,

One

of

is

one

tions

the

modulate

roots

in in

the the

been

steroid

proposed

receptors

proteolysis and

hypotheses

suggesting

perhaps

have

of

that

has

the in

process 31).

subunit

alterations

attracted

of

intact

The

cells.

effects

steroid origin of

of

These

aggregation in

receptor.

considerable

attention

phosphorylation-dephosphorylation

transformation initial

explain

receptor,

conformational that

to

(reviewed

receptors of

this

molybdate

in

reac-

vitro,

hypothesis

observed

on

or has the

t r a n s f o r m a t i o n of SRs. Toft

and

such

as m o l y b d a t e ,

coworkers

first

reported

tungstate,

and

that

phosphatase

vanadate

stabilized

inhibitors the

avian

11 PR a n d

blocked

binding

form

its

thermal

( 59,

78).

activation These

a c t i v a t e d SRc.

Furthermore,

reported

stimulated

line

to

phosphatase

suggested of

be

the

80).

that

Alternatively, on

SRs

are

dephosphorylation ported such

by

as

the

a

laboratory

that form

had and

In

studies,

these

inhibited

high

a

ions;

proposed

of

the

suggest

field

SR

is

via

their

manner

or

that

action

of

steroid

of

is

a

receptor

some

may

be

of w h i c h m a y

the receptor p r o c e s s i n g or d o w n r e g u l a t i o n of 5. W h a t The of

is the role of

last SR,

few y e a r s

some

amino a c i d

brought

appear

to

highly

focus

the

thought

to c o n t a i n

the h o r m o n e b i n d i n g d o m a i n

301 and

552

Occupancy

logic

conditions Using

group

found

a

region

is

that

E

is

of

receptors

hydrophobic

of h o r m o n e

thought

series

DNA-

whether

for

trans-

developments in a

in

sequential in

associated

the with

functional

domains

(85).

primary

s e q u e n c e of h u m a n a n d c h i c k e n E R a p p e a r

regions;

DNA.

of

to

receptors.

conserved

distinct

(86).

be

hormone?

have

of w h i c h

as

involved

be

83). to

transition

extraction

occur

of

(82,

seen

to

this

effect

corequisite

may

from

these

new

trans-

receptor

SRs

was

question

or

phosphorylations

hormones,

with

sup-

phophatase

direct

GR

Interesting

(79,

inhibitors,

results

a

in

was

was

receptor

with

The

pre-

view

nontransformed to

it

molybdate

alkaline

activated

phosphorylation

multiple

of

effective

open.

block

interaction

receptors.

that

phosphatase to

clues

of

were

still

of

was

alka-

p h o s p h o r y l a t ion-

latter

Furthermore,

first

binding

that

other

79).

dephosphorylation

formation

The

failed

of G R

uterine

dephosphorylation

involve

that

the

component(s)

not

the

latter

a

have

(81-84).

on

observations,

concentrations

or nuclear

calf

studies

preincubation

the

cellulose-bound

with

involves

may

ATP-Sepharose effect

transformation

and

78,

DNA

or no

regulatory

conversion

(59,

tungstate

via

SRc

fluoride,

provided

molybdate

receptor

or

had

these

some

mechanisms

to d e m o n s t r a t e

transformed

metals

other

on

of

of

observations

and

w e r e used

or

direct

levamisole

formation,

Based

itself

a DNA

incubation

transformation

receptor

actions

the rate of

by

(79).

to

compounds

to

human

induce ER

containing

in

The

to c o n t a i n

character

between amino

binding domain the

deletion large

and

receptor mutants, deletions

at to

six is

acids

physiobind

to

Chambon's within

the

12 hormone was

binding

suggested

domain that

failed

the

for g e n e a c t i v a t i o n , binding domain may

to a c t i v a t e

hormone

and

that

binding the

responsive e l e m e n t , or to p r e v e n t

SRc

that

sence

of

vi vo not

stimulates hormone

(Green

et

supported

termining vi t ro

(88,

bility

al.,

regions

or

DNA

function

GR

of

hormone

in

glucocorticoid

presence unable

gene

in

of

to

the

(91).

are

Beato to

response and

role

of

have

to

in

element

the

receptor,

hetero-oligomer proven

that

to

occupancy for

the

to m a s k i n g

shown

unable

to

full

in

ste-

reported

that

appears

hormone

vivo

has

been

different

a

of

domain

(92,

appears

to the

therefore,

in the

unoc-

view

of

SR

is to be

cannot

Chambon's

be

group

domain

are

At

least

be

nondispensable

differs

without

to

target

subunit

binding

(85).

a

in

for

that

presence

ER,

it

of

is

hor-

93). binding

of m a n y

unoccupied

partition

hormone

in-

require

appears

domain

domain.

however,

transcription

latter

binding

binding

transcription

steroid

target

that

hormone

DNA

Truncated GR,

mone binding domain

observed

of

the

nuclear

of

may,

binding

if the

that

to

GR

elements

Even

possi-

protein-DNA

hormone-free

non-hormone

90.

binding

stimulate

of

a

deleted

gene

steroid

Significance

the

mutants

transcription.

possible

role of

stimulate of

by

such as hsp

correct,

limited has

probably

in

for

be to u n m a s k a p r e f o r m e d D N A b i n d i n g d o m a i n e c l i p s e d cupied

the

and

responsive in

de-

PR

modulate

vivo

is

in

involved

DNA,

in

and

raised

(88) be

GR

necessary

MMTV

could

role

of

pre-

however,

no

of

al.(90)

the

activation

view,

plays

binding

et

but

gene

latter

However,

specific

The

hormone estrogen-

to a c t i v a t i o n

specifically

and

hormone,

to

unclear for

which

not

receptor.

recognize

vivo

GR

vi vo

teraction the

the

DNA

specifically

of

be

the

Willman

binds

partitioning in

the

are

binding

hormone

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Fig. 1 ( L e f t ) . E f f e c t s of hormone presence on the sedimentation r a t e of PR. Chick oviduct c y t o s o l was mixed with 1 )M C o r t i s o l and 0 . 3 nM HVEF and divided into two groups. Cytosol from group I was carplexed with 20 nM [^HJprogesterone ( [ % ] P ) f o r 2 h a t 0°C (+hormone), w h i l e group II c y t o s o l (-hormone) ronained on i c e without hormone a d d i t i o n . A l i q u o t s from both groups were incubated with d i f f e r e n t concentrations of ATP a t 0°C f o r 40 min. At the end of the 40-min p e r i o d , sodium molybdate (20 nM) was added t o a l l m i x t u r e s . Group II (-hormone) sanples were then incubated with 20 nM [3h]P f o r 2 h a t 0°C. A l i q u o t s ( 0 . 2 ml) were layered on separate 10-35% g l y c e r o l g r a d i e n t s containing 20 nM Na^Wo04 and 0 . 1 5 M KC1. The d i r e c t i o n of sedimentation i s from r i g h t to l e f t . The two arrows i n d i c a t e sedimentation p o s i t i o n of the i n t e r n a l mark e r s , peroxidase ( 3 . 6 S ) and glucose oxidase ( 7 . 9 S ) . A/a, C y t o s o l , 40 min 0°C + 20 nM Na^Vto04; B/b, c y t o s o l + Na2Mo04; C/c-F/f, + 2, 5, 10, 15 nM ATP. (Right). A l i q u o t s frcm both groups (^ hormone) were incubated a t 0°C w i t h 10 nM ATP (top) o r 0 . 3 M KC1 (middle) f o r the time p e r i o d s shown. One s e t of a l i q u o t s was brought to 23°C (bottern). Each point r e p r e s e n t s the percentage of the t o t a l a r e a of the r a d i o a c t i v i t y peak sedimenting in the 4S region. A 100% t r a n s f o r m a t i o n means that the e n t i r e peak of t^H]P corresponded t o the 4S region (Taken with permission f r c m R e f . 7 ) .

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F i g u r e 5b E f f e c t of t e s t o s t e r o n e on t h e p r o t e a s e i n h i b i t o r p r o m o t e r in (a) m o u s e p r o s t a t e e p i t h e l i a l c e l l s (MPYK) a n d (b) h u m a n b r e a s t t u m o u r c e l l s (ZR-75-1). Fusion genes were c o n s t r u c t e d w i t h D N A s e q u e n c e s c o d i n g for C A T in the a b s e n c e of a p r o m o t e r (SP6) o r w i t h p r o m o t e r s e q u e n c e s f r o m R o u s S a r c o m a V i r u s (RSV), M M T V (LTR) and the p r o t e a s e i n h i b i t o r g e n e , as follows: a 5 Kb D N A f r a g m e n t u p s t r e a m of the gene w a s i n s e r t e d in the c o r r e c t (pl2) a n d o p p o s i t e (21p) o r i e n t a t i o n r e l a t i v e to the C A T g e n e . A f t e r t r a n s f e c t i o n , cells w e r e g r o w n in the p r e s e n c e (+T) o r a b s e n c e ( - T ) of t e s t o s t e r o n e for 2 days a n d c e l l free e x t r a c t s w e r e then p r e p a r e d for C A T a s s a y s . 4.

Concluding

remarks

A f t e r g e n e t r a n s f e r , the t r a n s c r i p t i o n of M M T V b u t n o t the

cloned

p r o s t a t e g e n e s is r e g u l a t e d by a n d r o g e n s in a w i d e v a r i e t y

of

cells, i n c l u d i n g p r o s t a t e cell l i n e s .

In c o n t r a s t to v i r a l

p r o m o t e r s it is c o n c e i v a b l e that the p r o m o t e r s of c e r t a i n g e n e s are a c t i v e o n l y in the p r e s e n c e of transcription

cellular

cell-specific'

f a c t o r s a n d / o r o t h e r f a c t o r s r e q u i r e d to o v e r c o m e

the a c t i o n of r e p r e s s o r s e q u e n c e s .

S u c h f a c t o r s m a y be absent

i n a c t i v e in cell l i n e s w h i c h in g e n e r a l do n o t m a i n t a i n d i f f e r e n t i a t e d p h e n o t y p e in v i t r o a n d this c o u l d a c c o u n t

or

their for the

256 lack of expression of endogenous genes such as PBP and the protease inhibitor gene in prostate cell lines.

In the absence of

differentiated cell lines, it may be necessary to separate potential androgen response elements from putative repressor sequences or positive regulatory sequences which are inactive in the absence of specific transcription factors.

Thus, we are

fusing potential cellular androgen response elements to the constitutive thymidine kinase promoter in place of the homologous promoter.

In parallel, we are also synthesising oligonucleotides

which are complementary to regions of the prostate genes which exhibit a high degree of homology with the androgen response elements identified in MMTV and testing their ability to confer androgen responsiveness on the thymidine kinase promoter.

An

alternative strategy would be to use transgenic animals and utilize the entire gene with appreciable flanking sequences to ensure that the full complement of sequence elements are present in the gene constructs tested.

This approach also offers the

advantage that it may provide appropriate cell-cell interactions and systemic factors, which may be essential for gene expression.

References 1.

Darbre, P., C. Dickson, G. Peters, M. Page, S. Curtis, R.J.B. King. 1983. Nature 303, 431.

2.

Darbre, P., M. Page, R.J.B. King. 1986. Mol.Cell.Biol. 6, 2847.

3.

Parker, M.G., P. Webb, M. Needham, R. White, J. Ham: J.Cell Biochem. (in press)

4.

Cato, A.C.B., R. Miksicek, G. Schutz, J. Arnemann, M. Beato. 1986. EMBO J. 5, 2237.

5.

Yamamoto, K.R. 1985. Ann.Rev.Genet. 19, 209.

6.

Rushmere, N.K., M.G. Parker, P. Davies: Mol.Cell.Endocrinol. (in press)

7.

Higgins, S.J., M.G. Parker. 1980. In: Biochemical Actions of Hormones (G. Litwack, ed.). Academic Press, New York, p.287.

257 8.

P a r k e r , M . G . , G.T. Scrace, W . I . P . M a i n w a r i n g . 170, 115.

1978.

9.

H e y n s , W . , B. P e e t e r s , J. M o u s , W. R o m b a u t s , P . D e M o o r . E u r . J . B i o c h e m . 89, 181.

1978.

10.

H e y n s , W., P . D e M o o r . 1977. E u r . J . B i o c h e m .

11.

P a r k e r , M . , M. N e e d h a m , R. W h i t e , H. H u r s t , M . P a g e . N u c l e i c A c i d s Res. 10, 5 1 2 1 .

12.

Hurst, H.C., M.G. Parker.

13.

P a r k e r , M . G . , R. W h i t e , J.G. W i l l i a m s . 1 9 8 0 . 255, 6 9 9 6 .

14.

Zhang, Y . - L . , M . G . P a r k e r . 1985. M o l . C e l l . E n d o c r i n o l . 151.

15.

P a g e , M . J . , M . G . P a r k e r . 1982. M o l . C e l l . E n d o c r i n o l . 343.

16.

Chamberlin, L.L., O.D. Mpanias, T.Y. Wang. B i o c h e m i s t r y 22, 3 0 7 2 .

17.

M i l l s , J . S . , M. N e e d h a m , T.C. T h o m p s o n , M . G . M o l . C e l l . E n d o c r i n o l . (in p r e s s )

18.

H i i p a k k a , R . A . , C. C h e n , K. S c h i l l i n g , A. O b e r h a u s e r , A. S a l t z m a n , S. L i a o . 1984. B i o c h e m . J . 2 1 8 , 5 6 3 .

19.

L a s k o w s k i , M., I. K a t o . 1980. A n n . R e v . B i o c h e m .

20.

P e r r y , S . T . , D . H . V i s k o c h i l , K . - C . H o , K . F o n g , D.W. S t a f f o r d , E . M . W i l s o n , F . S . F r e n c h . 1985. In: R e g u l a t i o n of A n d r o g e n A c t i o n (N. B r u c h o v s k y , A. C h a p d e l a i n e , F . N e u m a n n , e d s . ) . C o n g r e s s d r u c k R. B r u c k n e r , B e r l i n , p . 1 6 7 .

21.

P a r k e r , M., H . H u r s t , M. P a g e . 1984. J . S t e r o i d B i o c h e m . 67.

22.

V o n d e r Ahe, D., S. J a n i c h , C. S c h e i d e r e i t , R. G. S c h u t z , M. B e a t o . 1985. N a t u r e 313, 706.

23.

Y a t e s , J., R . J . B . K i n g . 1981. J . S t e r o i d B i o c h e m . 14,

24.

Page, M.J., M.G. Parker.

25.

S h a i n , S.A., R . I . H u o t , L . S . G o r e l i c , G . C . S m i t h . C a n c e r Res. £ 4 , 2 0 3 3 .

26.

K u b o t a , Y . , E . B . G e h l y , K . H . L i n k , C. H e i d e l b e r g e r . In V i t r o 17, 965.

27.

T s c h e s c h e , H . , S. K u p f e r , R. K l a u s e r . 1 9 7 6 . P r o t i d e s of B i o l o g i c a l F l u i d s 23, 255.

1983. E M B O J. 2,

1983. Cell 32,

78,

Biochem.J.

221. 1982.

769. J.Biol.Chem. 43,

27,

1983. Parker:

49, 593.

20,

Renkawitz, 819.

495. 1984. 1981. the

REGULATION OF EXPRESSION OF XENOPUS VITELLOGENIN GENES BY ESTROGEN

O.K. Tata Laboratory of Developmental Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

Introduction During oogenesis in invertebrates and oviparous vertebrates the gradual deposition of yolk proteins, a process known as vitellogenesis, has been commonly used as an index of egg development. With the rapid introduction of methods of recombinant DNA, monoclonal antibodies and in situ localization of macromolecules, the study of vitellogenesis has yielded important information on the developmental and hormonal regulation of gene expression.

This

article will deal with some aspects of the nature of genes encoding yolk proteins (vitellogenin genes), the characteristics of their expression, and how the latter is hormonally regulated.

It will

be largely based on studies on Xenopus vitellogenin genes in the author's laboratory, with particular emphasis on their activation de novo in primary cultures of hepatocytes from male animals ana on the role played by estrogen receptor in regulating gene expression.

The reader is referred to recent reviews on Xenopus

vitellogenin gene expression (1-7).

Vitellogenin The vertebrate egg yolk proteins, phosvitin and lipovitellin, are not individually synthesized but are derived from a common precursor called vitellogenin.

The term vitellogenin was first used for

insects to refer to all plasma precursors of egg yolk proteins (8). Vitellogenins are almost exclusively synthesized in the liver or fat body of all oviparous animals, secreted into the blood and transported to the ovary where they are cleaved and processed into the yolk components.

Recent Advances in Steroid Hormone Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

260 The characteristics of plasma vitellogenins are highly similar in most species, as for the chicken, Xenopus and locust.

In all

species, with the exception of lower insects (mosquito, Drosophila), each of the two subunits is 210 - 250 kDa in size and has a comparable composition in carbohydrate, lipid and phosphorus (1,9). In Xenopus blood multiple vitellogenins have been detected which are encoded by four mRNAs (10-12).

This finding reflects.the

multiplicity of genes of the Xenopus vitellogenin family (see below).

The sequencing of full-length cDNA to vitellogenin mRNA,

as an extension of the analysis reported for chicken vitellogenin (13), will eventually provide accurate information on the organization of phosvitin and lipovitellin within the vitellogenin molecule.

Whatever the internal organization of the vitellogenin

molecule, large parts of the molecule have been conserved during evolution as revealed by immunologic cross-reaction and DNA-DNA and RNA-DNA cross-hybridization.

Hormonal Regulation of Vitellogenin Synthesis The primary signal for the initiation and regulation of egg development originates in the central nervous system in response to environmental stimuli (14).

These are conveyed by neurohormones

to endocrine glands such as the ovary and prothoracic gland in vertebrates and insects, respectively, which synthesize and secrete hormones that directly act on che sites of egg protein synthesis. In vertecrates, estrogen is the only hormone that obligatorily induces and maintains tne syntnesis of vitellogenin in che liver. It is worth noting that a single administration of estrogen to male oviparous vertebrates causes the synthesis and secretion into tne blood of large quantities of yolk proteins and lipids.

The initial

vitellogenic response to a single dose of estrogen dies away upon metabolism or "withdrawal" of the hormone.

A second dose produces

a more rapid and massive formation of yolk proteins and lipids, a phenomenon common to many inducible responses. The possibility of inducing the de novo synthesis of vitellogenin with a single administration of estrogen to male animals offers a unique opportunity to study the activation of permanently silent

261 genes.

Since one can easily study both the induction and de-

induction processes, induced vitellogenesis in the male allows one to better define the roles of such factors as hormone receptors, changes in gene configuration, translational efficiency, secretory mechanisms, etc., underlying hormonally regulated developmental processes.

In Xenopus the physiological primary and secondary

inductions have been fully reproduced in primary cell cultures, which represents another major advantage of this system in analyzing the early events leading to hormonal regulation of gene expression. At the same time, neither the primary nor secondary induction at early stages is complicated by overlapping changes in cell proliferation or DNA synthesis.

The Xenopus Vitellogenin Gene Family Thanks to the work of Wahli's group (2,3,15-20), more is known about the structure and organization of vitellogenin genes of Xenopus than of any other vertebrate species, although an increasing amount of information is becoming available for chicken genes.

As shown in

Fig. 1, the four actively expressed vitellogenin genes of Xenopus laevis fall into two groups and are termed Al, Bl, A2 and B2 (15). There is 80% homology of coding sequence between the two groups and 95% between two member genes of each group.

Each is made up of 34

similar exons but the size of the entire gene varies from 16 to 21 kbp of DNA because of large variations in intron sizes.

The Al

and Bl genes are known for some time to be linked with a 15 kbp of DNA, but it was not known until very recently how the other two genes are organized (17).

Using gene segregation analysis and

restriction fragment length polymorphism, Wahli's laboratory (21) now show that the gene A2 is also linked to genes Al and Bl but the length of the linking spacer is not known.

However, gene B2

segregated quite independently and is not therefore linked to the other three genes. major implications.

This arrangement within a gene family has two First, the evolution of the present day

Xenopus vitellogenin gene family must have involved an unusual pathway of gene and genome duplication with selective loss of one or more genes.

Second, it raises the question of whether or not

they are coordinately expressed when under hormonal control.

262 Xenopus Vitellogenin Genes

I 80% Homology

Genes

Genes

I

I

95% Homology .

A2A2

^ ^ 95% H o m o l o g y g ^

-A1

A1 -un ii i m i i UL.miuj-

? Fig. 1. Schematic representation of Xenopus laevis, their classification and B groups and their linkage. The sequence homology between individual (Data adapted from Refs. 15, 21.)

5'

" B1

-mini iimi '• un ni i—3'

B2 II llll I mil « I 111 III I 17.5kb

3'

the four vitellogenin genes in as two members each of the A figures as % denote the coding genes and the two groups.

Sequencing of the 5' upstream flanking regions (19,20) has revealed both similarities and differences within the gene family, which provide clues to the possible regulatory regions with which the estrogen receptor complex may interact (see pp. 2 7 4 ,

275).

Comparison of vitellogenin genes of Xenopus laevis with those of the more ancient X. tropicalis has shed some interesting light on the evolution of these genes.

The genome size of X. tropicalis is

half that of X. laevis, with three instead of four vitellogenin genes (two of A and only one of B type).

It can be argued that

following a very early duplication of the primordial vitellogenin gene there took place a more recent duplication of the whole genome resulting in four genes in modern Xenopus species (22).

It will

?

263

be interesting to know how the arrangement of the three genes in X. tropicalis corresponds to the A2-A1-B1 linkage in X. laevis.

A

parallel comparison of the vitellogenin genes, their mRNAs and protein products (restriction endonuclease digestion analysis of the DNA and peptide mapping of the plasma vitellogenins) in the more closely related X. laevis and X. borealis and the distantly related X. tropicalis (12) revealed both similarities and differences in the organization of the genes.

Thus, there must have occurred

significant rearrangement within the vitellogenin gene family during evolution. As regards comparisons with other species, the coding and 5' flanking sequences of Xenopus and chicken vitellogenin genes exhibit a high degree of overall sequence conservation

(18-20,23).

What is remarkable is the similarity in gene size, structure and cross-hybridization one observes in vitellogenin genes in insects, nematodes, birds, amphibia and fish (3,24,25).

Small stretches of

DNA sequences in the 5' upstream flanking regions have also been conserved in the more actively expressed Xenopus A1 and B1 genes and one of the three chicken (Vtg II) genes (19).

This indicates

the important role of upstream regions that are involved in their regulation by estrogen (see pp. 274, 275).

Hormonal Induction of Vitellogenin Genes in Whole Animals The complete absence of any vitellogenin-like material or its mRNA throughout its life in the adult male vertebrate liver or blood makes it easier to establish the early stages of hormonal induction of yolk proteins in the male than in the female.

As in the rooster

and other oviparous male vertebrates, a prolonged administration of large doses of estradiol to male Xenopus causes the accumulation of such vast quantities of yolk phosphoprotein that it is the major protein in the blood (26,27). A characteristic feature of steroid hormone induction of egg proteins is the "memory" effect when one compares primary and secondary responses (28).

The first detailed study of the memory effect for

yolk protein was undertaken in roosters in which a time-course analysis indicated that the secondary response had a shorter lag

264

and reached a higher magnitude than after primary hormonal response (29,30).

With the availability of specific antibodies against

Xenopus vitellogenin, it was possible to demonstrate the memory effect in male Xenopus in a more quantitative fashion (31).

After

the primary response had died away, the second administration of estradiol caused a rapid and massive build-up of circulating vitellogenin.

Not only did it greatly exceed the level of cir-

culating albumin, but there was a virtual disappearance of albumin from the blood.

This phenomenon of de-induction of albumin

synthesis that accompanies estrogen induction of vitellogenesis in Xenopus liver has received much attention recently (32-34). The synthesis of cDNA to vitellogenin allowed a more thorough study of the characteristics of hormonal induction of vitellogenin synthesis at the gene level.

From a careful hybridization analysis,

Baker and Shapiro (35) established that no vitellogenin mRNA could be detected in the male Xenopus liver and that the differences in primary and secondary responses seen in circulating vitellogenin were preceded by parallel changes in total vitellogenin mRNA in male Xenopus liver.

It was also shown that these mRNA levels

exceeded those of albumin and that the kinetics and extent of accumulation of vitellogenin mRNA during secondary induction in male and female liver were very similar.

However, these studies

in whole animals have serious drawbacks,if one is to address the central issue of the mechanisms underlying the role of hormonereceptor interactions with the induced gene, during the onset of gene activation.

For these reasons, the author's laboratory has

devoted much attention to studying the regulation of vitellogenin gene expression in primary cell cultures.

Regulation of Expression of Vitellogenin Genes in Primary Cultures of Male Xenopus Hepatocytes Among the many advantages of cell culture over whole a-nimals in studying hormonal regulation of gene expression are: to control accurately hormone concentration;

a) the ability

b) better analysis of

the early events associated with hormone-receptor interaction with regulatory elements of the gene;

c) rapid reversibility of

induction by removal of the hormone, thus enabling the study of the

265

de-induction process;

d) the analysis of single cell types in

heterogeneous tissues pooled from many animals, thus reducing variability and interference from non-competent cells. A major disadvantage of primary cell culture is the low level, or even absence, of response during a "refractory" period in freshly prepared cultures (36).

This problem, also termed as 'culture

shock 1 , has been studied in detail in the author's laboratory. These investigations are described below in some detail because they have enabled the full reproduction of the physiological regulation of Xenopus vitellogenin gene expression in tissue culture.

The Culture Shock Phenomenon and the Hormonal Response in vitro Many workers have observed that freshly prepared primary cell cultures respond poorly to various stimuli, including hormones, nutrients, drugs, etc. (36).

Several genes have also been found to

be transcribed at a much lower rate in freshly plated rat hepatocytes in primary culture than in the intact liver (37).

The

response to external stimuli, as measured by the inducibility of specific cellular products, usually improves after the first 2-3 days in culture but the cause of this refractoriness remained unknown until quite recently.

In the course of work on activation

of vitellogenin genes in primary cultures of Xenopus hepatocytes by estrogen, it was noticed that the stress of isolation of cells from various tissues results in a large accumulation of stress or heat shock proteins (hsp), particularly hsp 70 (38).

The synthesis of

stress proteins declines with time in culture and that the ability of estrogen to activate vitellogenin genes in cultured male Xenopus hepatocytes was a reciprocal function of the amount of hsp's in the cells . In other studies (40), the experimental induction of hsp's by thermal shock was exploited as a tool in manipulating various parameters of activation and regulation of vitellogenin gene transcription and of estrogen receptor levels (see pp. 276, 277). But it is the recognition of the culture shock phenomenon that made it possible to reproduce in primary cell cultures the de novo activation of vitellogenin genes and to sustain the accumulation of

266

06r

Stimulated with 10 M Estradiol.

0.5< 0.4z er

«

o

0.30.2-

cc E ^ 0.1-

400 Time( Hours) Fig. 2.

Kinetics of vitellogenin mRNA accumulation during primary and secondary induction in the same batch of cultured cells. Hepatocyte cultures were prepared from 8 hormonally unstimulated male Xenopus livers and stimulated with 10 _ ^M estradiol in culture for the periods indicated by the hatched bars below the graph. Total cellular RNA was extracted from the cells after 1-16 days in culture and the vitellogenin mRNA content was measured by disc hybridization to plasmid E7 carrying Xenopus vitellogenin cDNA insert. ( • ), Cells exposed to estradiol continuously after 2 days in culture; ( A ) , after 4 days of primary stimulation, the cultured cells were washed and withdrawn from hormone stimulation for 6 days, after which 10 Mestradiol was added to the medium (secondary induction); ( • ; , estrogen was first added after 12 days in culture. (Data adapted from Ref. 39.) vitellogenin mRNA at high rates for several days in the continued presence of estrogen.

Northern blot analysis and a disc hybridiz-

ation assay (39,41), the latter depicted in Fig. 2, both showed that the physiological characteristic of primary and secondary inductions are also reproduced in culture if the cells are allowed sufficient time to recover from the stress of setting them up in culture.

267 Coupled Regulation of Transcription and Stability of Vitellogenin mRNA It has been known from whole animal studies that, following a single administration of estrogen or upon withdrawal of the hormone, vitellogenin in blood or vitellogenin mRNA in liver rapidly disappear as the hormone is degraded or metabolized

(31,35).

Although it is most likely that the rapid build-up and decay of vitellogenin mRNA during primary and secondary induction in male hepatocytes were due to a combined effect of the hormone on transcription and stability of the mRNA, the contribution of each process could not be easily assessed in whole animals.

They were,

however, accurately determined in primary hepatocyte cultures. The steady-state levels of vitellogenin mRNA induced de novo by a single administration of estrogen in cultures of Xenopus hepatocytes, as shown in Fig. 2 (39,41,42), was initially due to transcription under conditions in which this mRNA is highly stable.

When the

stability of•vitellogenin mRNA was directly measured in the continuous presence or after withdrawal of estrogen from cultures, it was found that the presence of the hormone stabilized vitellogenin mRNA.

Thus, as shown in Fig. 3A, whereas the tjj of vitellogenin

mRNA in the continuous presence of estrogen was >48 hr, the removal of the hormone caused this value to fall to A1 > A2 - B2 following addition of hormone to both adult male and female Xenopus hepatocytes.

This

pattern was flexible in that the differential activation was enhanced or attenuated by varying the period of exposure to the hormone or its concentration.

That the differential rate of

accumulation of mRNA at the onset of induction was a reflection of unequal rates of transcription was verified by directly measuring the absolute rate of transcription of the individual vitellogenin genes.

The same pattern of B1 > A1 > A2 - B2 was again observed.

This was compatible with earlier findings, both in primary cultures (41) and in nuclear run-off transcription and DNase I sensitivity measurements in whole liver (49), that the B group genes were activated to a higher extent than were A group genes. How early in development is this pattern of differential activation within a gene family seen in adult cells established?

It was known

that vitellogenin could be detected immunologically in the blood at

270

Fig. 4.

Kinetics of accumulation of transcripts corresponding to the individual vitellogenin genes in male Xenopus hepatocyte cultures after primary induction with estradiol-17 B. Vitellogenin mRNA corresponding to genes A1 ( A ), A2 ( A ), B1 ( • ), and B2 ( O ) was quantitated by filter disc hybridization to 3 2 p _ nick translated HindiII excised cDNA. (A) 1 0 - 6 M estradiol was added once at time zero; (B) 10~®M estradiol was replenished in the culture medium every hour over 12 hr. (Data adapted from Ref. 48.) late metamorphic stages of Xenopus tadpoles or froglets immersed in water containing estrogen

(50,51).

Hybridization analysis revealed

that larval hepatocytes acquired competence to synthesize

vitello-

genin mRNA in response to the hormone by Nieuwkoop-Faber stage 61, i.e. estrogen receptor was present in hepatocytes at least by late metamorphosis

(48).

Measurement of individual vitellogenin gene

transcripts in metamorphosing tadpole liver revealed the same relative pattern of expression as in adult hepatocytes, i.e. gene B1 > A1 > A2 - B2, at the earliest stages of activation of these dormant genes.

Thus, the unequal pattern of expression is main-

tained throughout life, although the absolute rate of transcription of each gene increases rapidly between late metamorphic and froglet stages.

271

The most likely explanation for the above differential activation of the individual members of the vitellogenin gene family may be different promoter strengths or variable intensities of interaction between estrogen receptor or other transcription factors and gene sequences bearing regulatory elements.

It is therefore most

relevant that, while all four Xenopus vitellogenin genes have one or two blocks of the palindromic sequences GGTCAMNNTGACC between -310 and -375 bp in the 5' upstream region, the linked genes A1 and B1, which are more strongly expressed than the pair A2 and B2 , have an additional element further upstream at -663 and -554 bp. Recently, the groups of Wahli and Ryffel have studied the estrogenregulated expression in human MCF-7 breast cancer cells of a hybrid gene formed by fusion of the promoter regions of Xenopus vitellogenin genes B1 and A2 and the coding region of the bacterialCAT gene (52,53).

Transfection studies and deletion mapping showed

that deletion of the 13 bp element at position -334 was essential for hormonal inducibility.

It is worth noting that this element is

also present in the 5' upstream region of other liver-specific estrogen-inducible genes, including the chicken vitellogenin gene Vtg II (19).

The latter, which is more strongly expressed than the

other two vitellogenin genes, also has three such elements at similar location 5' upstream from the transcription initiation site. Differential expression of vitellogenin genes may arise from the cooperative interaction between

more than one regulatory element

in the upstream flanking sequences and positive transcription regulatory factors, including estrogen receptor.

Estrogen Receptor and Vitellogenin Gene Activation There is now substantial evidence that the level of steroid hormone receptor determines the kinetics of regulation of transcription of specific genes in the hormonal target cell (54).

Much of it is,

however, based on transformed or neoplastic cells or in untransformed cells in which the gene is already expressed at a low level in the absence of the hormone.

These studies, however, do not bear

directly on the de novo activation of transcription nor do they establish a close relationship between the number of nuclear receptors and activation of gene expression under normal

272 physiological conditions.

The fact that estrogen can activate de

novo the permanently silent vitellogenin genes in male Xenopus hepatocytes in culture and that the process can be reversibly reproduced in primary cell cultures, offers a unique opportunity to test directly the relationship between hormone receptor and gene transcription. Adult male Xenopus liver has low levels of estrogen receptor, comprising only 200-500 molecules tightly bound to the nucleus per cell (55-57).

Treatment of naive male Xenopus with estrogen

causes a 5-10 fold increase in high-affinity liver nuclear receptors to reach levels found in female liver (56-58).

This

elevated level of receptor in male hepatocytes persists for several weeks so that it may also explain the more rapid and extensive response to the hormone during secondary induction, in addition to any long-lasting changes in the conformation of vitellogenin genes. As regards the latter, an irreversible or long-lasting demethylation of CpG residues of both chicken and Xenopus vitellogenin gene sequences has been offered as one of the explanations for the 'memory' effect (59,60).

Interestingly, the demethylation sites

relevant to vitellogenin gene expression have been found not only in the 5' upstream and coding sequences but also in the 3' downstream regions.

However, such correlations should not be taken as

evidence of a cause-effect relationship nor as definitive evidence for a role for demethylation in gene expression.

For example,

Burch and Evans (61) found in chick embryos injected with estrogen that the hormone-induced DNase hypersensitive sites and demethylation disappeared but the vitellogenin gene memory effect persisted for up to 25 weeks after hatching. Equally important is the demonstration of upregulation by estradiol of its own receptor in primary cultures of male Xenopus hepatocytes and its association with the absolute rate of transcription of vitellogenin genes as a function of time (57).

Such an accurate

analysis could only have been possible in cell cultures, and the results depicted in Fig. 5 clearly show an almost stoichiometric relationship between nuclear receptor and activation of the dormant genes.

Stimulation with the hormone caused the receptor level in

naive male cells to rise to those found in female cells, accompanied by similar enhancement of vitellogenin gene transcription to rates

273

Fig. 5.

A: Correlation of nuclear estrogen receptor levels ( • ) with absolute rates of vitellogenin gene transcription ( • ) in male Xenopus hepatocytes as a function of time (hr) after the addition of estradiol. B: Relationship between nuclear estrogen receptor and absolute rate of transcription of vitellogenin genes in male Xenopus hepatocyte cultures following various periods of primary exposure to the hormone as in (A). (Data adapted from 57.) observed in female hepatocytes.

Experiments with cycloheximide

added at different times of culture showed that the small amount of receptor residing in male liver nuclei at the start of the experiment accounted for the activation of gene transcription in the first 4 hr after which the increase in transcription required continuing protein synthesis for both processes.

They also demonstrated the

reversibility of the relationship when the high receptor levels previously elevated by the hormone were depleted rapidly in the presence of the inhibitor of protein synthesis.

In addition to

changes in the number of receptor molecules, one has also to consider the more short-term modulations of receptor activity caused by reversible co-valent modifications upon hormone addition or

274

withdrawal.

The most likely modification is phosphorylation and

de-phosphorylation, as has been demonstrated for estradiol receptor in the uterus (62).

Steroid Receptor and Regulatory Gene Sequences Considerable progress has been made recently with nuclease protection or 'foot-printing' procedures for determining regions around steroid regulated genes that interact with the relevant receptor. Thus, DNA sequences located between -100 and -700 bp upstream from the transcription initiation site have been implicated as the site of regulation by many steroid hormone receptors of a variety of genes, such as ovalbumin, lysozyme, uteroglobin and MMTV

(54,63-66).

A consensus sequence located at -458 to -725 bp upstream in the 5' flank of the chicken vitellogenin gene II was found to be a site of interaction with estrogen receptor, as judged from DNase I protection assays (67).

Similar core sequences have been detected in

the 5' flank of all four Xenopus vitellogenin genes (19).

Also,

the chicken apoVLDL gene which is estrogen-regulated, but not induced de novo, in the liver

has two similar sequences at around

-300 bp upstream but not around -600 bp.

Burch (68) has also

reported the presence of an SV40-like enhancer core sequence at a 5' upstream nuclear hypersensitive site in the chicken vitellogenin II gene.

Four 7-9 bp sequence elements were also found in this gene

and apoVLDL gene, as well as in three estrogen-induced egg white protein genes in the oviduct.

In an ontogenic study in chick

embryo liver, the apoVLDL and vitellogenin genes were not however simultaneously activated by estrogen (69).

Besides these relatively

proximal 5' upstream sequences thought to be involved in interaction with steroid receptors, one has to also consider the possibility of more remote sequences.

For example, Chambon 1 s laboratory have

recently shown that estrogen and progesterone receptors, in conjunction with tissue-specific transcription regulatory factors, induce DNase I-hypersensitive sites up to 6 kbp upstream from transcription site of the chicken ovalbumin gene (70). An important recent development in the area of receptor-gene interaction is the cloning of several steroid hormone receptor genes

275 (71-73).

From sequencing of the cloned genes, it has emerged that

all receptors exhibit a high sequence homology with the DNA-binding cystein-rich domain of the

v-erb A proto-oncogene.

Interestingly,

glucocorticoid and progesterone receptors have been shown to bind to the same sites in the promoter regions of MMTV and chicken lysozyme genes, as revealed by exonuclease III foot-printing studies (74).

It is unlikely that these oncogene-related DNA

binding sites would account for the gene regulatory function of steroid receptors and there is already some evidence that other proteins interacting both with the receptor and other DNA sites must also be involved

(70,75,76).

It is, however, important to realize in drawing conclusions from DNA sequences alone that we know virtually nothing as yet about the influence of such factors as the higher order organization

of

genes in chromatin and the effect of distribution of different members of a gene family on different chromosomes on the regulation of gene activity.

For example, estrogen-sensitive genes when

activated by the hormone have been shown to be preferentially enriched in the nuclear matrix fraction in contrast to the quiescent state of the genes, including vitellogenin genes (77-80).

At the

same time, estrogen receptor has also been found to be similarly enriched in the matrix fraction (77,81).

In another study, the

amounts of some non-histone proteins were elevated and the modified composition of matrix proteins persisted for a long time after primary induction of vitellogenesis by estrogen had been reversed in rooster liver (82).

It was suggested that the long-lasting

changes associated with the nuclear matrix may be associated with the 'memory' effect which allows a more rapid secondary induction. Such associations can only be considered as tentative, since the exact significance of the localization of specific genes and putative

regulatory proteins in the nuclear scaffold structure,and

how genes could be possibly translocated into and away from sucn structures by hormonal and other regulatory signals, still remain to be elucidated.

276 Table 1.

Effect of Heat Shock on Total and Nuclear Estrogen Receptor Levels in the Presence or Absence of Estradiol in Cultured Male Xenopus Hepatocytes.

Receptors/cell

Treatment None E2 alone Heat shock alone Heat shock with E 2

Receptors in nuclei

1200

420

1200

1200

0

0

1200

E 2 for 1 hr, then heat shock with E 2

900

Heat shock alone, then recovery at 26°C for 4 hr

200

Heat shock alone, then recovery at 26°C for 20 hr

600

500 680 120 480

After 3 days in estrogen-free medium, male hepatocyte cultures were incubated for 6 hr with 10~^M estradiol (E 2 ). The cells were transferred to hormone-free medium and allowed to incubate for 12 hr to enable the cells to metabolize completely any remaining estradiol. The cells were then incubated in estrogen-free or 10 M estrogen-supplemented medium at normal temperature of 26°C or at the heat shock temperature of 34°C, as indicated, for 12 hr. (Data adapted from Ref. 40.)

The Exploitation of Heat Shock Phenomenon in Elucidating the Role of Estrogen Receptor in Vitellogenin Gene Expression We have already seen how the competence of fresh primary cultures of Xenopus hepatocytes in their vitellogenic response to estrogen is affected by culture shock or the accumulation of hsp-like proteins induced by cellular stress in setting up the cultures ( pp.

265,266

)•

The experimental application of thermal shock to

naive male Xenopus hepatocytes modifies the accumulation of vitellogenin mRNA induced by estrogen (40).

Both the transcription and

stability of the mRNA are influenced by the temperature and duration of heat shock, although the two effects can be dissociated.

A brief

pulse of heat shock at 31°C affects only transcription, whereas above that temperature, or for thermal shock at 31°C exceeding 2 hr, the vitellogenin mRNA already accumulated was rapidly degraded, even in the presence of estrogen which protects the induced mRNA

277

against degradation.

Both the inhibition of transcription and

accelerated breakdown of vitellogenin mRNA can be directly correlated to the amount of newly-synthesized hsp 1 s in the cell cultures. The marked inhibition of vitellogenin gene transcription by heat shock was also correlated with a striking effect on estrogen receptor level or activity in hepatocyte nuclei.

Experiments

described in Table 1 show how heat shock applied in the absence of the hormone causes a total loss of estrogen receptor in male cells in which the receptor had previously been upregulated by the hormone. The presence of the hormone during heat shock or exposure to it just prior to the application of the stress substantially protects the receptor.

The exact cause or significance of this phenomenon of

protection against thermal shock is not clear.

Whatever these may

be, heat shock can be a valuable tool in manipulating the level of steroid receptor and steroid-induced mRNA in studies designed to understand more fully the role of hormones in regulating gene expression.

Switching on Transcription of Silent Vitellogenin Genes in Nuclear Preparations Recently, there have been successful attempts at mimicking the induction of gene transcription with soluble extracts of nuclei or cytoplasm added to nuclei or nuclear extracts.

These include the

activation of transcription of genes encoding silk fibroin, globin, heat shock proteins and adenovirus (83-85) by extracts of cells in which these genes were actively transcribed.

However, in all these

systems the test gene was already transcribed at a low rate in vivo, i.e. it was in an "open" configuration, and that the soluble extracts modulated the rate of transcription.

The completely

silent vitellogenin genes in adult male Xenopus liver offer a more suitable model to study the process of switching on of silent genes in vitro. In order to test the roles played by estrogen receptor and other positive transcriptional factors, the author's laboratory has recently analyzed the effect of tissue extracts on the de novo

278

Table 2.

S-100 Extract from Estrogen-treated (E2) Male Xenopus Liver confers Hormone-specific de novo Transcription of Vitellogenin Genes in Control (C) Male Liver Nuclei.

S-100

Heparin

32 P-RNA Hybridized (ppm) Vitellogenin Albumin

Vg/Alb

C

-

0.5

93

0.005

C

+

0.7

86

0.01

E2

-

22.0

61

0.37

E2

+

4.0

70

0.06

Nuclei from untreated male Xenopus liver were pre-incubated, then incubated with S-100 extracts, with and without heparin, and transcription of vitellogenin mRNA then measured (86).

activation of vitellogenin genes in isolated male Xenopus hepatocyte nuclei (86).

By paying particular attention to the amount of a

soluble extract termed S-100 (100,000 g supernatant of postnuclear fraction) added to the nuclei and to the period (45-90 min) of preincubation of nuclei and S-100, it was possible to obtain a specific switching on of silent vitellogenin genes.

As shown in Table 2,

pre-incubation of hormonally untreated male liver nuclei with homologous S-100 led to the transcription of only albumin but not vitellogenin genes.

However, pre-incubation and incubation of the

same nuclei with S-100 fractions from estrogen-treated male Xenopus induced the transcription of vitellogenin mRNA.

Since vitellogenin

genes are fully dormant in these nuclei, their transcription must represent a de novo activation, which was also corroborated by incubation of nuclei in the presence of heparin, an inhibitor of initiation of transcription. The above process of switching on vitellogenin genes in vitro was to some extent tissue-specific.

The S-100 from male liver cells

failed to activate vitellogenin mRNA synthesis in nuclei from erythrocytes and oviduct, the latter being also a major estrogenregulated tissue involved in egg protein synthesis.

In other

experiments, the liver nuclear transcripts were analyzed for an uncharacterized Xenopus oviduct-specific estrogen-inducible mRNA,

279 Table 3.

Tissue Specificity of Activation of Vitellogenin and 6G Genes as seen by Co-incubation of Nuclei and S-100 Fractions from Xenopus Oviduct and Male Liver^

Nuclei

S-100

Male liver

Male liver

Male liver

Oviduct^

Oviduct

Male liver + E-

Rate of Transcription Vitellogenin

(ppm)

0 0 75

Nuclei and S-100 were pre-incubated for 90 min before the nucleotides were added and the transcription reaction carried out for a further 45 min. ^6G Refers to a messenger RNA coding for an as yet unknown protein that is expressed in Xenopus oviduct but not liver and inducible with estrogen (87). 3The oviduct S-100 strongly inhibited (by about 75%) overall transcription in liver nuclei; the values are therefore corrected for this inhibition. termed "6G" (87).

Thus, as shown in Table 3, the hormonally

competent liver S-100 failed to activate the dormant vitellogenin genes in oviduct nuclei, although gene 6G continued to be transcribed.

Conversely, an S-100 from adult Xenopus oviduct, in which

6G is expressed, failed to induce its transcription in male liver nuclei.

Since both Xenopus liver and oviduct have estrogen

receptors, it is unlikely that the receptor is the only active component in switching on the dormant vitellogenin genes, but that some other tissue-specific transcriptional factor(s) must also be involved.

The Involvement of DNA-binding Proteins and Estrogen Receptor Many transcriptional factors have DNA-binding properties (88) and, as already mentioned, steroid receptors bind to specific sequences flanking the genes they regulate (54).

The above studies on

switching on of silent vitellogenin genes in isolated nuclei were therefore extended to determining the participation of DNA-binding proteins and estrogen receptor in the process.

280 By using a procedure based on DNA partition chromatography, whereby species-specific DNA-binding proteins were separated from nonspecific proteins, it was possible to show that Xenopus DNA-specific proteins from estrogen-treated liver S-100, but not from control tissue, strongly activated vitellogenin mRNA synthesis (89).

The

procedure also allowed a substantial enrichment of the active factor(s) in the crude S-100 extracts.

Proteins with a high

affinity for low copy no. Xenopus DNA, but not for repetitive DNA, caused this gene activation in isolated nuclei.

By using defined

cloned genomic DNA fragments to fractionate these partially enriched extracts further, it should be possible to both identify DNA sequences involved in this activation as well as to obtain purified transcriptional factors. As regards estrogen receptor, since it is easily released from the nucleus upon cell disruption (90,91), the S-100 fraction from estrogen-treated livers should contain a significant amount of receptor.

In order to test the possibility that estrogen receptor

was involved in the activation of dormant vitellogenin genes in isolated male liver nuclei, the effect of monoclonal antibodies to estrogen receptor was investigated in the same studies.

Incubating

the competent S-100, or an enriched DNA-binding protein fraction derived from it, completely abolished its ability to switch on vitellogenin genes without affecting the normal transcription of albumin genes.

Jost et al. (92) showed that the transcription of

a hybrid chicken vitellogenin II gene added to embryonic chicken liver nuclei was enhanced by the addition of a preparation containing estrogen receptor.

The same group also showed a 40% inhibition

of secondary stimulation of chicken vitellogenin genes in isolated liver nuclei by nuclear and cytoplasmic extracts treated with antibodies to estrogen receptor (93).

Total inhibition of

vitellogenin gene activation by antibody to estrogen receptor does not imply that estrogen receptor is the sole component in the S-100 extracts responsible for the activation.

Rather the specific

switching on of vitellogenin genes is more likely to require the combined participation of estrogen receptor and tissue-specific transcription factors.

In further experiments it was also shown

that exposure of nuclei from estrogen-treated liver to receptor antibodies did not affect the rate of ongoing transcription of

281

vitellogenin genes already activated in vivo.

This preliminary

finding raises the possibility that the major role of the receptor may be to initiate transcription but not to interact with preformed functionally active transcription complexes.

There is now growing

evidence that multiple protein factors regulate gene conformation and transcription and that in order to exert a gene regulatory function, these must interact among themselves as well as with multiple DNA sites (94-96).

While these ideas of complex inter-

actions are still at an early stage of development, there is some indirect evidence that as yet undefined proteins associated with estrogen receptors may play some role in the regulation of transcription of vitellogenin genes (97,98).

Further analysis of the

roles played by tissue-specific transcription factors, DNA-binding proteins and steroid receptors will necessitate reconstitution experiments with cloned genomic DNA sequences and soluble cell-free extracts.

General Conclusion The fact that estrogen induces the de novo synthesis of the yolk protein precursor vitellogenin in hepatocytes of male oviparous vertebrates has allowed a better analysis of the initial stages of gene activation than for estrogen-regulated genes in female tissues, such as the oviduct.

Furthermore, as has been extensively discussed

in this article, there is

an

additional advantage of studying the

process of vitellogenesis in Xenopus, since in primary hepatocyte culture specific gene activation can be studied in the absence of any cellular proliferation.

Thus, it has been possible to demon-

strate the unequal expression of the four vitellogenin genes in this species, which in turn may be useful in understanding the role of interaction between estrogen receptor and regulatory gene sequences in the action of steroid hormone. Another special feature of induction of vitellogenesis in male oviparous vertebrates is the low level of estrogen receptor in hepatocytes and the substantial up-regulation of receptor level produced by the hormone.

This has rendered it possible to demon-

strate a stoichiometric relationship between receptor number and

282 absolute transcription rate of the induced gene in vivo.

The

inactive configuration of vitellogenin genes in male hepatocytes and the low receptor number has also proved highly advantageous in devising an assay for transcription regulatory factors in vitro. The specific switching on of the dormant genes in male hepatocyte nuclei provides a simple test system for characterization and isolation of factors conferring expression on genes.

Ultimately,

it will be of utmost importance to explain the high degree of tissue-specificity of regulation of gene expression by steroid hormones, as illustrated by the selective activation of different genes in Xenopus liver and oviduct by estrogen.

Whatever the out-

come of future studies, it is clear that work on the regulation of Xenopus vitellogenin genes has already made a significant contribution to our understanding of steroid hormone action and regulation of eukaryotic gene expression.

Acknowledgement I am grateful to Mrs. Ena Heather for expert help in the preparation of this article.

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ESTROGEN RECEPTOR REGULATION OF VITELLOGENIN AND RETINOL PROTEIN GENE EXPRESSION

D . J . S h a p i r o , M . C . B a r t o n , J. B l u m e , L. G o u l d , M . J . L e w , D . M . M c K e a r i n , D . A . N i e l s e n a n d I.J. W e i l e r D e p a r t m e n t of B i o c h e m i s t r y , 61801

U n i v e r s i t y of

BINDING

Keller,

D.

Illinois, Urbana,

IL

Introduction In t h i s c h a p t e r w e w i l l regulatory

focus on w h a t we have

learned

s t r a t e g i e s by w h i c h a e u k a r y o t i c cell r e s p o n d s

s t e r o i d h o r m o n e s by s p e c i a l i z i n g a m o u n t s of s p e c i f i c m R N A s .

to p r o d u c e

hepatic mRNAs coding vitellogenin

i n d u c t i o n of

for the egg yolk p r e c u r s o r

(reviewed

In X e n o p u s , v i t e l l o g e n i n a n d r e t i n o l

protein

(RBP) a r e p r o m i n e n t m e m b e r s of a c l a s s of in t h e l i v e r u n d e r e s t r o g e n c o n t r o l ,

the s e r u m a n d u p t a k e a n d s t o r a g e

mRNA.

results

In f u l l y

of v i t e l l o g e n i n m R N A

half

is a c h i e v e d b o t h t h r o u g h

The

synthesis

liver

vitellogenin mRNA

accumulation

transcriptional

selective cytoplasmic

T h e s p e c i f i c a c t i v a t i o n of v i t e l l o g e n i n

t r a n s c r i p t i o n and the s t a b i l i z a t i o n / d e s t a b i l i z a t i o n vitellogenin mRNA are unaffected

b y i n h i b i t i o n of

protein

The estrogen

R B P m R N A a l s o r e p r e s e n t s a d i r e c t e f f e c t of

estrogen.

Recent Advances in Steroid Hormone Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

gene

of

(8,10), and therefore are properly c h a r a c t e r i z e d

d i r e c t o r p r i m a r y e f f e c t s of e s t r o g e n .

into

oocyte.

liver cells, vitellogenin

the c e l l ' s m R N A .

s t a b i l i z a t i o n of v i t e l l o g e n i n m R N A a g a i n s t (8,9).

for export

a m o u n t s of

a c t i v a t i o n of t h e v i t e l l o g e n i n g e n e s a n d by degradation

proteins

to p r i m a r y c u l t u r e s of X e n o p u s

induced Xenopus

represents approximately

binding

binding

in t h e d e v e l o p i n g

in t h e i n d u c t i o n of m a s s i v e

for

the

in 1 - 6 ) a n d for t h e s e r u m r e t i n o l

(7).

A d d i t i o n of e s t r a d i o l - 1 7 $

large

protein,

protein

synthesized

the

to

and accumulate

The s y s t e m s we e m p l o y as m o d e l s

estrogen regulated gene expression are the

cells

about

as

induction

of

290 The a c t i v a t i o n of v i t e l l o g e n i n g e n e t r a n s c r i p t i o n

is

accompanied

by the i n d u c t i o n of a d d i t i o n a l n u c l e a r e s t r o g e n r e c e p t o r . estrogen receptor

in X e n o p u s l i v e r c e l l s a p p e a r s to be

n u c l e a r e v e n in the a b s e n c e of e s t r o g e n

(11).

largely

In o r d e r to

to e v a l u a t e the role of the X e n o p u s e s t r o g e n r e c e p t o r

receptor

(12).

begin

(xER)

the r e g u l a t i o n of g e n e e x p r e s s i o n , w e h a v e i s o l a t e d a n d a cDNA clone encompassing

The

in

sequenced

the e n t i r e p r o t e i n c o d i n g r e g i o n of

C o m p a r i s o n of the a m i n o a c i d s e q u e n c e s of

the

the

xER, the f i r s t s t e r o i d h o r m o n e r e c e p t o r to be s e q u e n c e d from a cold b l o o d e d o r g a n i s m , and h u m a n a n d a v i a n ERs to d r a w some g e n e r a l c o n c l u s i o n s c o n c e r n i n g d o m a i n s of these g e n e r e g u l a t o r y

R e s u l t s and

(13-15) a l l o w s

the

us

functional

proteins.

Discussion

E s t r o g e n r e g u l a t i o n of v i t e l l o g e n i n g e n e

transcription

The v i t e l l o g e n i n e x p e r i m e n t a l s y s t e m is d e s c r i b e d in s t u d i e s we did a few y e a r s ago in w h i c h we u s e d q u a n t i t a t i v e R N A d o t h y b r i d i z a t i o n to m e a s u r e the k i n e t i c s of v i t e l l o g e n i n accumulation

in p r i m a r y X e n o p u s l i v e r c u l t u r e s .

s h o w n in Fig. 1.

mRNA

These data

less t h a n 1 m o l e c u l e / c e l l

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

50,000

molecules/cell.

In o r d e r to i d e n t i f y the r e g u l a t o r y s t r a t e g i e s e m p l o y e d X e n o p u s l i v e r c e l l s to a c h i e v e this i m p r e s s i v e m e a s u r e m e n t s w e r e m a d e of the a b s o l u t e transcription

are

Estrogen induces vitellogenin mRNA from m u c h

direct

rate of v i t e l l o g e n i n

in p r i m a r y X e n o p u s l i v e r c u l t u r e s .

The d a t a

T a b l e 1 s u m m a r i z e the r e s u l t s of o u r w o r k in this a r e a In the a b s e n c e of e s t r o g e n , there

by

induction,

is no d e t e c t a b l e

(8-10).

transcription

of the v i t e l l o g e n i n g e n e s d o w n to a level of 98%) of a m i n o a c i d s e q u e n c e h o m o l o g y

the p u t a t i v e DNA b i n d i n g r e g i o n of the X e n o p u s , h u m a n a n d ERs is p a r t i c u l a r l y s t r i k i n g

for two r e a s o n s .

The

in

avian

evolutionary

lines l e a d i n g to m a m m a l s a n d a m p h i b i a n s are t h o u g h t to h a v e diverged approximately

350 m i l l i o n y e a r s ago.

The

great

e v o l u t i o n a r y d i s t a n c e among these o r g a n i s m s is e m p h a s i z e d by the

AMINO ACID NUMBER

Fig. 5 H o m o l o g y b e t w e e n the x E R and the h u m a n a n d a v i a n ERs. The amino a c i d s e q u e n c e of the x E R b l o c k s of TO is c o m p a r e d to the s e q u e n c e s of the h u m a n (• •) and a v i a n (o o) ERs a n d the p e r c e n t h o m o l o g y of e a c h 10 a m i n o a c i d block is p l o t t e d . The e s t r o g e n r e c e p t o r d o m a i n s s u g g e s t e d by this d a t a are s h o w n b e l o w the figure using the n o t a t i o n of C h a m b o n a n d h i s c o l l e a g u e s (15, from [27] ) .

302 p r e s e n c e of 44 s i l e n t n u c l e o t i d e s u b s t i t u t i o n s r e g i o n s of x E R and h u m a n ER.

in the DNA

In a d d i t i o n , the x E R m u s t

w i t h a t a r g e t DNA s e q u e n c e at a t e m p e r a t u r e of a l t e r the K d of the x E R - D N A c o m p l e x .

This m i g h t

The p r e s e n c e of a h i g h l y

r e g i o n in the e s t r o g e n r e c e p t o r s m a k e s

s e e m p r o b a b l e that the e s t r o g e n r e c e p t o r s y s t e m arose o r g a n i s m s w h i c h d o not t h e r m o r e g u l a t e . for r e g u l a t e d

interact

approximately

18°C, w h i l e the h u m a n a n d a v i a n ERs f u n c t i o n at 37°. conserved DNA binding

binding

it

in

It m a y r e t a i n its

ability

i n t e r a c t i o n w i t h t a r g e t DNA s e q u e n c e s over a b r o a d

temperature range.

The n e a r l y i d e n t i c a l a m i n o a c i d s e q u e n c e s

the DNA b i n d i n g d o m a i n of the X e n o p u s , h u m a n and a v i a n

in

ERs

s u g g e s t s that a l l three r e c e p t o r s m a y b i n d to a s i m i l a r

DNA

sequence. The DNA b i n d i n g r e g i o n of the x E R The x E R , in c o m m o n w i t h o t h e r s t e r o i d h o r m o n e r e c e p t o r s ,

contains

a r e g i o n rich in C y s , Lys a n d Arg r e s i d u e s w h i c h is r e l a t e d

to

the "zinc f i n g e r " s t r u c t u r e p r o p o s e d for the 5S gene transcription factor TFIIIA

(23).

T h i s r e g i o n of the x E R is >98%

h o m o l o g o u s w i t h the h u m a n a n d a v i a n ERs and 52% h o m o l o g o u s

with

the r e g 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 h i c h is s u f f i c i e n t b i n d i n g to the DNA s e q u e n c e of the g l u c o c o r t i c o i d element

(24).

A s e q u e n c e c o m p a r i s o n of this r e g i o n of the

and the c o r r e s p o n d i n g

r e g i o n s of the h u m a n

(25,26), c h i c k e n p r o g e s t e r o n e (29,30) r e c e p t o r s

C o m p a r i s o n of

s e q u e n c e s a l l o w s us to d e r i v e a c o n s e n s u s s e q u e n c e steroid receptors.

these f i n g e r s of

The n u m b e r a n d l o c a t i o n s of c y s t e i n e is h i g h l y c o n s e r v e d in the four

(c-erb-A)

(Fig. 6, C O N S )

for the s t r u c t u r e of the two p u t a t i v e m e t a l - b i n d i n g histidine residues

and

receptors.

The n u m b e r a n d l o c a t i o n s of the b a s i c a m i n o a c i d s l y s i n e a r g i n i n e are also e x t r e m e l y w e l l c o n s e r v e d among the

and

receptors.

This a r e a of the r e c e p t o r s m a y be c o n v e n i e n t l y d i v i d e d into potential metal binding elements. residues

The f i r s t e l e m e n t

(Fig.

180-200) c o n s i s t s of two p a i r s of Cys r e s i d u e s ,

s e p a r a t e d by two a m i n o a c i d s

xER

glucocorticoid

(27,28) a n d h u m a n t h y r o i d

is s h o w n in Fig. 6.

for

response

(as in T F I I I A ) and f l a n k i n g

two 6,

each 13

amino

303

xER

CAVCSDYASGYHYGVWSCEC-CKAFFKR

HGR

CLVCSDEASGCHYGVLTCGSCKVFFKR

cPR

CUCGDEASGCHYGVLTCGSCKVFFKR

C-EREA C-ERBA

CVVCGDKATGYHYRCI TCEGCKGFFRR

CONS

C - v C D AsG HYGV-TC-CK-FFKR

C-ER5A

CON 5 Fig. 6 C o m p a r i s o n of t h e a m i n o a c i d s e q u e n c e s of t h e D N A b i n d i n g r e g i o n s of h o r m o n e r e c e p t o r s . The conserved Cys, Lys, Arg rich region containing the p u t a t i v e DNA b i n d i n g d o m a i n of the xER, human glucocorticoid receptor, chicken progesterone receptor, and human C-erbA (thyroid receptor) are shown. The s e q u e n c e s have been a r b i t r a r i l y d i v i d e d into two c o n t i n u o u s e l e m e n t s on two lines. C y s a n d H i s r e s i d u e s a r e s h o w n in l a r g e l e t t e r s . Amino a c i d s c o n s e r v e d in a l l f o u r h o r m o n e r e c e p t o r s a r e s h o w n in b o l d letters. In t h e c o n s e n s u s s e q u e n c e ( C O N S ) , a m i n o a c i d s c o n s e r v e d in a l l f o u r of t h e r e c e p t o r s a r e s h o w n in l a r g e l e t t e r s a n d a m i n o a c i d s c o n s e r v e d in t h r e e o f t h e f o u r h o r m o n e r e c e p t o r s a r e s h o w n in s m a l l l e t t e r s . R e s i d u e s not c o n s e r v e d are d e n o t e d by a d a s h ( f r o m [12] ). acids w i t h a c o n s e r v e d His near the center. hydrophobic

a n d is d e v o i d of

the b a s i c amino acids g e n e r a l l y

at t h e i r c o n t a c t p o i n t s o n D N A . receptors are more homologous receptor.

residues

interest as

it is e x t r e m e l y

Lys and Arg

residues.

entire region

carboxy

seen

(Fig.

The region

6, r e s i d u e s

basic and contains in at l e a s t t h r e e of

the

The d i s t r i b u t i o n of h y d r o p h o b i c amino r e g i o n of

the second e l e m e n t ,

His

222-240) seven

steroid

thyroid

encompassed is o f

conserved in

the

four

hormone

acids

t h e f i r s t e l e m e n t , a n d of b a s i c a m i n o a c i d s

terminal

proteins

In t h i s r e g i o n t h e t h r e e

Each of the b a s i c a m i n o a c i d s

is c o n s e r v e d

residues

in

T h e s e c o n d e l e m e n t c o n s i s t s of o n e c o n s e r v e d

by t h e l a s t f o u r C y s

throughout

relatively

to e a c h o t h e r t h a n to t h e

residue and five conserved Cys residues.

receptors.

This

region contains eight Val, Ala, Tyr, and Trp

in

represents

the an

304 aspect of the structure of steroid hormone receptors which does not appear to have been previously noted.

The amino acid

distribution raises the possibility that the two fingers interact with different structural features on the DNA. This sequence comparison supports the view that the steroid receptors and the closely related thyroid hormone receptor are members of a supergene family (31), and maintain a common protein structure for binding to DNA which is related to, but not identical to, the zinc finger elements seen in several eucaryotic proteins. The xER mRNA family The RNA blot shown in Fig. 7 indicates that 4 major mRNAs, which are approximately 9, 6.5, 2.8 and 2.5 kb in length, hybridize to the xER clone under conditions of high stringency.

One possible

explanation for the multiple xER mRNAs is that the polyadenylation signals in the xER genes are unusually weak and that a number of different polyadenylation sites are used. Hybridization of MCF-7 cell mRNA at high stringency with a human ER cDNA clone reveals only the single 6.7 kb ER RNA (Fig. 7, panel C-2).

Additional RNAs related to estrogen receptor RNA are

revealed by low stringency hybridization of human RNA with the xER cDNA clone.

These RNAs range from approximately 3 to

approximately 12 kb in length (Fig. 3, lane C-l).

It has

recently been proposed that the steroid hormone receptors arose from a single primitive gene and are members of a supergene family (31).

These cross-hybridizing RNAs, which are related to

estrogen receptor mRNA, might represent other members of the supergene family and may encode other hormone receptors. Estrogen regulation of xER mRNA levels Previous work from our laboratory indicated that the induction of additional estrogen receptor protein is required for the efficient induction of vitellogenin mRNA (10).

Since there were

no data in any system indicating that estrogen receptor mRNA was

305

Fig. 7 B l o t h y b r i d i z a t i o n of e s t r o g e n r e c e p t o r m R N A s . Poly(A)+ RNA w a s p r e p a r e d f r o m 14 d a y e s t r o g e n i n d u c e d X e n o p u s l i v e r , f r o m a v i a n l i v e r and from h u m a n M C F - 7 c e l l s . The figure is a c o m p o s i t e of s e v e r a l d i f f e r e n t g e l s r u n u n d e r s i m i l a r conditions. In p a n e l A , total RNA w a s f r a c t i o n a t e d and h y b r i d i z e d to a v i t e l l o g e n i n cDNA c l o n e u n d e r s t a n d a r d c o n d i t ions. Panel B s h o w s the a u t o r a d i o g r a m r e s u l t i n g from the h i g h s t r i n g e n c y h y b r i d i z a t i o n of X e n o p u s p o l y ( A ) m R N A w i t h the pxER4 insert. Panel C-l is a low s t r i n g e n c y h y b r i d i z a t i o n of + h u m a n p o l y ( A ) m R N A to the pxER4 insert. Panel C - 2 is a h i g h s t r i n g e n c y h y b r i d i z a t i o n of h u m a n p o l y ( A ) R N A to a h u m a n ER c l o n e ( g e n e r o u s l y p r o v i d e d by G. G r e e n e [13]). The s i z e of v i t e l l o g e n i n m R N A is 6.5 kb (3). The l o c a t i o n of the 18S and 28S rRNA s t a n d a r d s is s h o w n for the three s a m p l e s . The a m p h i b i a n r i b o s o m a l RNAs are s m a l l e r than the h u m a n r R N A s . R e s i d u a l rRNA in the s a m p l e s s o m e t i m e s p r o d u c e s c o m p r e s s i o n of a d j a c e n t R N A s . T h i s is r e s p o n s i b l e for the b a c k g r o u n d b a n d s b e l o w 28 and 18S in lane C - 2 (from [12]). i n d u c i b l e , we d e c i d e d to q u a n t i t a t e estrogen receptor mRNA.

the l e v e l s of X e n o p u s

A d m i n i s t r a t i o n of e s t r a d i o l - 1 7 g

X e n o p u s l a e v i s induces h e p a t i c e s t r o g e n r e c e p t o r approximately

laevis to m a l e

mRNA

18 fold, from 0 . 1 - 0 . 1 5 m o l e c u l e s / l i v e r

cell

in

c o n t r o l a n i m a l s to 2.3 m o l e c u l e s / c e l l in e s t r o g e n - s t i m u l a t e d x E R m R N A is i n d u c e d by e s t r o g e n to levels a n i m a l s (Table 4).

306 Table 4 Estrogen Induction of xER mRNA a

Time After Estrogen days

xER mRNA

Induction

molecules/cell

fold

Whole Animals 0

0.13

1

1

0.9

7 6

2

0.8

4

0.4

3

8

1.2

9

10

1.4

11

12

17

14

18

Primary cultures 0

0.4

3

1.5

6

3.4

a

Poly(A) RNA was isolated from Xenopus liver at the indicated times, and xER mRNA levels determined by quantitative RNA dot hybridi zation (7). Fold induction represents the increase in xER mRNA content relative to the control. (Summarized data from M. Barton and D. Shapiro, manuscript in preparation.) approximately 3 fold higher than are observed in normal female liver, both in vivo, and in primary liver cultures.

Blot

hybridization of liver RNA suggests that all of the different species of xER mRNA are induced by estrogen.

During induction

xER mRNA accumulates at a rate of approximately 0.15 molecules/cell/day both in vivo and in primary cultures. The basal level of xER mRNA (0.1-0.15 molecules/cell) or one molecule of mRNA per 7-10 cells is perhaps the lowest mRNA level to be quantitated in a higher eucaryotic cell.

The extremely low

basal level of xER mRNA and protein which are approximately 100 fold lower than typical levels of glucocorticoid receptor may

307 e x p l a i n w h y i n d u c t i o n of xER is r e q u i r e d for an e f f i c i e n t r e s p o n s e to e s t r o g e n .

The i n d u c t i o n of x E R m R N A by the

ligand

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

For

g l u c o c o r t i c o i d s d o w n r e g u l a t e the level of receptor mRNA

glucocorticoid

(32).

E s t r o g e n r e g u l a t i o n of v i t e l l o g e n i n m R N A Estradiol-176

of

example,

stability

induces a s p e c i f i c s t a b i l i z a t i o n of

mRNA against cytoplasmic degradation

vitellogenin

([8], see Fig. 2).

The

selective, s t a b i l i z a t i o n of v i t e l l o g e n i n m R N A is a r e v e r s i b l e c y t o p l a s m i c e f f e c t of e s t r o g e n .

R e m o v a l of estradiol-17(5

from

the c u l t u r e m e d i u m s h i f t s the half l i f e of v i t e l l o g e n i n m R N A 500 to 16 h o u r s .

R e a d d i t i o n of e s t r a d i o l - 1 7 5

c y t o p l a s m i c v i t e l l o g e n i n m R N A to a half life of 500

hours.

A l t h o u g h the r e g u l a t i o n of v i t e l l o g e n i n m R N A d e g r a d a t i o n e s t r o g e n is a p a r t i c u l a r l y

s t r i k i n g e x a m p l e of this

c o n t r o l of m R N A s t a b i l i t y has b e e n o b s e r v e d eucaryotic and procaryotic systems

process,

These

data

is a f l e x i b l e

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

by

in at l e a s t 20

(Table 5).

indicate that r e g u l a t i o n of m R N A s t a b i l i t y

from

the

restabilizes

and

complement

controls.

S e q u e n c e s near b o t h the 3' end and in the 5 ' - u n t r a n s l a t e d

region

h a v e e m e r g e d as m a j o r s i t e s for the c o n t r o l of m R N A s t a b i l i t y several systems.

In o r d e r to a n a l y z e the c o n t r o l of

m R N A s t a b i l i t y we h a v e p r e p a r e d D N A c o n s t r u c t s

in w h i c h p a r t s of

the v i t e l l o g e n i n g e n e s are fused to the CAT g e n e . c o n s t r u c t s are t r a n s f e c t e d

The

resulting

into c u l t u r e d h u m a n l i v e r c e l l s

G2 c e l l s ) and a s s a y e d for h o r m o n e m e d i a t e d m R N A

r e g i o n of

v i t e l l o g e n i n m R N A can c o n f e r the a b i l i t y to be s t a b i l i z e d observations).

(Hep

stabilization.

P r e l i m i n a r y d a t a s u g g e s t that the 3 ' - u n t r a n s l a t e d e s t r o g e n o n the C A T g e n e

in

vitellogenin

(D. N i e l s e n a n d D. S h a p i r o ,

by

unpublished

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m t. X X 0) ® •o -o L, L, z è 13810-13817. 45. Seamon, K.B., W. Padgett and J.W. Daly. 1981. Proc. Natl. Acad. Sei., U.S.A. Zfi, 3363-3367. 46. Gehring, U. and P. Coffino. 1977. Nature 2 M , 167-169. 47. Smith, A.C., M.S. Elsasser and J.M. Harmon. 1986. J. Biol. Chem. ¿¿1, 13285-13292. 48. Hollenberg, S.M., C. Weinberger, E.S. Ong, G. Cerelli, A. Oro, R. Lebo, 635-641. E.B. Thompson, M.G. Rosenfeld and R.M. Evans. 1985. Nature 49. Okret, S., L. Poellinger, X. Dong and J.-A. Gustafsson. 1986. Proc. Natl. Acad. Sei., U.S.A. H , 5899-5903. 50. Nielsen, C.J., J.J. Sando and W.B. Pratt. 1977. Proc. Natl. Acad. Sei., U.S.A. 24, 1398-1402. 51. Wheeler, H.H., K.L. Leach, A.C. La Forest, T.E. 0'Toole, R. Wagner and W.B. Pratt. 1981. J. Biol. Chem. 256, 434-441. 52. Singh, V.B. and V.K. Moudgil. 1985. J. Biol. Chem. 2 M , 3684-3690. 53- Weigel, N.L., J.S. Tash, A.R. Means, W.T. Schräder and B.W. O'Malley. 1981. Bioehem. Biophys. Res. Comm. 1Ö2., 513-519. 54. Woo, D.D.L., S.P. Fay, R. Griest, W. Coty, I. Goldfine and C.F. Fox. 1986. J. Biol. Chem. 2£1, 460-467. 55. Migliaccio, A., A. Rotondi and F. Auricchio. 1984. Proc. Natl. Acad. Sei., U.S.A. £1, 5921-5925. 56. Dougherty, J.J., R.K. Puri and D.O. Toft. 1982. J. Biol. Chem. 252, 14226-14230. 57. Miller-Diener, A., T.J. Schmidt and G. Litwack. 1985. Proc. Natl. Acad. Sei., U.S.A. £2, 4003-4007. 58. Sanchez, E.R. and W.B. Pratt. 1986. Biochemistry 25, 1378-1382. 59. Mendel, D.B., J.E. Bodwell, B. Gametchu, R.W. Harrison and A. Munck. 1986. J. Biol. Chem. 261, 3758-3763. 60. Holmgren, A. 1985. Ann. Rev. Bioehem. 51, 237-271. 61. Grippo, J.F., W. Tienrungroj, M.K. Dahmer, P.R. Housley and W.B. Pratt. 1983. J. Biol. Chem. 25£, 13658-13664. 62. Grippo, J.F., A. Holmgren and W.B. Pratt. 1985. J. Biol. Chem. 2fi&, 93-97. 63. John, J.K. and V.K. Moudgil. 1979. Bioehem. Biophys. Res. Comm. 20., 1242-1248.

335 64. Barnett, C.A., T.J. Schmidt and G. Litwack. 1980. Biochemistry 19, 5446-5455. 65. Wränge, 0., J. Carlstedt-Duke and J.-A. Gustafsson. 1986. J. Biol. Chem. 2£1, 11770-11778.

STEROID HORMONE RECEPTOR PHOSPHORYLATION.

Kanury V.S. Rao and C. Fred Fox Department of Microbiology and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90024

INTRODUCTION.

Beginning with the Coris1 observations on phosphorylase b (1) and the discovery of cAMP-dependent protein kinase by Krebs and coworkers (2), protein phosphorylation has become recognized as a common biochemical event in regulation of cellular processes.

In

recent years receptor-mediated phosphorylation has received considerable attention as a mechanism for intracellular signalling (3).

Receptors for several polypeptide hormones including

epidermal growth factor (EGF) (4), platelet-derived growth factor (PDGF) (5), insulin (6) and somatomedin C (7) are tyrosine-specific protein kinases which are activated by hormone binding to undergo self-phosphorylation.

The kinase activities associated with

purified insulin and EGF receptors can act on a variety of substrates (3); suggesting that their tyrosine-specific phosphorylating capacities regulate the activities of selected proteins in vivo.

A major impetus for this area of research came

from a realization of the close relationship between sequences of certain growth factors or their receptors and sequences encoded in specific cellular oncogenes.

At least six identified cellular

genes encode tyrosine-specific protein kinases, and there are indications of more (8) .

The r.-sis proto-oncogene encodes PDGF and

the EGF receptor has strong homology with gp 65er^ B , the transforming protein of avian erythroblastosis virus (AEV) (9). From the standpoint of steroid receptors, it is of interest to note that the sequences coding for the DNA-binding domain of the glucocorticoid (10), progesterone (11,12) and estrogen (13)

Recent Advances in Steroid Hormone Action © 1987 Walter d e Gruyter & Co., Berlin • N e w York - Printed in Germany

338 receptors are homologous with sequences within the AEV v-erb-A oncogene. Recent evidence that steroid receptors are phosphoproteins supports the idea that receptor phosphorylation may play roles in regulating steroid hormone action. Steroid receptors generally are intracellular proteins which are activated upon ligand binding. In the original two-step model of steroid hormone action (14) , steroid receptors were thought to be cytoplasmic in the absence of hormone and thus incapable of influencing gene expression. Ligand binding stimulated translocation to the nucleus making receptors available at primary sites of action. Though conceptually attractive, this hypothesis is being reevaluated. Recent findings indicate that the estrogen (15,16) and progesterone (17,18) receptors are primarily nuclear in localization, even in the absence of ligand. Nevertheless, a basic aspect of the 'activation' mechanism still holds true. Binding of a steroid hormone to its intracellular receptor confers upon receptor a property, termed either 'activation' or 'transformation'. Operationally, steroid receptor 'activation' is a change in state characterized by an increased affinity for cation exchangers such as phosphocellulose and DNA, and occurring in response to treatment with ligand, high ionic strength or heat. This change in state has been postulated to influence the ability of receptor to alter gene expression in target cells (14). A priori then, functional roles for receptor phosphorylation could be invoked at the level of influencing ligand binding, activation/transformation or interaction with DNA or chromatin. In this connection it is pertinent to note that the complete complementary DNA sequence of the estrogen receptor contains information for four protein tyrosine kinase consensus sequences that are potential sites for phosphorylation (13,19). Of these, two are in the region required for binding of the hormone-receptor complex to DNA and another in the hydrophobic, steroid-binding domain. Tyrosine residues at positions 43, 184, 219 and 526 of the human MCF-7 breast carcinoma cell estrogen receptor sequence have both basic and acidic amino acids residing within 7 residues in the direction of the NH2-terminus (19). This pattern is characteristic

339

of tyrosine residues that are phosphorylated in several cellular proteins and which specify sites for phosphorylation by several protein tyrosine kinases (20).

In addition, there are two serine

residues at positions 236 and 305 in the sequence B-B-X-Ser, where B is a basic amino acid.

Both these serine residues represent

potential cAMP-dependent phosphorylation sites (19) since cAMP-dependent protein kinase has a strong preference for serines that have multiple basic amino acids within 2-5 amino acid residues in the direction of the NH2-terminus (21). In this article we summarize currently available information on steroid hormone receptor phosphorylation.

It is intended more as

an overview, than as a comprehensive survey of the literature.

STEROID RECEPTORS AS PHOSPHOPROTEINS. Steroid receptors generally are phosphoproteins.

The accumulated

evidence favors the view that their phosphorylation has functional consequences in steroid receptor action. Progaatarona receptor.

Direct evidence for progesterone

receptor phosphorylation was obtained from ia. vivo experiments on phosphorylation of the chicken oviduct progesterone receptor by Toft and his associates (22,23).

In initial studies, incubation of

32

an oviduct tissue mince with [ P]orthophosphate, followed by purification in a protocol involving affinity, gel-filtration and ion exchange chromatographies, gave evidence for two major phosphoproteins with molecular weights of 90 and 104 kDa (22).

In

addition, a minor phosphoprotein band at a slightly higher molecular weight was detected also.

All these were phosphorylated

on serine residues. The 90 kDa component was originally identified as receptor, but subsequent studies showed that it was a non-steroid binding component of the 8S progesterone receptor complex (23).

Based on sedimentation studies in high salt

gradients and photoaffinity labelling with the synthetic progestin R5020, the progesterone receptor subunits were identified as having molecular weights of 75 and 110 kDa.

Purification from a

340 [32P]orthophosphate labelled oviduct tissue mince indicated that both receptor subunits were phosphorylated (23). Similar results were obtained by Logeat £i. al., who immunopurified rabbit uterine progesterone receptor from either 32Pi-labelled slices or following intraperitoneal administration of 32 Pi to animals (66) . Weigel £t al. have shown that the B subunit of chick oviduct progesterone receptor is phosphorylated on serine (24). The dissociated, 4S form of the B subunit component was purified on a large scale and examined by amino acid analysis. Phosphoserine was the only phosphoamino acid detected and was present at about 0.6 moles per mole of peptide. In recent studies we observed that the progesterone receptor from cultured human breast carcinoma cells is also phosphorylated (Rao, K.V.S. and Fox, C.F., unpublished observations). [32P]Orthophosphate labelling of intact T47D cells and immunoprecipitation of the progesterone receptor with monoclonal antibodies, supplied by Dr. G.L.Greene (Univ. of Chicago), gives, on SDS—PAGE, a single 32Pi-protein band at a molecular weight of 120 kDa. Upon phosphoamino acid analysis under conditions where phosphotyrosine hydrolysis is minimal, phosphoserine was the only phosphoamino acid detected. Glucocorticoid receptor. After injecting adrenalectomized rats with radioactive orthophosphate, Singh and Moudgil purified the liver glucocorticoid receptor and demonstrated that it is a phosphoprotein (25). They characterized the 32P-protein peak as glucocorticoid receptor by comparing its chromatographic behavior with that of receptor complexed with [3H]dexamethasone or covalently labelled with [3H]dexamethasone-21-mesylate. In a related study, Litwack and his associates identified a 32P-labelled component that copurified with glucocorticoid receptor and migrated as an additional phosphorylated band upon SDS-PAGE (26). The mobility of this band indicated a molecular weight of 24 kDa. They speculated that it might represent a polynucleotide, perhaps RNA, which was tightly associated with purified, untransformed glucocorticoid-receptor complex. Glucocorticoid receptor phosphorylation also has been demonstrated

341

after [32P]orthophosphate labelling of intact mouse fibroblasts in culture and affinity purification on deoxycorticosterone-agarose (27) . In this case, Housley and Pratt showed that phosphorylation occurred exclusively on serine residues. Recent evidence from our laboratory indicates that the human glucocorticoid receptor is also phosphorylated on tyrosine residues (Rao, K.V.S. and Fox, C.F., submitted). Labelling of intact, breast-derived HBL100 cells with 32 Pi followed by immunoprecipitation of glucocorticoid receptors and subsequent phosphoamino acid analysis yielded approximately 10% phosphotyrosine and 90% phosphoserine, but no detectable phosphothreonine. Immuneprecipitation of glucocorticoid receptors from 32Pi-labelled mouse thymoma WEHI-7 cells with a monoclonal antibody raised against rat liver glucocorticoid receptor yields an additional, non-steroid binding phosphoprotein of molecular weight of 90 kDa (28). This 90 kDa protein has been characterized as a heat shock protein and appears to be a common component of the untransformed, molybdate-stabilized, heteromer complex of all steroid receptors (29). Upon thermal transformation of the receptor, no 90 kDa phosphoprotein was immunoprecipitated (28). Estrogan receptor• Direct evidence for estrogen receptor phosphorylation was obtained recently by Auricchio and coworkers (30). Incubation of rat uteri with 32 Pi followed by affinity purification yielded a 32P-labelled receptor. Upon phosphoamino acid analysis, phosphotyrosine was the sole phosphoamino acid detected. Further evidence of tyrosine phosphorylation was obtained through demonstration of high affinity binding of phosphorylated receptor to anti-phosphotyrosine antibodies. Androgen receptor. Though there has been no direct demonstration of androgen receptor phosphorylation, hormone binding activity of crude androgen receptor preparations from rat ventral prostrate cytosol is enhanced by ATP (31). This correlates well with observations for other steroid receptors where there is a well established relationship between receptor phosphorylation and increased ligand binding activity (see the following section on Receptor phosphorylation and ligand binding activity). While these

342

data support a case for androgen receptor phosphorylation, direct evidence is required in order to draw a definitive conclusion. In summation, avian and mammalian steroid receptors are generally phosphoproteins. The progesterone receptor is phosphorylated on serine, and the estrogen receptor on tyrosine. The glucocorticoid receptor is phosphorylated primarily on serine residues with some contribution at tyrosine residues. In addition, at least one non-steroid binding component of the untransformed, heteromeric complexes of steroid receptors, the 90 kDa heat shock-induced protein, is also phosphorylated.

STEROID RECEPTORS AS KINASES. A logical sequel to the finding that steroid receptors are phosphoproteins is studies designed to gain an understanding of the origin of that phosphorylation. Phosphorylation of a steroid receptor could arise either via action of cellular protein kinases or by self phosphorylation. Progesterone receptor. Several early reports indicate that steroid receptors are protein kinases. Baulieu and coworkers reported evidence for autophosphorylation of purified components of the chicken oviduct progesterone receptor (32). Using preparations purified to near homogeneity, this group showed that, in the presence of divalent cations, radioactive phosphate was incorporated from [y -32P]ATP into both the 90 and 110 kDa subunits by a heat sensitive enzymatic activity. While phosphorylation of the 90 kDa component depended only on Ca2+' the 110 kDa component was phosphorylated only in the presence of Mg 2+ . The authors suggested that Ca 2+ dependent phosphorylation of the 90 kDa subunit may represent a very selective protein kinase activity, while Mg 2+ dependent 110 kDa subunit phosphorylation resembled that of several described protein kinases. Addition of calf thymus histones to the incubation mixtures resulted in histone phosphorylation with the 110 kDa subunit preparation, but not with the 90 kDa subunit preparation. The authors speculated that the 110 kDa subunit of the progesterone receptor might exert its action

343

by phosphorylating chromatin proteins, thereby modulating expression of specific genes. Eatrogaa racaptor. Incubation of immune-complexes formed by extracts of human breast carcinoma MCF-7 cells and an estrogen receptor-specific monoclonal antibody with [ y -32 P] A TP revealed, upon subsequent SDS-PAGE, three major radioactive polypeptides of molecular weights 57, 47 and 43 kDa. These comprised over 98% of the radioactively phosphorylated products (33). Phosphorylation required ATP, as opposed to GTP and depended on Mg 2+ as opposed to Ca 2+ and Mn . Based on as yet unpublished results, the authors have stated that the kinase activity in these preparations was serine-specific ( which is at odds with the observation of Auricchio's group that phosphotyrosine is the sole phosphoamino acid detected in rat uterine estrogen receptor). Cells lacking estrogen receptor were not characterized by this activity. Kinase activity present in the preparation also phosphorylated phosphatidylinositol and phosphatidylinositol-4-phosphate, but not 1,2-diacylglycerol. Phosphatidylinositol was converted in part to 32 P-phosphatidic acid and phosphatidylinositol-4-32P while phosphatidylinositol-4-phosphate was phosphorylated to the 4,5-biphosphate. This phospholipid kinase activity was not detected in cells lacking estrogen receptor, suggesting an association with the presence of estrogen receptor. Preparations containing the estrogen receptor isoforms I and II displayed different abilities in phosphorylating exogenous lipids, and the authors suggested a cooperative mechanism between the two in regulating phosphoinositide metabolism. Glucocorticoid racaptor. An association of glucocorticoid receptor with protein kinase activity was first demonstrated by Kurl and Jacob (34). They showed that glucocorticoid receptor purified from the soluble fraction of rat liver homogenates incorporated phosphate from [y-32P]ATP in the presence of Mg 2+ . Receptor purification was achieved using a dexamethasone-affinity column and a single band was displayed at a molecular weight of 90 kDa upon Coomassie staining of SDS-polyacrylamide gels. The phosphorylated preparation also displayed a single band at 90 kDa upon autoradiography. However the latter samples were first

344

chromatographed on DEAE-cellulose prior to electrophoresis and minor phosphorylated contaminants, not detectable by Coomassie staining, could have been removed by this process. Singh and Moudgil showed that, in the presence of [y-32P]ATP and divalent cations, purified rat liver glucocorticoid receptor preparations phosphorylated calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase (35) . Enhanced phosphorylation was observed in the presence of hormone. No aut©phosphorylation of glucocorticoid receptor was, however, detected. Miller-Diener al. (36) reported that only the activated form of highly purified rat hepatic glucocorticoid receptor was capable of autophosphorylation. This required hormone and was stringently dependent on Ca 2+ . An HPLC analysis of hydrolysates indicated that phosphorylation was primarily on threonine residues [though no phosphothreonine is observed on receptors phosphorylated in vivo in mouse fibroblasts (27) or human breast epithelial cells (Rao and Fox, submitted)]. Based on 32 P: 3 H ratio of the purified receptor, there was a stoichiometry of ten phosphates per ligand molecule bound (26,36) . Receptor phosphorylation was blocked by analogues of ATP such as 8-azido-ATP and fluorosulfonylbenzoyl adenosine; demonstrating the presence of an ATP binding site on the kinase responsible for receptor phosphorylation(36). Addition of exogenous histones resulted in their phosphorylation in the presence of Mg 2+ , but not Ca 2+ . Enhanced histone phosphorylation was observed when divalent cations were excluded from the incubation mixtures (36). In contrast with the above reports, Sanchez and Pratt (37) found no evidence to support the concept that mouse L-cell derived glucocorticoid receptor is a protein kinase. They immunoprecipitated glucocorticoid receptor from the soluble fraction with a polyclonal antiserum and incubated the complex with [y-32P]ATP. This resulted in phosphorylation of receptor in the presence of Mg 2+ but not Ca 2+ . Phosphorylation was observed regardless of whether receptor was occupied, transformed or untransformed. On the other hand when monoclonal antibodies were used, no glucocorticoid receptor phosphorylation was observed in the resulting immune-complex in the presence of either Ca 2+ or Mg 2+ .

345

From this the authors concluded that, while L-cell cytosol contains protein kinase activity(ies) that phosphorylate(s) receptor, neither the glucocorticoid receptor nor the 90 kDa receptor associated heat shock-induced protein have intrinsic protein kinase activity. The kinase activity observed with the polyclonal antiserum immune-complex could have been due to nonspecific adsorption of a cytosolic kinase. Alternatively , the L-cell glucocorticoid receptor may have been highly phosphorylated and required receptor dephosphorylation in order to participate in ¿a. vitro autophosphorylation. These studies employed a single monoclonal antibody that was raised against the rat liver receptor, and one cannot rule out, prima facie, that these antibodies might have somehow rendered kinase activity inherent in the receptor inactive. Hapgood fit al- have also suggested that purified rat liver glucocorticoid receptor is not a protein kinase (38). Their purified receptor preparation was homogenous by criteria such as Coomassie staining of SDS-polyacrylamide gels. However, on incubation with [y-32P]ATP, autoradiographic analysis of SDS polyacrylamide gels revealed the presence of several phosphorylated proteins in addition to the glucocorticoid receptor receptor band. Furthermore, preparations from mock purifications containing no glucocorticoid receptor protein, also displayed kinase activity. On photoaffinity labelling with 8-azldo- [y-32P] ATP, a 48 kDa protein was affinity labelled but no radioactivity appeared in the glucocorticoid receptor band. This implies that glucocorticoid receptor is devoid of an ATP binding site, a necessary prerequisite for kinase activity. Similar conclusions were also drawn for the progesterone receptor (39). In contradiction to their earlier report (32), Baulieu and coworkers recently reported that most of the Mg2+-dependent protein kinase activity that co-purified with both the oligomeric and monomeric forms of the chick oviduct receptor belonged to an enzyme distinct from any currently known receptor components (39) . This enzyme was partially separable from the progesterone receptor B subunit by DEAE-Sephacel chromatography and had physicochemical characteristics, e.g., Stokes radius and isoelectric point, that

346 distinguished it from progesterone receptor subunits. Weigel also found that the kinase activity contained in partially purified preparations of the A and B subunits of chick oviduct progesterone receptor was chromatographically separable from the receptor subunits (40) . Based on the more recent evidence, it is uncertain if protein kinase activity is inherent to steroid receptors. In some cases, activity that was initially thought to represent self-phosphorylation is now known to have arisen from contaminants in the receptor preparations. Clearly, more in-depth analysis is needed before a protein kinase activity can be ascribed to steroid receptors. Future attempts to demonstrate a steroid receptor associated kinase activity would first have to rigorously rule out a possible contribution from contaminants. In addition, none of the studies cited above have included demonstrations of kinetic criteria that must be satisfied to establish autophosphorylation. One necessary requirement for self-phosphorylation is that the slope of a plot of log receptor phosphorylation rate versus log receptor concentration must equal 1.

STEROID RECEPTORS AS SUBSTRATES FOR KINASE ACTION. Progesterone receptor. Weigel et. al. (41) demonstrated that purified avian progesterone receptor is an in vit.ro substrate for cAMP-dependent protein kinase. Incubation of purified A or B subunit preparations of hen oviduct progesterone receptor with apparently homogenous bovine heart cAMP-dependent protein kinase, in the presence of [y-32P]ATP and Mg 2+ ' resulted in their cAMP-dependent protein phosphorylation. No characterization of the number of sites phosphorylated or stoichiometry or kinetics of the process however was described. Ghosh-Dastidar fit showed that purified chick oviduct progesterone receptor subunits are high affinity substrates for phosphorylation by biochemically homogenous, human A431 cell epidermal growth factor (EGF) receptor in a reaction requiring divalent cations and EGF (42). Phosphorylation occurred

347

exclusively on tyrosine residues and the Kj,, for the process was 100 nM; several orders of magnitude lower than values reported for other in vitro substrates for the EGF-EGF receptor complex. Tryptic digestion and phosphopeptide analysis revealed two major and at least five minor phosphate acceptor sites common to both the A and B subunits. The identical peptide maps for both the A and B subunits showed that they were phosphorylated primarily in regions of sequence homology. This contrasted a with previously reported lack of homology between the A and B subunits for regions containing tyrosine residues in general (43) . Birnbaumer £t al. found that peptide maps of 125I-labelled progesterone receptor subunits revealed homology in only four of the nearly fifty 125 I-labelled peptides resolved (43). Woofit.al. recently showed that purified progesterone receptor also was phosphorylated by purified insulin receptor kinase but not by purified platelet derived growth factor (PDGF) receptor (44). Both the EGF and insulin receptors phosphorylated progesterone receptor subunits at high affinity, exclusively on tyrosine residues and with maximal stoichiometries that were greater than 1. The turnover number with EGF receptor exceeded 100 min -1 . While the EGF-activated receptor phosphorylated both subunits to an equal extent, the insulin-activated receptor elicited a preference for the B subunit. Fingerprinting of trypsin-produced phosphopeptides revealed that the EGF and insulin receptors phosphorylated an identical major site on both A and B subunits, but differed in their specificities for other sites (44). A caveat to these findings is that in recent experiments, we have not observed EGF-stimulated tyrosine phosphorylation of progesterone receptor in intact cultured human breast carcinoma cells (Rao, K.V.S. and Fox, C.F., unpublished results). This does not rule out the possibility that the progesterone receptor is phosphorylated by EGF receptor in avian cells. Estrogen receptor. Phosphorylation of the estrogen receptor has been studied extensively by Auricchio and coworkers. In initial studies they observed that the ligand binding activity of purified calf uterine estrogen receptor was inactivated by incubation with nuclei from calf uterus (45). Inactivation was enhanced by

348

dithiothreitol and inhibited by phosphatase inhibitors such as fluoride, molybdate and 4-nitrophenyl phosphate; suggesting a role for dephosphorylation in this inactivation. The estrogen receptor inactivating activity was purified partially from nuclear extracts by ion-exchange chromatography and had a high affinity for both ligand occupied and unoccupied receptor (45). Subsequent to this, these workers identified in the soluble fraction of tissue homogenates, an ATP-dependent activity that reactivated ligand binding of the inactivated receptor (46). This activity was purified partially by ion-exchange chromatography and had a high affinity for inactivated receptor. Maximal activation of estrogen receptor ligand-binding activity was obtained in the simultaneous presence of Mg 2+ and Ca 2+ (4 6) . More recently this group showed that the activity of this partially purified kinase was dependent on Ca 2+ and calmodulin (47,86) and phosphorylated the purified estrogen receptor exclusively on tyrosine residues (47). Glucocorticoid receptor. Recently Singh and Moudgil (25) showed that purified rat liver glucocorticoid receptor is a substrate for phosphorylation by cAMP-dependent kinase in the presence of Mg 2+ . A smaller proteolytic fragment of the receptor (MW = 45 kDa) which retained the steroid binding site also was phosphorylated. As described in a previous section on Steroid receptors as kinases, purification of steroid receptors often results in copurification of intracellular kinases (37,38,39) that can phosphorylate receptors, in a reaction usually requiring Mg 2+ . The nature of these enzymes is not known presently and awaits characterization. In summary, there is now evidence that steroid receptors, in vitro, are substrates for a variety of kinases and could therefore be subject to regulation by phosphorylation. However, studies with intact cells are required to test the significance of these findings. That protein kinases have markedly relaxed specificities in vitro is well known (48). While steroid receptors appear to be substrates for phosphorylation by cAMP-dependent kinase in vitro, there has been no demonstration, to date, that this phenomenon occurs in intact cellular systems. EGF does not appear to stimulate progesterone receptor phosphorylation in intact human

349 breast carcinoma cells, though the avian receptor is a highly effective in. vitro substrate.

RECEPTOR PHOSPHORYLATION AND LIGAND BINDING ACTIVITY. The earliest clue that steroid receptor phosphorylation might influence steroid binding was demonstrated by Munck and coworkers. They showed, by adjusting glucose and oxygen concentration in the culture medium, that the uptake of Cortisol by rat thymocytes correlated with the cellular ATP level (49).

Subsequently it was

suggested that ATP is involved in activation of inactive receptor protein to a glucocorticoid-binding form (50,51).

Similar

conclusions were obtained for androgen receptors where ATP and GTP enhanced ligand binding activity of the soluble fraction containing receptor from rat ventral prostrate (31).

Progesterone receptor.

Ligand binding activity of the chick

oviduct progesterone receptor is stabilized by phosphatase inhibitors such as fluoride and molybdate (52).

However, this

stabilization is observed only with the undissociated oligomeric receptor complex and it is not clear whether stabilization is due to inhibition of phosphatase activity or to some other effect of these agents on an oligomeric receptor state. Recent studies on progesterone binding to chick oviduct receptor showed the presence of distinct, high and low affinity, classes of binding sites (53).

Both appeared to be associated with the same

receptor molecules as shown by their copurification and chromatographic properties.

Both the A and B subunits were

characterized by high and low affinity binding.

No cooperativity

between the two sites was detected from either rate or equilibrium binding studies and the low and high affinity sites were present at a ratio of about 2:1 in a preparation with equal quantities of the A and B forms.

The hormone binding activity of the low affinity

site (Kd= 25 nM) was inactivated by treatment in vitro with alkaline phosphatase with no corresponding change in either receptor number or dissociation constant of the high affinity

350 component (Kd= 1 nM). Satrogan racaptor. As described in the preceding section on Steroid receptors as substrates for kinase action, Auricchio and coworkers have isolated, from calf uterine cytosol, a calcium and calmodulin dependent kinase that phosphorylates purified estrogen receptors on tyrosine residues (47). In the presence of 1.0 (J.M Ca 2+ and 0. 6 |iM calmodulin, receptor phosphorylation by the kinase increased three-fold. Under these conditions, there was also a three-fold increase in the estradiol binding capacity of the preparation, suggesting a causal relationship between enhanced phosphorylation and ligand binding activity. Glucocorticoid racaptor. Much of our current knowledge of the role of receptor phosphorylation on ligand binding activity comes from the systematic and elegant studies by Pratt and his associates. From their observations it appears that ligand binding activity is dependent on receptor phosphorylation and, in addition, requires that the receptor be maintained in a reduced state. Initially they found that purified calf intestinal alkaline phosphatase inactivated the glucocorticoid binding capacity of soluble preparations from mouse fibroblasts (54). Inactivation was not due to proteolysis of receptor and depended strictly on the activity of phosphatase. Prior heat inactivation of the phosphatase or inclusion of phosphatase inhibitors such as molybdate or fluoride blocked inactivation. Only unoccupied receptor was inactivated; ligand-bound receptor was unaffected. In a subsequent study with cell free systems from either mouse L-cells, rat thymocytes or rat liver, the particulate fraction sedimenting between 4 x 105 and 45 x 105 g.min contained an enzyme capable of inactivating glucocorticoid binding activity. Inactivation required unoccupied receptor; the hormone binding activities of occupied glucocorticoid or estrogen receptors were not affected. Inactivation of unoccupied glucocorticoid receptor was blocked by phosphatase inhibitors (55). Inactivation of rat thymocyte glucocorticoid receptor was reversible in that it was reactivated by a factor (or factors) present in the cytosol of

351

mouse L-cell fibroblasts (56).

This factor(s) was heat stable and

estimated to have a molecular weight between 5 and 20 kDa.

More

recently, through use of polyclonal antibodies, this heat stable factor was identified as thioredoxin (57).

In order to understand

the mechanism by which phosphorylation stabilized ligand binding activity, Pratt and coworkers studied the decay of glucocorticoid binding capacity in the cytosol of rat liver thymocytes (58). half life for this decay was 4 hr at 0° and 20 min at 25°.

The

While

phosphatase inhibitors had only a minimal effect on this inactivation, dithiothreitol had a significant stabilizing effect at 0°, but only a small effect at 25°.

Addition of molybdate along

with dithiothreitol totally prevented inactivation at either temperature, and, receptors inactivated at 25° in the presence of molybdate recovered all their binding activity upon subsequent addition of dithiothreitol.

Enhanced ligand binding was observed

if ATP was added also or upon addition of the heat stable factor. Maximal activation was obtained on addition of ATP, dithiothreitol, molybdate and the heat stable factor (thioredoxin). Based on these observations, the authors postulated that for ligand binding activity to occur, receptor must be phosphorylated and maintained in a reduced state (58,59).

While it was proposed initially that

molybdate stabilization could proceed

through a complex involving

a phosphate moiety on receptor (60), subsequent observations that receptor inactivation in the presence of molybdate was reversible while inactivation in the absence of molybdate was not, led these workers to suggest that molybdate may act by complexing with sulfhydryl groups on receptor (61).

Receptor dephosphorylation

facilitated oxidation of a sulfhydryl moiety on the receptor molecule leading to irreversible loss in binding activity. Molybdate, by complexing with sulfhydryl groups, prevented this oxidation and permitted binding activity to be restored upon dithiothreitol addition (61).

RECEPTOR PHOSPHORYLATION AND SUBCELLULAR DISTRIBUTION. Early data in the literature imply a role for steroid receptor phosphorylation in subcellular distribution, but some of these data may need reinterpretation.

Immunocytochemical (16,17,18) and cell

352 enucleation (15) experiments have indicated that the estrogen (15,16) and progesterone (17,18) receptors are primarily nuclear in localization in undisrupted cells. Steroid receptor distribution observed in both the soluble and particulate fractions of cell homogenates may, therefore, not be indicative of that in intact cells. Sstrogon receptor. Auricchio and coworkers found that [3H]estradiol binding activity in either mouse or calf uterine cytosol could be inactivated partially by incubation with the particulate fraction from corresponding tissue homogenates (45,62). The inactivating activity associated with the particulate fraction appeared to be specific for estrogen-target tissues in that particulate fractions from either mouse liver or quadriceps muscle had no effect on estrogen binding in mouse uterine cytosol (62). While addition of phosphatase inhibitors such as molybdate, fluoride, zinc or 4-nitrophenyl phosphate quantitatively prevented this inactivation, protease inhibitors had no effect; suggesting that a dephosphorylation process is involved (35,62). In order to further characterize this phenomenon, mice were injected with 17-B-estradiol (63). At various times they were sacrificed and estrogen binding in the soluble and particulate fractions of uterine tissue homogenates examined. Fifteen minutes after hormone injection, there was a decrease in estrogen binding capacity in the soluble fraction of cell extracts with a corresponding increase in that of the particulate fraction. After 1 hr, lowered estrogen binding was observed in both soluble and particulate fractions, which by 4 hr was 50% that in control mice (63). Interestingly, when the soluble fraction was incubated with ATP, enhanced estradiol binding was observed. When mice were sacrificed 24 hr subsequent to hormone administration, total recovery of estrogen binding capacity was observed in uterine homogenates. Incubation of the soluble fraction with ATP had no additional effect (63). Based on these observations a model was proposed wherein cytosolic estrogen receptor is translocated to the nucleus upon ligand binding. In the nucleus, receptor is dephosphorylated by a nuclear phosphatase leading to a loss in ligand binding activity and subsequent release of inactive receptor into the cytoplasm. It was

353 suggested that a cytoplasmic ATP-dependent activity, possibly a kinase, could reactivate inactive receptor (46,63). An ATP-dependent activity with the requisite properties was purified partially by ion-exchange chromatography and shown to enhance estradiol binding activity in preparations of either crude cytosol (46) or highly purified estrogen receptor (47) that were first inactivated by phosphatase treatment. For the purified receptor, maximal activation was obtained in the presence of Ca 2+ and calmodulin (47). Under these conditions estrogen receptor phosphorylation on tyrosine was also stimulated (47). Progesterone receptor. In a similar vein, Garcia ££. al. observed that, while progesterone receptors in the soluble fraction of chick oviduct homogenates were phosphorylated, receptors immunoprecipitated from extracts of the particulate fraction were not (64). Different methods such as salt extraction, micrococcal nuclease treatment and NaDodS04 denaturation were employed and in no case were particulate fraction-associated receptors found to be phosphorylated. In contrast to this, Milgrom and coworkers report that cytosolic rabbit uterine progesterone receptor is a phosphoprotein and is further phosphorylated upon ligand binding. This polyphosphorylated form of the receptor corresponds to the particulate bound, putatively active form of the receptor (66). Glucocorticoid receptor. For the glucocorticoid receptor, using pulse chase experiments, Ishiifit.al.. have observed what they interpret as a redistribution of receptor in L-cells in a ligand dependent manner (51). Following binding with radioactive ligand and then a 'chase' with a thousand-fold higher concentration of nonradioactive ligand, ligand binding activity fractionating with the soluble fraction of cell homogenates declined rapidly with a half life of 30-40 minutes, while binding activity in the particulate fraction increased by a comparable amount. This process was temperature dependent, occurring at 37° but not at 0 . Energy deprivation with 2,4-dinitrophenol also reduced bound steroid in the supernatant fraction and increased it in the particulate fraction of cell homogenates. This effect was reversed by the addition of glucose. The authors concluded that upon binding of hormone, the soluble, ligand-binding component was

354 translocated to the particulate fraction, from where it was eventually released back to the soluble fraction. The latter step appeared to be energy dependent, as energy deprivation resulted in accumulation of glucocorticoid binding activity in the particulate fraction. Unfortunately there is little direct information on intracellular receptor distribution, let alone the role of phosphorylation as a determinant. The information available so far on influence of phosphorylation on steroid receptor distribution may need to be reevaluated in the light of evidence that the estrogen and progesterone receptors may be primarily nuclear in localization.

PHOSPHORYLATION IN NUCLEAR INTERACTIONS. Several earlier reports suggest that steroid receptors possess an intrinsic protein kinase activity capable of phosphorylating histones in vitro (32,34,35,36). However,and as discussed in a previous section on Steroid receptors as kinases, recent evidence favors the view that steroid receptors are not protein kinases and that the activities observed may be due to impurities copurifying with receptors (38,39,40). Phosphorylation has been implicated in receptor activation. An ATP-dependent activation of the glucocorticoid receptor has been noted (65,87). Similarly, activation of the estrogen receptor can be achieved at low temperature by incubating with ATP (67). Barnett £t al. have shown that calf intestinal alkaline phosphatase stimulates activation of the glucocorticoid receptor-ligand complex to a DNA binding form, as measured by binding to DNA-cellulose (68). Phosphatase inhibitors blocked this change of state. Based on these observations it was inferred that glucocorticoid-receptor activation involves a dephosphorylation mechanism. In an effort to reconcile observations that ATP and other low molecular weight phosphorylated compounds such as AMP, p-nitrophenyl phosphate etc., also stimulate activation (65,67,68,87) these authors suggest that activation of the glucocorticoid receptor complex involves both phosphorylation and dephosphorylation of different components of

355 the activation complex (68). These data should be interpreted prudently, especially where no interaction with a specific DNA sequence has been demonstrated. The simple process of removing a negatively charged phosphate group from the receptor molecule could by itself lead to increased, albeit nonspecific, binding to the DNA phosphodiester backbone. Miller and Toft have shown that activated progesterone and estrogen receptors show enhanced binding to ATP-Sepharose (69). They suggest that ATP may be biologically important in some function of the activated receptor. However, as receptor activation operationally indicates an enhanced affinity for negatively charged ion-exchangers such as phosphocellulose or DNA, enhanced ATP binding might simply represent a manifestation of this acquired property.

BIOLOGIC RESPONSES TO STEROIDS. Biologic responses to steroid hormone administration include modification of gene expression and regulation of cell growth (70). While progestins are generally growth inhibitory (71), estrogens stimulate growth in target cells (72,73). Recent studies from Lippmann's laboratory suggest that the estrogen effect is indirect and mediated via estrogen-dependent release of growth factor-like activities (74). The mitogenic response to glucocorticoids is varied; stimulation of growth is observed in some cells (75) while growth of others is inhibited (76,77). In the latter case it appears that glucocorticoids act by arresting cells in specific stages of the cell cycle, presumably by inhibiting transcription of specific genes which ordinarily confer upon these cells an autocrine-regulated growth inducing capacity (78,79,80). Addition of extraneous growth factors overcomes this inhibition (78,79). Receptor phosphorylation may play central roles in steroid hormone action. It is well documented that receptor phosphorylation is, at least in some cases, crucial for ligand binding. Less well documented is the evidence that phosphorylation may influence

356

subcellular distribution of receptors, and in some instances receptor activation.

The biologic effects of steroids may also be

regulated by phosphorylation of steroid receptors by cellular kinases.

In this connection, studies on receptor mediated

interactions between steroid hormones and growth factors are of interest.

PHYSIOLOGICAL RELATIONSHIPS BETWEEN GROWTH FACTOR AND STEROID ACTIONS. There is now an accumulating body of data suggesting a physiological relationship between steroid hormone and growth factor actions, possibly at the level of their respective receptors. Dexamethasone can enhance EGF binding to quiescent human diploid foreskin fibroblasts under certain assay conditions (75).

Maximal

enhancement was obtained after 24 hr of dexamethasone treatment and was inhibited by cycloheximide.

Maximal enhancement was observed

when EGF was present at low concentrations as

125

I-EGF (0.2 ng/ml).

The extent of enhancement decreased with increasing concentrations of

125

I-EGF.

From this, the authors concluded that dexamethasone

induced a qualitative change in cell surface EGF receptor properties; possibly increasing their affinity. also enhanced

125

Glucocorticoids

I-insulin binding to Swiss mouse fibroblasts and

this up-regulation did not appear to involve insulin receptor synthesis (81).

A three- to five-fold increase in insulin binding

was observed 12 hr after glucocorticoid administration at which time insulin-induced receptor down regulation was markedly reduced. Similarly, progestins also increased EGF (82) and insulin (83) binding to cultured breast carcinoma cells.

Incubation of adherent

T47D cells with progestins resulted in enhanced EGF binding with no change in binding affinity (82).

Maximal stimulation was obtained

after 10 hr of progestin treatment.

Physiological relationships of

androgen or estrogen receptors with growth factors have not not been described, though estrogens stimulate secretion of growth factor-like activities into the medium of cultured MCF-7 cells (72,74) and androgens stimulate transcription of the nerve growth

357 factor gene in mouse submaxillary gland (84). Recent studies in our laboratory show that addition of growth factors results in rapid decreases in progestin (85) or glucocorticoid (Rao,K.V.S. and Fox, C.F., unpublished results) binding in cultured human breast cells. Incubation of T47D, MCF-7 or ZR75-1 cells with 3 nM EGF or insulin for 60 min at 37° resulted in a 30 to 50% decrease of subsequently measured progestin binding with no change in progestin binding affinity (85). EGF also caused a decrease in glucocorticoid binding in HBL100 cells without altering the apparent dissociation constant for dexamethasone binding ( Rao, K.V.S. and Fox, C.F., unpublished results). Interestingly, inhibition of cell proliferation due to addition of these steroid hormones was overcome by addition of growth factors to the culture medium. Growth factor-steroid receptor relationships are suggested to have important implications for endocrine therapies (83).

CONCLUSIONS. The phosphoprotein nature of steroid receptors is well established. They are phosphorylated on a variety of residues; demonstrating the absence of a global mechanism (Table 1). The estrogen receptor is phosphorylated on tyrosine residues, and the progesterone receptor is phosphorylated on serine residues. The glucocorticoid receptor is phosphorylated primarily on serine, with some contribution on tyrosine residues. It will be of interest to determine the functional roles played by these receptor phosphorylations. We note correlations wherein ligand binding to the tyrosine phosphorylated estrogen receptor is associated with a mitogenic response; the action of serine phosphorylated progesterone receptors is generally growth inhibitory. Glucocorticoids, acting via receptors that are phosphorylated both on serine and tyrosine residues, can have growth inhibitory or stimulatory effects depending on cell type and circumstances. Phosphorylation can be crucial for the ligand binding activity of steroid receptors. This has been shown rigorously by Pratt and his

358

Table 1. Summary of amino acid residues phosphorylated in various steroid receptor systems. Receptor

System

Amino acid phosphorylated* in situ

Progesterone

Glucocorticoid

Estrogen

Avian Oviduct Slices

serine 22 ' 24

Cultured Human Breast Carcinoma Cells

serine®

Purified Chick Oviduct PR + Purified EGF-R

-

Purified Hen Oviduct PR +cAMP-dependent kinase

-

Cultured Mouse Fibroblast

serine27

Cultured Human Breast Epithelial Cells

serine, tyrosine5

Purified from Rat Liver

-

Purified Rat Liver GR +CAMP-dependent kinase

-

Rat Uterine Slices

tyrosine 30

in vitro

tyrosine 42 nt 41 -

threonine36 nt 25 -

Human Breast Carcinoma Cell Extracts

-

serine33

Purified Calf Uterine ER +Ca2+/Calmodulin Dependent Kinase

-

tyrosine 47

•Numbers in superscript correspond to citiations in the list of references. ^Unpublished results described in this report, nt, not tested.

359

group who demonstrated that phosphorylation assists in stabilizing glucocorticoid receptors in a conformation favorable for ligand binding. Steroid receptor phosphorylation appears to result from the action of intracellular, cytosolic protein kinases (37,46), which are yet to be characterized in detail. Auricchio and coworkers have isolated one such cytosolic, Ca 2+ and calmodulin dependent kinase that phosphorylates the purified estrogen receptor on tyrosine residues (47). This enzyme may be responsible for in vivo tyrosine phosphorylation of estrogen receptor (30). Pratt and coworkers have recently identified a NADPH-dependent, thioredoxin-mediated protein reducing system that seems necessary for maintainance of steroid binding by glucocorticoid receptors (57). The relationship between this protein reducing system and receptor phosphorylation, both of which are apparently critical for ligand binding activity, is not known. It remains to be seen whether these two processes represent independent systems, which confer dual regulation of cellular responses to glucocorticoids. Much of the literature on receptor phosphorylation in steroid hormone action has not led to definitive conclusions. The case for steroid receptor-associated kinase activity is now in doubt since recent findings with the progesterone (39) and glucocorticoid (37,38) receptors do not confirm their protein kinase activities. The estrogen receptor associated kinase activity observed by Witliff and coworkers (33) represents a single report and has not been confirmed by others. Further, that kinase appears to be serine-specific, thus contrasting with the observations of Auricchio and his group that the estrogen receptor is phosphorylated exclusively on tyrosine residues (30). Protein kinases generally are self-phosphorylated at the same residues on which they phosphorylate substrate proteins. While purified steroid receptors are phosphorylated by growth factor-activated protein tyrosine kinases and serine-specific, cAMP-dependent protein kinase, the significance of these studies rests on results of experiments with intact cellular systems. This is particularly true in light of the fact that protein kinases can have significantly relaxed specificities in vitro. For example,

360 though we have found that purified avian progesterone receptor is a high affinity substrate for phosphorylation by human EGF receptor kinase at high rate, we have been unable to demonstrate phosphorylation of progesterone receptor in human cells stimulated with EGF. Another area of controversy is the relationship between phosphorylation and subcellular distribution of steroid receptors. Milgrom and coworkers reported that a ligand dependent phosphorylation of the progesterone receptor gives rise to a polyphosphorylated particulate-bound form (66). In contrast , Garcia £t al. observed that the immuneprecipitated particulate-bound progesterone receptor is not phosphorylated (64). The latter is in keeping with findings of Auricchio's group that the ligand-bound estrogen receptor is dephosphorylated in the nucleus (45,62,63). Similarly, Pratt and coworkers have observed a glucocorticoid receptor dephosphorylating activity in the particulate fraction from mouse fibroblasts, rat thymocytes and rat liver (55). This phosphatase activity, however, is specific only for the unoccupied receptor, and the implications of this observation are not clear. There is much to be learned about the precise role of phosphorylation in steroid receptor function and future research should resolve at least some of the current controversies. With the availability of highly specific monoclonal antibodies, such studies should not be long in forthcoming, providing valuable insight into this exciting new aspect of steroid hormone research. The need for a consensus on the molecular mechanism by which ligand binding ultimately leads to steroid receptor-mediated modifications in gene expression is equally important. The current controversy on whether these receptors are cytoplasmic or nuclear-localized in the absence of hormone requires resolution. Given rational models that are firmly based in fact, one could then systematically test possible roles for receptor phosphorylation in the various steps involved. Of interest also is more recent evidence for relationships between growth factor and steroid hormone activities. Further exploration

361

in this area could provide valuable information directed at therapeutic strategies (83).

A significant inverse relationship has

been observed for estrogen and EGF receptor contents in primary breast tumors (88). ABBREVIATIONS. SDS—PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EGF, Epidermal growth factor; PDGF, platelet derived growth factor.

ACKNOWLEDGEMENTS. Studies from our laboratory were supported by ACS Grant No. BC-473 and by USPHS Grant No. AM25826.

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THE RAT UTERUS AS A MODEL FOR STEROID RECEPTOR AND POSTRECEPTOR CHANGES DURING AGING R.S. Chuknyiska Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224. *Present Address: The Johns Hopkins University, School of Medicine, Department of Surgery, Francis Scott Key Medical Center, Baltimore, Maryland 21224.

Introduction It appears that changes in the steroid hormone-receptor and hormone-receptor-acceptor interactions are a general phenomenon of the aging process as they have been reported for many receptors and target tissues (1-11). It is well documented that when estradiol binds to the nuclear acceptor in the cell of a target tissue one of the first events which is observed after the message to the genome has been delivered is stimulation of RNA synthesis, followed by an increase in protein synthesis (12,13). It is widely accepted that age associated deficits in hormone-stimulated gene expression (2,14,15) are due to both loss of uterine estradiol receptors (16-19) and impaired association of receptor estradiol complexes with nuclear acceptors (20-23). However, some steroid-sensitive tissues show no loss of receptor number with age, despite a decrease in sensitivity (10,24,25). In this chapter we focus our attention on various post-receptor events such as nuclear receptor-acceptor binding and stimulation of nuclear RNA polymerase II, both of which are critical events in transmission of the steroid message to the cell (13,26,27). It is proper to mention that differences which are observed in nuclear binding and enzyme activity in the rat uterus could serve as a model for analyzing the effects of changes in the endocrine status and subsequent events which occur in the human after menopause. Moreover, it has been reported that there is a decrease in the binding capacity of estrogen receptors in postmenopausal women (28). Thus it was of interest to determine if nuclei and cytoplasmic receptors derived from uteri of senescent rats differed from those obtained from mature rats in their ability to support nuclear binding. The end points mentioned above are discussed in this chapter more as an attempt to analyze the results obtained in

Recent Advances in Steroid Hormone Action © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

368 the p r o c e s s of s e a r c h i n g for some age r e l a t e d d e p e n d e n c y b e t w e e n d e c r e a s e d nuclear b i n d i n g and i m p a i r e d nuclear f u n c t i o n s in rat uterus rather than a c o m p r e h e n s i v e r e v i e w already e x i s t i n g d a t a .

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 E s t r a d i o l R e c e p t o r s i Age R e l a t e d D i f f e r e n c e s D e t e c t e d by S u c r o s e G r a d i e n t A n a l y s i s Uterine Cytosol Receptor Complexes

of

of

P r e v i o u s r e p o r t s have s u g g e s t e d that q u a l i t a t i v e d i f f e r e n c e s b e t w e e n c y t o s o l s t e r o i d r e c e p t o r s in v a r i o u s t i s s u e s are m a n i f e s t e d as d i f f e r e n c e s in ligand b i n d i n g , nuclear b i n d i n g or 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 , a l t h o u g h the latter is still c o n t r o v e r s i a l (6,29,32). It has r e c e n t l y been shown that t r a n s f o r m a t i o n can occur in c o m p l e t e l y p u r i f i e d receptor p r e p a r a t i o n s (33-35). F o l l o w i n g 3535 a m m o n i u m s u l f a t e p r e c i p i t a t i o n of c y t o s o l f r a c t i o n s , b i n d i n g to the nuclear a c c e p t o r i n c r e a s e s five f o l d , p o s s i b l y due to e l i m i n a t i o n of factors w h i c h may i n h i b i t n u c l e a r b i n d i n g (36). T h e r e f o r e in our e x p e r i m e n t s c y t o s o l r e c e p t o r s were i s o l a t e d by 35% a m m o n i u m sulfate p r e c i p i t a t i o n of 105000 g s u p e r n a t a n t s of h o m o g e n i z e d rat uteri f o l l o w e d by a s l i g h t m o d i f i c a t i o n of the p r o c e d u r e as was already r e p o r t e d (22,37). Whether or not we e l i m i n a t e some p o s s i b l e f a c t o r s r e s p o n s i b l e for d e c r e a s e d a c c e p t o r - r e c e p t o r b i n d i n g is not yet known. N e v e r t h e l e s s , the m a j o r d i f f e r e n c e s in b i n d i n g c a p a c i t y of m a t u r e and s e n e s c e n t uteri p e r s i s t under these c o n d i t i o n s as d i s c u s s e d f u r t h e r . One a p p r o a c h to i n v e s t i g a t e m e c h a n i s m s that m i g h t a c c o u n t for the d i f f e r e n c e s b e t w e e n m a t u r e and s e n e s c e n t s t e r o i d r e c e p t o r - a c c e p t o r b i n d i n g is to c o m p a r e the 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 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 o b t a i n e d by h i g h s p e e d c e n t r i f u g a t i o n of p a r t i a l l y p u r i f i e d c y t o s o l s l a y e r e d on sucrose gradients. C h a n g e s in the s e d i m e n t a t i o n p r o f i l e of the r e c e p t o r s as a f u n c t i o n of c h a n g e s in the salt c o n c e n t r a t i o n of the e x t r a c t i o n buffer or of the i n c u b a t i o n t e m p e r a t u r e have been s t u d i e d e x t e n s i v e l y in the past y e a r s (38-45). It was of i n t e r e s t to c o n s i d e r p o s s i b l e d i f f e r e n c e s in the s t r u c t u r e of e s t r a d i o l r e c e p t o r s o b t a i n e d from mature and senescent rats. In our i n i t i a l a t t e m p t to e l u c i d a t e these c h a n g e s , we have e x a m i n e d the e f f e c t s of aging on the c o n v e r s i o n of rat u t e r i n e e s t r a d i o l receptor from 8S to 4S and 5S, p r e r e q u i s i t e s for n u c l e a r b i n d i n g (38-40). Although recent e v i d e n c e s u g g e s t s that e s t r a d i o l r e c e p t o r s may n o r m a l l y be found in a s s o c i a t i o n with the n u c l e u s (46), it is clear that much tighter b i n d i n g o c c u r s after p h y s i c o c h e m i c a l c h a n g e s that alter r e c e p t o r s e d i m e n t a t i o n p r o p e r t i e s from 8S form to a

369 state with high affinity (43,44). Such changes can be e l i c i t e d in vitro by salt treatment, dilution or heating (12,41,4817 I l l

(A)

4000 CL

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100

-

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2

RECEPTOR—E2 COMPLEX BOUND (fmol/mg DNA)

Fig. 11. Effect of nuclear acceptor site occupancy on RNA polymerase II activity. Portions of the same nuclei and receptors prepared for the experiment shown in Fig. 1 were u t i l i z e d , except that receptors were incubated with unlabeled estradiol to prepare complexes. These were then used to activate RNA polymerase II. Polymerase activity is plotted as a function of nuclear acceptor site occupancy (as d e t e r m i n e d for Fig. 10) for each individual value. Slopes were c a l c u l a t e d by linear regression analysis. Various symbols represent individual e x p e r i m e n t s for mature (o) and senescent (•) p r e p a r a t i o n s . Correlation coefficients were 0.81, 0.93, and 0.87 for the regressions of the m a t u r e , senescent, and combined groups, respectively.

F u r t h e r m o r e , this relationship was confirmed when cytosol receptors were then divided into two portions and simultaneously used to assess nuclear binding and the stimulation of RNA polymerase activity. When different c o n c e n t r a t i o n s of nonlabeled estradiol are incubated with increasing c o n c e n t r a t i o n s of labeled estradiol and 3 H - U T P

394 increase in incorporation of 3 H - U T P into the nuclei and decrease of the L^HJestradiol bound to the receptor occurs (Fig. 12).

> E

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Structure of Different Estratrlenes with Various Substitutions

H

449 has been reported (31) as has estratrien-3-ol

(32).

Other estrogens

utilized (estratriene, Ej, E2, E 3 , estradiol-16a, estradiol-17a and 6-ketroestradiol) were purchased from Research Plus, Inc. (Bayonne, New Jersey). Cytosolic estrogen receptor (E2R) preparation (33) and assays were carried out according to classical Scatchard (34) and competitive binding (35) methods utlizing dextran-coated charcoal (DCC).

Progesterone

receptor (PgR) was determined in DCC assays under conditions which eliminated "Type II" PgR from the results (36).

Results

Affinity of substituted estratrienes for E?R.

Affinity experiments were

carried out utilizing the established competitive binding assay (35) in which a range of competitor concentrations were examined in experiments with 4 nM 3hE£.

Data from these studies are presented in Tables 1-4.

The estratriene nucleus did not compete with ^HEg for binding sites over a 2000 fold range (Table 1).

Furthermore a keto function placed at

position-17 yielded a ligand with an affinity too low to measure in the assay.

As has been previously reported (2-4) the 3-phenolic hydroxyl

proved to be the most important function on the Eg molecule for bindinq to receptor (i.e., estratrien-3-ol has 40% of Eg's affinity for receptor whereas the estratrien-17s-ol Table 1.

retained only 8% of the maximum affinity).

Relative Binding Affinity of Substituted Estratrienes Relative

Compound

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Binding Affinity*

Estratriene

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516 we w i l l try to h i g h l i g h t the points which deserve comment and to draw some general

conclusions.

a) Adsorption step The adsorption of the macromolecule to be p u r i f i e d on the b i o s p e c i f i c matrix can be performed using the batch or the column procedure.Several parameters can be considered f o r a r a t i o n a l choice between these two p o s s i b i l i t i e s . F i r s t the batch adsorption can be recommended when the macromolecule of i n t e r e s t

is

present at a very low concentration in the s t a r t i n g medium, i s unstable and binds to the immobilized ligand with a high a f f i n i t y ( 4 3 ) . When r e s o r t i n g to batch adsorption, a l a r g e sample of the solution from which the macromolecule needs to be extracted can be adsorbed on a small amount of b i o s p e c i f i c adsorbent. The " d i l u t i o n " of the s p e c i f i c ligand during t h i s step i s by no means a drawback since owing to the high a f f i n i t y of the system the macromolecule w i l l rapidly bind to the beads. Moreover in the batch procedure a l l the macromolecules of the sample w i l l s t a r t incubating with the a f f i n i t y matrix at the same moment and the adsorption can be pursued f o r hours, i f needed, to obtain complete binding. On the other hand when r e s o r t i n g to column adsorption a large sample can be applied onto a l i t t l e amount of a f f i n i t y g e l only i f binding of the macromolecule to the a f f i n i t y matrix i s rather rapid

the

(obtai-

ned a f t e r a contact time of a few minutes or l e s s ) , but even in this case the l a s t part of the sample w i l l wait several hours b e f o r e reaching the gel beads and can t h e r e f o r e be i n a c t i v a t e d during t h i s time. However the column procedure gives e x c e l l e n t r e s u l t s when the macromolecule to be p u r i f i e d i s quite s t a b l e . Moreover the gel is maintained in a compacted form during the e n t i r e procedure and i s t h e r e f o r e not submitted to manipulations which can r e s u l t

in

some loss of material as in the case of the batch process.

Owing to the i n s t a b i l i t y of the unbound g l u c o c o r t i c o i d receptor and in order to obtain a b e t t e r saturation of the a f f i n i t y matrix we chose batch adsorption f o r the p u r i f i c a t i o n of both the rat (19, 25) and the rabbit (26)

receptor.

As depicted in f i g u r e 6 the adsorption k i n e t i c s of the receptor on the a f f i n i ty matrix c l e a r l y depended on the cytosol volume/gel volume r a t i o . When this r a t i o was increased from 5 to 30 a complete adsorption of the receptor was no longer obtained, but the adsorption y i e l d remained higher than 70 % a f t e r a 5h incubation time, and, above a l l , the s p e c i f i c saturation of the g e l was g r e a t l y increased ( f i g u r e 6, panel B ) . Increasing t h i s s p e c i f i c saturation is h i g h l y

517

B)

Cytosol vol./ ge ,

vo.

Figure 6. Binding of the cytosolic glucocorticoid receptor from rat liver to the nonylamino dexamethasone adsorbent (NANOFAc Sepharose). Aliquots of gel (0.5 ml) were incubated with increasing volumes of cytosol. Binding activities in the supernatant and in a control cytosol were measured at different times by incubating with 20 nM [^Hj dexamethasone at 4°C for 16h. a) Specific adsorption is expressed as the ratio of binding in the supernatant (B) to the binding in the control (B 0 )during the course of the adsorption for different cytosol/gel ratios (5, 10, 20 and 30). b) Specific saturation of the gel is expressed as percentage of ligand sites effectively occupied by receptor molecules as a function of adsorbent loading (expressed by the ration cytosol/gel).

518 desirable to obtain a better degree of purification of the receptor as it increases the ratio of specific versus n o n specific binding on the gel. M o r e over it allowed receptor elution in a concentrated form b y using a small v o l u m e of elution solution, and this concentrated receptor could then be more easily handled for subsequent experiments, for example ultimate purification b y high-performance size exclusion chromatography, in vitro activation and use for mouse immunization. For the purpose of increasing the specific

saturation

of the affinity gel a prepurification step performed before the affinity

step

and allowing a first concentration of the receptor seemed v e r y attractive since a cytosol volume/gel volume ratio greater than 100 could not be reasonably envisaged. Thus the u n b o u n d cytosolic receptor was first submitted to protamine sulfate precipitation, which resulted in a five-fold volume

reduction

and a eight-fold purification w i t h excellent yield (19). The protamine extract was then adsorbed on the affinity matrix with a protamine

sulfate

sulfate

extract/gel ratio of 50 (v/v), i.e. corresponding to 250 for the cytosol. The gel volume used for routine purification using 15 to 50 adrenalectomized rats was in the range 0.5-2 ml. Optimization of the cytosol/gel ratio permitted h i g h receptor load on the gel

: 2-4 nmol receptor/ml wet gel versus 0.025-0.16

nmol for the gels u s e d by others for the purification by affinity graphy of the glucocorticoid and progesterone receptors

chromato-

(see b e l o w tables II

and III).

The column adsorption procedure was preferred by Renoir (14, 15) for the p u r i fication of the chick oviduct progesterone receptor using the gel we designed and prepared for him. Here a rather large amount of gel was u s e d w i t h a cytosol/ gel ratio in the range 5-20 (v/v). As a consequence the specific

saturation

of the gel was rather low and the receptor was eluted in a diluted form. However a h i g h specific saturation of affinity gels can be attained by using the column procedure, as exemplified in the work of Greene (12) who was able to adsorb as m u c h as 10 nmol of estrogen receptor per ml of packed adsorbent after passing about 1 liter of cytosol on a 2 ml column of estradiol

affinity

matrix. W h e n using the column procedure a determination of the efficiency and of the theoretical capacity of the gel could also be performed

(49).

b) Washing step

Washing of the loaded gel before elution is crucial for the purity of the final product. This is specially true in the case of the steroid hormone re-

519

ceptors for w h i c h the theoretical purification factor to be attained is very high. Even w h e n a good specific saturation of the adsorbent is obtained we should be reminded that we are dealing with an extremely low macromolecule concentration and that a few micrograms of contaminating p r o t e i n might represent the m a i n part of the eluted proteins. Thus rapid and efficient w a s hing procedures displaying the m a x i m u m stringency possible without loss or damage to the receptor have b e e n developed. In the case of the glucocorticoid receptor the following sequence was applied : adsorption buffer at 4°C, low salt buffer at 4°C, high salt buffer at 4°C, high salt b u f f e r at 25°C for a short time, then back to the adsorption b u f f e r at 4°C. The entire procedure lasted less than an hour and involved 230 gel b e d volumes

(25) . The gel can be

w a s h e d in a column using either a strict or a m i x e d column procedure. In the latter case the gel was suspended in the wash solution by mechanical

agita-

tion in the column at the beginning of the procedure in order to accelerate the cleaning of the beads (a very h i g h flow rate was applied at this moment) which was then persued using conventional column washing after gel

sedimenta-

tion. The progesterone receptor w h i c h is more stable than the glucocorticoid receptor was submitted to a more drastic treatment including a washing of the affinity gel w i t h 2.5-3.0 M urea resulting in a 3 fold improvment in the purity of the eluted receptor (15, 50).

c) Elution step

Biospecific elution with a highly receptor-specific labeled steroid displaying a h i g h affinity for this macromolecule is in fact recommended. The eluting ligand is generally u s e d at a micromolar concentration in buffer solution (1 to 4 gel volumes). The elution was performed at 0°C with 5 y M tritiated triamcinolone acetonide for the rat liver glucocorticoid

receptor

(19, 25) and at 20°C for the rabbit receptor, which dissociated very

slowly

from the immobilized ligand at 0°C (26). Similarly, elution of the chick oviduct progesterone receptor from our NADAc-Sepharose matrix was easily obtained by using 1.0-2.0 JJM solutions of tfitiated progesterone at 0°C The low derivatization of our gels (0.15-0.5 ymol of immobilized

(14).

steroid/ml

of gel) was probably responsible for this easy elution since we h a d already observed

that with highly derivatized gel (2.0 to 5.0 ^mol steroid/ml)

the

biospecific elution was obtained in only extremely poor yield (19). However rather than resorting to chaotropic agents like sodium thiocyanate, or to o r ganic solvents, like dimethylformamide, to help the dissociation of the

520

receptor f r o m a too derivatized matrix, an expedient already u s e d b y others

(12,

44), we preferred to design appropriate adsorbents which avoid the employment of such methods. These agents m a y indeed cause some damage to the receptor and result in some n o n biospecific elution of some of the contaminating m a c r o m o lecules adsorbed on the gel (a perfect gel and/or washing step do not

simply

exist) and could therefore jeopardize the efficiency and yield of the p u r i f i cation. With our gels 300-2100 fold purification factors and 20-40 % yield were obtained for the glucocorticoid receptor and 935-2600 fold purification factors and 24-49 % yield for the progesterone receptor (14, 15, 19, 25, 26 and tables II and III).

d) Final purification step

Owing to the high value of the purification factor necessary to o b t a i n steroid receptors in an homogeneous state, the affinity chromatography step, even in the best case, was unable to produce a completely purified protein. W h e n using our gels the affinity eluates corresponded to a theoretical purity of 10-30 % on the basis of the molecular mass of the steroid binding unit (14, 15, 25, 26). Thus a further purification step was required, even if it was complicated b y the w e l l k n o w n instability of highly purified steroid-receptor complexes and the difficulty of handling very dilute p r o t e i n solutions without significant

loss.

Conventional techniques such as ion exchange and gel filtration chromatography on open-bore columns were used for the progesterone receptor

(14, 15). H o w -

ever, in the case of the glucocorticoid receptor, decisive progress was made b y resorting to high-performance size-exclusion chromatography on a T S K G3000 SW column (25, 26). This step, w h i c h allowed a further 2-5 fold purification, was performed with 90 % yield in less than 40 min. and appeared particularly suitable for the final purification of the small affinity eluate sample

(1-5

m l , injected as 0.5 ml aliquots, i.e. 10-50 pg protein) containing a rather concentrated receptor given by our affinity procedure

(see the previous

dis-

cussion about the optimization of the adsorption step in part Ilia of this chapter). Moreover high-performance ion exchange chromatography was also u s e d with very satisfactory results for analytical and preparative work on affinity eluates. Thus owing to their excellent resolution and speed both these h i g h performance techniques resulted in clearly better separations than conventional chromatography and undoubtely afforded a significant improvment in our previous purification procedure

(19). However the obtainment of good results

when applying high-performance protein chromatography to the study of

steroid

521 hormone receptors is b y no means ascertained by the possession of correct equipment : m i n i m u m consideration needs to be paid to the technical aspects of the use of this methodology and to the optimization of the chromatographic procedure. Preparative SDS polyacrylamide gel electrophoresis represents an alternative final step to which w e resorted in order to obtain highly p u r i fied glucocorticoid receptor preparations, thereafter used for rabbit

immuni-

zation (Richard et al., in preparation).

The fact that the affinity eluate contains some components which are distinct from the steroid binding subunit of the receptor immediately raises the following question : h o w can we distinguish the other putative subunits of the receptor complex which must be present in the eluate from n o n specific c o m ponent artefactually copurified with the receptor ? The simplest w a y to answer this crucial question is to perform a "mock" purification which consists in loading the gel with a receptor sample previously saturated with a n excess of unlabeled steroid. In this case no specific b i n d i n g of

either the receptor

or its various components will be observed whereas n o n specific binding and elution of the contaminants can be studied. This straightforward control was successfully utilized to prove that the 90 K heat shock p r o t e i n is a n effective component of the molybdate stabilized form of the chick oviduct progesterone receptor purified by using our NADAc affinity matrix, and not a n artefact due to the affinity procedure (51). Similarly, we are currently studying the eventuality of the association of a specific RNA molecule with the glucocorticoid receptor (Sablonniere et al., unpublished data).

e) Assay of the purified receptor

The purified receptor is generally obtained in the affinity eluate as a dilute solution containing a low amount of protein (1-50 ng/ml) in presence of an excess of tritiated eluting ligand. Since steroid receptors are hydrophobic and rather unstable proteins their accurate assay in the purified form is not easy and needs to be performed using at least two distinct methods. We usually employ a dextran-coated charcoal assay performed in the presence of serum albumin as receptor-stabilizing agent, a hydroxylapatite adsorption assay and fast size exclusion chromatography o n a T S K GSWP column (7.5 x 75 mm, LKB). For protein determination various methods can be used, like the assay of Bensadoun and Weinstein (52), the coomassie blue assay of Bradford and its variants

(53,

54) or the Amidoschwarz method of Schaffner and W e i s s m a n (55). The fact that

522 all these methods y i e l d results

largely dependent on the protein composition

of the sample must b e stressed. Thus the experimentator need only choose the most convenient assay for his purpose and be aware that, w h e n using a calibration curve established w i t h bovine serum albumin solutions the calculated results can differ from the actual protein concentration in the sample by a 2-4 fold factor according to the nature of the protein considered (54 and references cited herein). This fact must be k e p t in mind w h e n calculating

the

receptor purification factor from the specific activity of the purified m a terial and the molecular weight of the steroid binding

subunit.

f) Conclusion

Here again it appears that after the design and synthesis of a suitable affinity matrix further efforts n e e d to b e spent for the optimization of the affinity chromatography procedure, and specially o n its adsorption and washing steps, the care afforded to w h i c h largely determine the quality of the receptor purification. The higher the specific saturation of a good matrix, the more efficient

the washing step and the better are the results.

Finally,

authentic analytical preoccupations m u s t be kept in m i n d w h e n performing the assay of the purified receptor and carrying out the final purification step.

IV. Application of our Affinity Matrixes for the Purification of Glucocorticoid and Progesterone Receptors! Comparative Results.

a) Comparative date concerning the affinity step.

By using a rational and systematic approach we were able to design two a f f i nity matrixes suitable for the purification of the glucocorticoid and the progesterone receptor respectively

: NANOFAc Sepharose, or N-9-amino-nonyl-3-

-

oxo-9-fluoro-11g,17a dihydroxy-16a-methyl-androsta-1,4-diene-17g-carboxamide coupled to Sepharose C L 4B, and NADAc Sepharose, or N-12-amino-dedecyl-3oxo4-androstene-17B-carboxamide. These gels are n o w u s e d by several groups and can be compared with the other affinity adsorbents already proposed for the same purpose, among which Sterogel, the marketed version of the adsorbent

ini-

tially described b y Grandics (21), is the most popular one (figures 7 and 8). The m a i n features of the u s e of our gels and of their competitors are in Tables II and III.

summarized

523 REFERENCES

GEL STRUCTURE

LIGAND

CO-MH-tCH^-HH-ftqOrosel Failla (16) Weisz

(23)

DOC

CO-NH-CHr-CH-CH.-O 'OH I