Evidence that natural vs synthetic steroid hormones bind to physicochemically distinct cellular receptors

BIOCHEMICAL

Vol. 73, No. 3,1976

EVIDENCE

AND BIOPHYSICAL

VS

THAT NATURAL

TO PHYSICOCHEMICALLY

SYNTHETIC

DISTINCT M.K.

INSERM Received

U 36,

RESEARCH COMMUNICATIONS

STEROID

CELLULAR

RECEPTORS

Agarwal

17 rue du Fer 2 Moulin,

September

HORMONES BIND

75005

Paris

24,1976

Natural (corticosterone) vs synthetic (dexamethasone) bound liver cytosols were fractionated on three chromatographic systems. Dexamethasone bound radioactivity eluted in positions different from those that were labelled with corticosterone filled receptors.

The initial drugs

step

is belived

to

cological

agent

with

special

significance

since

a study

available of

the

consist

of their

eukaryotic

(corticosterone,

(triamcinolone,

more

concentrations,

is

to the

of the constitute

and are with

it bind

hormones

bind intended

other

the

than

same cytoplasmic

as a caution

Copyright 0 1976 by Academic Press, Inc. All rights of reproduaion in any form reserved.

former,

these distinct

intepretation

mineralocorticoids. 767

of

gluco-

: the

naturally

synthetic latter

are

at equimolar both

receptor

that

to

viz

that

process.

few

and expression

the

accepted

specific

hormones

one of the

and the

the

proof

phanna-

assumes

Two groups

Although

physicochemically

viz

(1).

employed

generally

organ

hormones

remains

cortisol)

a formal to

This

organization

currently

active

the

to adrenocortical

genome

are

of most

of

receptor.

dexamethasone).

10 - 100 fold

binding

respect

to delineate

occurring

here

of the

of action

mode of action

hormones

initiation

mechanism

a cellular

corticoid

molecules

the

with

models complex

in

sorts

prior

to the

The results two groups receptor

of

presented of proteins

of data sex steroids.

Vol. 73, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3H r12‘k

FRikTl~i

NUMBER

Fig. 1 Discrimination between natural vs synthetic steroid binders in rat liver by ion-exchange chromatography. Male, Wistar rats (150-200 g) were bilaterally adrenalectomized at least 48 h before use, exsanguinated under ether perfusion with the initial buffer anaesthesia and, after by aortic cannulation, liver 105,000 supernates (4 ml) were incubated (60 min, 4'C) with 10' 9 M corticosterone (insert) or dexamethasone ; 2 ml blood serum was incubated with 0.25 pCi of 14C-corticosterone. The free radioactivity by additional incubation in presence was removed, separately, cell of activated charcoal (Sigma C 5260) of 100 mg ml-l which was thereafter eliminated by centrifugation (3000 g) and passage through glass wool. The cell sap alone (insert) or with serum was finally loaded on DEAE-cellulose-52 (Whatman) columns (1 x 25 cm) equilibrated with 0.001 M PO4, pH 7.5. After passage of 60-70 ml of this initial buffer (fraction vol 6-7 ml) protein was eluted (at arrow) with a linear gradient of 60 ml of 0.001 M and 60 ml of 0.2 M sodium phosphate pH 7.5, at a flow rate of 60 ml/h (fraction vol ca. 3 ml). All manipulations were carried out at 4'C. Aliquots (1 ml) were mixed with 10 ml Unisolve (Kochlight) and counted in a Packard Tricarb scintillation spectrometer with corrections spilling and background. A280 values were deterfor quenching, mined manually. 1,2, 3H-corticosterone (82 Ci/mM ; batch B12) was a product of Amersham, 1,2, 3H-dexamethasone (15 Ci/mM ; batch 66074) and 4-14C-corticosterone (lot 467 - 248 ; 57 mCi/ mM) were purchased from CEA, Saclay. All other chemicals were high purity rea ent grade. ------A2.30 ; 0-e 3H ; A. o--------o

768

BIOCHEMICAL

vol. 73, No. 3,1976

In earlier

receptor which

can be resolved 0.02

provided

proteins

into

one of which in the

in

; the

bind

to blood

serum

(insert,

Fig.

used

in place

(10e8M)

results

synthetic

steroids

in no instance

city

in 0.02

0.04

their

various

peaks

excess

of cold,

with

was further

homologous

corticosterone

did

the

could with

In

although

was totally

abolished

excess

of the

From Fig. to a protein followed

competing,

2 it eluted

by a broad

corticosterone

(insert

is

clear

in

the

peak

of Fig.

cross

113,000 free

of

in presence

steroid.

100 fold

binding versa)

of

a hundred

molecule.

dexamethasone molecular

the

competition

(and vice

2) was clearly

769

that

triamcinolone

heterologous that

The specifi-

fact

molecule. diminish

steroids

ones.

in presence

concentrations

However,

we observe

by the

(10m7M each)

fold

with

synthetic

natural

at equimolar this

was

concentrations

abolished

not

to

analogues

counterparts.

confirmed

were totally

contrary

was greater

natural

M PO4 region

M PO4 region

of binding

studies,

than

and could

when triamcinolone

M region

of

steroid

natural

At limiting

0.001

two sorts

(T)

and at no concentration

radioactivity or the

in the

evidence

synthetic

1) and other

of dexamethasone.

labelling

such

no labelling

transcortin

were obtained

corticoid

M PO4 prewash

corticoid)

does

Similar

0.001

(2-4)

M PO4 prewash)

labels

(where

a natural

(5).

the

unit

Conclusive

M PO4 region

with

corticosterone

1).

dexamethasone

eluted

0.04

in 0.001 a natural

Fig.

be found not

with

(see insert

specific

heterogeneous

GR1 (eluted

1 that

and double

glucocorticoid

M ~04 region)

in Fig.

other

competition

as a polymorphic,

as corticosterone

the

shown that

GR exists

and GR2 (in

is

by both

studies,

we have

labelling,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was bound weight

On the bound

region,

other to

hand,

3 sorts

Vol. 73, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

FRACTION NUMBER

Fig. 2 Discrimination between natural vs synthetic steroid binders by molecular filtration on Sephadex G-200. 2 ml liver c tosol was equilibrated with 3H-corticosterone (insert) or YH dexamethasone and loaded, without the charcoal on Sephadex G-200 (Pharmacia) columns (1 x130 cm) treatment, equilibrated and eluted with 0.01 M Na phosphate, pH 7.4, containing 0.1 M NaCl. Fractions (1.1 - 1.4 ml) were collected at a flow rate of lo-12 ml at 4'C. All other details as in ; 0-4 3H. legend to Fig. 1. --------A280

of

components

weight

In

region.

transcortin

bound

molecular

weight

on G-200 those

maximum double

labelling

in

labelling

region

4).

(2,

the

67,000

experiments

14C - corticosterone

molecular

(not

eluted

When cross

we obtained

was attempted,

in

shown),

the

63,000

competition

conclusions

similar

to

on DE 52 above. Lastly

in

with

the

0.18

(Fig.

M and 0.4

A-25 was attempted,

M regions

based

Transcortin

bound

under

conditions.

these

dexamethasone

3),

both

components

eluted

when chromatography on charge

corticosterone In

bound

eluted

separate

770

on sephadex

and molecular in

the

experiments,

0.6

weight.

M Xl

when liver

region,

BIOCHEMICAL

Vol. 73, No. 3,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3 2 c? R Q 1 I3

FRACTION NUMBER

Fig. 3 Analysis of natural vs synthetic steroid binders by chromatography on DEAE-Sephadex A-25 columns. 4 ml each of liver cytosol were incubated with 10a7M of either 3H-corticosterone or to 3H-dexamethasone ; 4 ml blood serum were incubated with 0.5 uCi 14C-corticosterone. After each liver cytosol was mixed charcoal treatment, separately, with 2 ml serum and loaded on the A-25 (Pharmacia) column (90 x 16 cm) equilibrated with 0.05 M Tris-HCl, pH 7.5, and eluted with a linear gradient between 150 ml each of 0 or 1~ XC1 in this initial buffer. Fractions (3 ml) were collected at a flow rate of 8-10 ml/h. All other details as in legend to Fig. 1. -------A280 ; .-------0 3Hi Q-----o 14c.

homogenate

was equilibrated

dexamethasone, to a mere bound

hump of

peak

required

clarity

on DE-52

It for

the

corticosterone, 0.4

coeluting

has previously

been

from

profiles

in place

M KC1 region

peak

elution

of elution

A-50

obtained

was reduced with

transcortin

shown that

gels,

greatly

with

of

presence

diminishes

phosphate

gradients

(4).

The results proteins

carrying

different

from

described

here

synthetic those

chromatographic

systems.

characteristics

in

studies

(6).

demonstrate

steroids

that

saturation earlier

in

a much larger

corticosterone.

of KCl, the

the

with

carry This

It

convincingly

migrate

in positions

natural

corticoids,

may explain

the

observed

during

has also

been

771

that

in

disparity

cross observed

three in‘

competition that

the

Vol. 73, No. 3,1976

synthetic more

BIOCHEMKAL

steroids

bind

than

natural

easily

tendency

to equate

receptor.

It

temptation

since

system

whereas

can not

a certain

steroid,

not

ontogeny to answer response via that

be said forms

or phylogeny.

to the

saturation had hitherto

glucocorticoid a subpopulation been

by association-dissociation,

various

deemed

in responses

secondarily

by the

structure

since

competition

of

proteins. the

animal

during

receptor

any event,

(5).

observed

that

steroids

either

is

required

physiological

would

of the

this

profiles

logical

unitary

set

different

hormones

the

need be especially

of the In

a limited

certain

synthetic

Purification

avoid

inactive

actually

more

with

unequivocally.

of

the

are

appears

come in contact

cultures

dictated

they

costs

from

a common unit

conformation

possibility

at all

can evoke

of

a common

glucocorticoid

are mostly

whether

or whether

this

cell

steroids

dexamethasone

still

The former does

natural

as the

one must

with

is

far

(5) or triamcinolone

inferences

; workers

are modified

assumes the

that

to draw universal

careful

here

binders

receptors

There

(7).

dexamethasone (9)

RESEARCH COMMlJNlCATlONS

to

corticoids

is obvious

of measurements

It

nonspecifically

the

or corticosterone

(8)

AND BlOPHYSlCAL

appear

polymorphic

through

studies

and Scatchard

to proceed receptor only analysis.

REFERENCES V.C. and O'Malley, B.W. 1. Smith, R.G., Irmain, C.A., Bultham, Nature, 253 : 271 (1975) 254 : 623 (1975) 2. Agarwal, TK.,Nature, 3. Agarwal, M.K.,FEBS Letters, 62 : 25 (1976) 4. Agarwal, M.K.,Biochem. J., ln : 567 (1976) 5. Lippman, :a. and Thompson, Gr, J. Steroid Biochem.,? : 461 (1974) R.E. and McEwen, B.S.,Biochim. Biophys. Acta, 6. De Kloet, (1976) 421 : 124-132 FEBS Letters (in press) 7. Grwal, M.K., 8. Mayer, M. , Kaiser, N., Milholland, R.J. and Rosen, F., J. Biol. Chem., 249 : 5236 (1974) X. and Fezlson, P., J. Biol. Chem., 247 : 7890 9. Beato, (1972) IO. Thanks are due to Monique Philippe for technical assistance. Supported in part by INSERM and the DGRST.