Identification of specific high affinity sites for the estradiol receptor in the erythrocyte cytoskeleton

vol. 103, No. 2,198l

BIOCHEMICAL

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RESEARCH

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30, 1981

November

IDENTIFICATION

OF SPECIFIC

HIGH AFFINITY SITES FOR THE ESTRADIOL RECEPTOR IN THE ERYTRROCYTE CYTOSKELETON

Giovanni

Alfred0

Puca and Vincenzo

Sica

First Medical School, University di Patologia generale, Via S. Andrea delle Dame, 2, 80138 Naples, Italy

Istituto Received

September

682-689

of Naples,

4,1981

SUMMARY. The possible relationship of the soluble, "cytosolic" estradiol receptor with complex membranous and cytoskeletal structures of the cell matrix has been studied using a model erythrocyte.system. Extraction of erythrocyte ghosts with a nonionic detergent (Triton X-100) under conditions that yield a cytoskeletal matrix reveals the presence of a limited number (less than 100) of specific sites having high affinity (kd lo-' M) for the estradiol-receptor complex. The interaction between the estradiol receptor and the cytoskeleton is critically dependent on temperature and it is improved by 25 mM KC1 or NaCl and by 2.5 mM MgC12. The data suggest that the estradiol receptor, which has been generally considered to be freely "soluble" in the cytoplasm, may actually be physiologically associated in an integral manner with a complex cytoskeletal network in the cell cytoplasm.

INTRODUCTION. as being target

Steroid

water-soluble cells,

hormone

macromolecules,

waiting

for

the arrival

of the hormone-receptor

complex

changes

the

This

which

determine

mechanism

and most

more than

of this

10 years

Recently this

the cytosol

since

(of

water-soluble Increasing

of a specific leads

dogma"

have been

interpreted

model

at least

target

cells)

molecule

(2-5).

of steroid

one of its of the

of the

receptor

intricate

0006-291X/81/220682-08$01.00/0 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

major

682

of their Formation

molecule. conformational

the

action

of this

or addressed (see Ref.

question

hormones, model.

during

and serious

points,

i.e.,

as an unrestrained,

nature

the

the

1).

the validity

action,

and complex

compartment.

of steroid

on the basis

which

considered

to the nuclear

proposed

have been published

awareness

for

was first

cytoplasm

to putative

have not been elucidated this

been

hormone

of the receptor

dogma of the mechanism concerning

in the

"central

thesis

much data

central

be raised

data

have generally

floating

presumably

transfer

has become the

experimental

Many aspects

receptors

of

doubtgmust presence

in

freely

of the ultrastruc-

Vol. 103, No. 2,1981

tural

BIOCHEMICAL

organization

of the

various

cytoplasmic

closely

related This

(6).

importance

in cellular

The present uterus with

interacts components

be considered

organized

functions

with

high

of more complex

steroid

of the

receptors

network

and functional

evidence

that

may be

of structural unit

elements

of fundamental

the estrogen

and in a specific

erythrocyte

cytoskeletal

model

eucaryotic

of integration

hormone

microscopic

affinity

as a simplified

degree

COMMUNICATIONS

(7-15).

provide

of the

that

a morphological

studies

RESEARCH

and the high

suggests

to a highly forms

BIOPHYSICAL

cytoplasm

elements

network

AND

system

of calf

and saturable

system; for

receptor

manner

the latter

exploring

may generally

cytoskeletal

properties

cells.

MATERIALS AND METHODS. 17B-Estradiol-2,4,6,7-t4 (84 to 110 Ci/mmol) was from Amersham. Cow blood erythrocyte ghosts and cytoskeleton were prepared as described by Bennett (16,17); these were resuspended with phosphate buffer at a protein concentration of 3 to 10 mg/ml, diluted 1:l (v:v) with 99% glycerol and stored at -70° until used. Calf uterine cytosol was prepared as previously described (18) except that only phosphate buffer (7.5 mM phosphate buffer, pH 7.5) instead of Tris-HCl EDTA and dithiothreitol buffer was used. The assay of specific binding activity of cytosol was performed by the Dextran-coated charcoal method (19). The binding of the [%]estradiol-receptor complex to erythrocyte ghosts and cytoskeletons was performed in duplicate. Calf uterine cytosol was preincubated for 10 min at 20° in a total volume of 0.4 ml of phosphate buffer with 2.5 pmoles of [3H]estradiol, 12.5 pmoles KC1 and 1.25 pmoles MgC12. The reaction was started by adding 0.1 ml of cytoskeleton or ghost suspension (0.25 to 2 mg of protein) and followed either for 2 hours at 20” or for 30 min at 30°. Parallel tests were performed in the presence of 1 x 10m6 M unlabeled estradiol. The reaction was stopped by adding 10 ml of ice-cold phosphate buffer. Samples were centrifuged at 30,000 x g for 20 min at 4O and the pellets were washed three times. The final pellet was resuspended with 1 ml of phosphate buffer and assayed for radioactivity. The values obtained in the presence of unlabeled hormone were subtracted from the total.

Specific

RESULTS. -in erythrocyte These

sites

(Fig.

1).

cyte

membranes, are only

membranes

with

erythrocyte the

ghosts

with This

but

detected

The maximal

concentrations. the

binding

number

1% Triton

sites only

or cytoskeleton

extraction

of binding X-100

(20).

estradiol of calf

appears

and does not

uterine

is used

the uterine

with

with

detergent

hormone

of erythro-

higher

operationally

cytosol

cytosol.

by treatment

increase

The tritiated

683

can be detected

of the membranes

sites

procedure

if

radioactive

in the presence

after

extraction

“cytoskeleton”

for

detergent

to prepare does not

is omitted,

if

associate the

Vol. 103, No. 2,198l

BIOCHEMICAL

AND

0.25

BIOPHYSICAL

0.50

0.75

1.00

TRITON

RESEARCH

1.25

X-100,

COMMUNICATIONS

1.50

%

Fig. 1. Efrect of extracting erythrocyte ghosts with Triton X-100 on the appearance of specific binding of [%]estradiol-receptor complex. Erythrocyte ghosts were extracted for 15 min at 0" with the indicated concentrations of Triton X-100. After extraction, the ghosts were centrifuged and washed four times with phosphate buffer. The pellets were resuspended at a final protein concentration of 12 q g per ml with phosphate buffer. Aliquots of 0.1 ml were then incubated for two hours at 20“ with 0.2 ml of calf uterine cytosol, previously labeled with [%l]estradgol.

cytosol

is

tissue

(e.g.,

(e.g.,

albumin,

for

inactivated liver,

Binding

(Fig.

of the

inherent

of calf

uterine

subsequent

at O" (dotted

decrease ionic

about

association with

cytosol

with

binding line,

2 hours

receptor

erythrocyte

30° cannot

estradiol-receptor

proteins sites

be considered

than

for

of other

therefore of the

from a non-target.

to be a

cytoskeleton.

is time

and temperature

be used easily

complex.

because

Preincubation

at 20° does not

[3H]estradiol-receptor

complex

in important

ways by KC1 or NaCl.

change

to the cytoskeleton

2).

is

affected

three-fold

higher

no specific

cytoskeleton

the hormone

of the Fig.

with

to the

higher

of the

a cytosol

amounts

components

Temperatures

if

similar

must

receptor

instability

the binding

strengths

This

estradiol

2).

The interaction increases

is used or if

interaction of the

(30 min at 600),

imraunoglobulins)

are used.

receptor-mediated

the

thymus)

ovalbumin,

estradiol

dependent

by heating

in the presence

but

not

than

to values 1 M (Fig.

below 3A).

684

of 25 mM KCl; those

higher

seen without

In the presence

Binding

concentrations KCl,

even at

of 25 mM KCl,

Ca++

BIOCHEMICAL

Vol. 103, No. 2.1981

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

MINUTES

Fig. 2. Time course of binding of estradiol-receptor complex to erythrocyte cytoskeleton. Erythrocyte cytoskeleton was incubated with 0.1 ml of calf uterine cytosol. Incubations were carried out at O" (a), lo0 (o), ZOO (A) and 30° (A). At the indicated times the reaction was stopped by adding cold phosphate buffer and the binding was measured. The dotted line represents the binding at 20°.

at

4O after

preincubation

of

the

estradiol-receptor

complex

for

2 hr

(up to 5 mM) has no stimulatory effect, while 5 mM Mn++ slightly increases the ++ binding. Mg stimulates the binding substantially, with an optimal concentration at 2.5 mM (Fig.

3B).

The binding

of the

the concentration ml,

provided

shown). that

the

affinity

suggest

the

(Fig.

presence

affinity

4A).

and quantity component

number

of these

high

that the

4B).

From the

1 mg of cytoskeletal cytoskeleton

affinity data

the

(Fig. is

about sites

described protein

from one erythrocyte

receptor

interaction analyses

of binding 4A).

is

is not is

without in figure corresponds appears

685

related

limiting

kept

appears

sites

modifying

to bind

while

markedly

constant

in

of the

substantially

the dissociation

5 x 10'

and

experiments

4B, and independently to about

per

to be saturable

differing

Mg++ increases

to

(not

constant

of such saturation

The dissociation

10ms M.

linearly

of 100 to 1000 pg protein

concentration

Scatchard

of two classes

affinity

(Fig.

cytoskeleton

increased,

higher

range

of estradiol

when the is

complex

in the

concentration

receptor

of high

their

of cytoskeleton

However,

of the

[%]estradiol-receptor

the constant determining

erythrocytes,

a maximum of 50 to 100

vol. 103, No. 2,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

I

I 100

I 200

I 300

COMMUNICATIONS

I

I

r

6

8

IO

I

I 400

2

4

KCI , mM

MgCl2

,mM

Fig.83. Effect of increasing concentrations of KC1 and MgClz on the binding A. Erythrocyte of [ Hlestradiol-receptor complex to erythrocyte cytoskeleton. cytoskeleton was incubated for 2 hr at 20° with 0.2 ml of calf uterine cytosol cytoskeleton and calf with the indicated concentration of KCl. B. Erythrocyte uterine cytosol were incubated in phosphate buffer containing 25 mM KC1 and the indicated concentration of MgClz.

molecules

of estradiol-receptor

is bound

per

forms

studied

separately,

the native

affinity

to the

lost

the ability

of estradiol

to these

of the

a discreet

Triton

shells.

sites, molecules

might to factors which

while

receptor

those

forms

uterine

to erythrocyte

lipid

bilayer

and finite

are

removed

of binding

of the erythrocyte

to steric

can be removed

(data

localization hindrance

by the

detergent.

686

fact not

controlled (18)

found ghosts

the

binding

shown). displays

almost

detergent,

associated to bind

or to their is

the

the estradiol binding

occupancy certain,

Triton with

of the putative

It

have

when many of the proteins

the nonionic is

and

great

that

complex

However, with

with

after

the

estradiol-receptor

be due to an internal related

unaltered

(18)

or by trypsin

despite

ghosts.

amount

The inability

is

binds

obtained

protease

cytoskeleton

are prepared

receptor

forms

uterine

to the

The calf

binding

X-100,

receptor

one mole of estradiol

receptor

8 S form of the

Ca++ -dependent

to bind

DISCUSSION.

and most

of the estradiol

cytoskeleton,

by the

no specific

that

mole of receptor.

When the various

proteolysis

assuming

complex,

by other

however,

that

BIOCHEMICAL

Vol. 103, No. 2,198l

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

‘H t,RfCtPTOR

COMPLEX BOUND ;lO%~

3H-EiRECEPTOR

COMPLEX

[lo-'M]

Fig. 4. Effect of increasing the concentration of [3H]estradiol-receptor complex on the binding of erythrocyte cytoskeleton. Erythrocyte cytoskeleton was incubated for 2 hr at ZOO with different amounts of calf uterine cytosol. The binding was carried out in the presence of 25 mH KC1 alone (A) or in the presence of 25 mM KC1 and 2 mM MgClz (B). Inserts: Scatchard plots of the data.

these

sites

membrane. also

are very The strong

suggests

temperature

the

facilitate

of the the

residual

changes,

or it

Alternatively, arrangements Saturation

dependence

is not

might

simply

analysis

since

binding

might

receptor

it

proteins

of the

modificatidn cytosol

at low temperature the need

might

induce

of the

receptor disrupt

receptor-cytoskeletal

This

organization.

heating

essential

687

on temperature

of a necessary

prior

may actively

of the erythrocyte

binding

cytoskeletal

reflect

access

of the

cytoskeleton

to cytoskeletons

components,

low temperatures of structural

an "open"

(21)

facilitate

the

a manifestation

requirement

membrane

with

of estradiol for

receptor

subsequent

The temperature

of the

associated

requirement

effect

or activation

2).

strongly

for

for

(Fig.

increased

necessary to its necessary

does not

fluidity

conformational binding

site.

organizational

binding. interaction

reveals

the

Vol. 103, No. 2,1981

presence

BIOCHEMICAL

of a very

sites

per

major

protein

ankyrin,

erythrocyte

4.1)

the

components

Similar

systems

estrogen

primarily the (24)

in

that

cytoplasm. cytoskeletal

cells.

very

Thus,

it

exist

constitutes

a highly approaches

interactions

cells,

for

is

model elsewhere to those

conceivable

Although

could of action

the

and high

explain

the hormone-dependent

high also

exist

cytosol

(3-5),

cytoskeletal

in

is

and that matrix

of the cell

analogous

adenylate

affinity

water-

in the

network

demonstrated

data hormones

traditional

and preparation of a complex

affinity

membrane.

here

found

the

++ Mg .

for

that

described

strength

manner,

of steroid

demonstrate

that

actin,

ionic

in part

microtrabecular

have recently

one of the

3, spectrin,

of specific

receptor

as part

organized

that

of the erythrocyte

homogenization

--in situ

binding

to be selective

which

8 S estradiol

of tissue

normally

matrix

similar

affinity

band

existence

the general

of the native,

Similar

the

COMMUNICATIONS

in a nonspecific

appears

to be published sites

an artefact

receptors

target

with

binding

nature

for

of high

the binding.

interaction

evidence

may exist

100)

(e.g.,

for

of binding

RESEARCH

the possibility

cytoskeleton

in the cytoskeletal

Results

target

than

excludes

may be responsible

contradictory

cytoskeletal

soluble

of the

provide

binding

22-31).

(less

This

enhancement

data

apparently

ghost.

BIOPHYSICAL

receptor-cytoskeletal

cation-dependent These

number

components

band

influences

(2-5,

limited

AND

cyclase

erythrocyte system

(32-34).

ACKNOWLEDGEMENTS. The authors thank Drs. Naji Sahyoun and Vann Bennett for their helpful advice. This work was supported by the Progetto Finalizzato: Controllo della Crescita Neoplastica, by a grant from the Wellcome Research Laboratories, RTP, North Carolina and the National Institute of Health Contract No. NOl-CB-64074.

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