hCG receptor in rat ovaries

Vol. 64, No. 4, 1975

BIOCHEMICAL

LABELING

AND BIOPHYSICAL

OF LH/hCG RECEPTOR IN RAT OVARIES

M.R.

Pandian*

, Qn P. Bahl* ** J. Segal

Sheldon *

Dept.

of Biochemistry,

and Division

State

University Buffalo,

** Received

May

The Population

1,

RESEARCH COMMUNICATIONS

of Cell

of New York New York

Council,

and

and Molecular

Biology

at Buffalo

14214

New York,

New York

10021

1975

SUMMARY Human chorionic gonadotropin (hCG) was found to stimulate the incorporation of [14C1 N-acetyl-D-glucosamine in rat ovary in vitro. Subcellular fractionation of the ovarian tissue revealed that the plasma membranes were stimulated maximally to the extent of 200 to 300% by the hormone indicating the stimulation of the synthesis of plasma membrane glycoproteins. In addition, an appreciable amount of the radioactivity was incorporated in the cell surface LH/hCG receptor. The evidence in support of the labeling of the receptor was derived from the behavior of the detergent solubilized receptor on Sepharose 6B column and on hCG-Sepharose The labeled receptor thus purified showed binding affinity affinity adsorbent. for cl2511 hCG. Thus, the hormone stimulates the synthesis of cell surface glycoproteins as well as the LH/hCG receptor.

Considerable

evidence

has accumulated

receptors

for

membranes

of the

isolation

and characterization

(6).

polypeptide

and glycoprotein

target

The availability

cells

its

investigation.

membrane

receptor

for

of cell

Since

surfaces,

glucosamine

glycoproteins

from hCG-primed

describes

are

One of the major presence labeled

Gonadotropins

was considered.

in the receptor

investigation

hormones

radioactively

luteinizing

it

which

has been its

of the

facilitate

(hCG) (7,8).

(l-5).

recently

under

an approach

and superovulated

hormone

in extremely receptor

are to introduce

low amounts

should

certainly

shown to stimulate

labeled

to label

the LH/hCG receptor

plasma

gonadotropin

some of the major

of gonadotropins.

rats.

the

in the receptor

the stimulus

immature

that

on the plasma

or human chorionic

and glycolipids feasible

located problems

have been (IH)

suggests

components

N-acetyl-DThe present in ovaries

Vol. 64, No. 4, 1975

MATERIAIS

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND METHODS

Sprague Dawley strain of female rats (18-21 days old) were used in all experiments. Commercial preparation of hCG (4300 IU/mg) from Organon, West Grange, N.J., was used to inject the rats. Nonionic detergent, mulphogene - BC 720 (ethoxy poly (Ethyleneoxy) ethanol) was a gift from GAP Corporation, New York. N-acetyl-l-[14C] D-glucosamine (41.7 mCi/mM) was purchased from New England Nuclear Corporation and N-acetyl-D-Cl14~1 glucosamine (58 mCi/m mole) from Amersham/Searle Corporation. L-[4,5-3H] leucine (46 Ci/m mole) and sucrose were obtained from Schwarz/Mann. Incorporation of [14c] N-acetyl-D-glucosamine and c3~] leucine in rat ovary: Administration of saline or hCG to rats was done subcutaneously. Superovulation of immature rats was induced as described (7). Ovaries from control, hCG treated or superovulated rats were removed and teased gently. Care was taken not to rupture the follicles. The tissue preparation was pre-incubated at 37O for lo-15 min. in 0.01% bovine serum albumin containing tris buffer medium (9). N-Acetyll-[14C1-D-glucosamine or N-acetyl-D-Cl14C1 glucosamine with or without [3H1 leucine (2 pC!i each/ml) was added and the incubation continued for 90 min. with constant shaking. Ovarian suspension was washed with incubation medium containing 10 mM N-acetyl-D-glucosamine and 10 mM leucine and homogenized in 50 mM tris-HCl buffer, pH, 7.2 containing 1 mM CaC12 in a glass homogenizer with teflon pestle. The homogenate or the 10,000 g pellet was precipitated with 10% trichloroacetic acid and the pellet was solubilized in soluene (Packard) before the addition of scintillation mixture (9). 14 Solubilization of the C- or 3H-labeled receptor and its chromatography on Sepharose 6B: Normally, 4 pairs of ovaries from control, hCG-primed or superovulated rats were incubated with the labeled precursors as above. After --in vitro homogenization and centrifugation at 10,000 g for 30 min, the resulting pellet at 144,000 was solubilized with 0.25% mulphogene at 25 o for 1 hr and centrifuged g for 1 hr. The supernatant was applied to a pre-equilibrated Sepharose 6B column (1.5 x 87 cm) and eluted at 4 o with 0.05 M tris-HCl buffer, pH, 7.2 containing 0.1% 2 ml fractions were collected. 0.1 ml Emulphogene, 0.1 M NaCl and 1 mM CaC12. Each fraction was tested for aliquots were dried and counted for radioactivity. binding activity by the soluble binding assay using polyethylene glycol as described (10). The fractions containing the receptor activity were pooled and concentrated by Amicon diaflo cell using PM-30 membranes (Amicon Corporation, Lexington, Mass.). Separation of the receptor by hCG-Sepharose affinity adsorbant: Sepharose 4B was activated by cyanogen bromide and was coupled directly with hCG (12,000 IU/mg) (11). The 14C-labeled receptor peak from Sepharose 6B, described above, was diluted with tris buffer to a final concentration of 0.01% Emulphogene and added to the affinity adsorbant. The suspension was kept for 16 hrs at 4O with gentle shaking and centrifuged. After washing the pellet repeatedly with 0.1 M NaCl in tris buffer, the radioactivity retained was eluted either with 0.5% mulphogene or 2 M guanidine hydrochloride. RESULTS AND DISCUSSION Administration resulted weight

in the proliferation and by specific

In addition, vitro

of hCG (100 IU twice

with

N-acetyl-1-c

of ovaries

incorporation

when the ovaries

a day for

3 days)

as seen by the

of N-acetyl-1-C

from saline

14 C] D-glucosamine

increase

and hCG treated

animals

female

in the

14 C] D-glucosamine

and c3~1 leucine,

1200

to immature

were

a marked

rats

tissue --in vivo

(12).

incubated

-in

increase

in

Vol. 64, No. 4,1975

BIOCHEMICAL

of 14 C - and 3H-label

the incorporation was observed.

This

of the ovarian

homogenate

organelle

which

increase

of the

tris

The crude

buffer. gradient

fraction 1) , while

were

for

In order

the ovaries [14cl

membrane

fraction

200-300%

ovaries

to investigate

from

control

out

not

glycoprotein

animals

sucrose

purified

g pellet

plasma over

subcellular containing

by sucrose

the

membrane control

(Table

show any variation by hCG.

indicating Similar

rats. on the plasma was one of the

were

The 10,000

animals

subcellular

synthesis

superovulated

10,000

isotonic

label

did

the receptor

and hCG-primed

in

purified

in the

primed

of the hormone,

was further

shown to be located

whether

rich

specific

influence

pellets

from

and C3~1 leucine.

D-glucosamine

(13)

increase

membrane

LH/hCG has been

the

The resulting

and nuclear

with

the

hormone

in membrane

was carried

(14).

of plasma

obtained

under

ovaries

to have

stimulation

Receptor 15).

labeled

the mitochondrial

specific results

stimulated

RESEARCH COMMUNICATIONS

from the

To determine

I).

centrifugation

was found

in ovaries

was more pronounced (Table

was being

fractionation

density

AND BIOPHYSICAL

incubated

g pellets

membranes labeled

with of the

(3,4,

components,

N-acetyl-lovaries

thus

TABLE I Effect

of hCG administration on the incorporation of N-acetyl-l-[14C1 D-glucosamine and c3~1 leucine in immature rat ovaries Incorporation of [14C1 Nacetyl-D-glucosamine Saline -hCG

Tissue Preparation Wary hanogenate 10,000 g Pellet membranes

*DPM/mg Protein. **Purified

plasma

**

52,?40+ 742-

71,790*+ 5850

12,400+

23,970+ 2080-

26,200~ 839

5,420 6,044

Values membranes

are

standard from

Saline

33,362*+ 1044

2891Plasma

Incorporation

sucrose

21,920 21,474

error

of

gradient

1201

the

mean. centrifugation

(14).

of c3Hl leucine -hG

-

94,260+ 1EEo45,350+ 503-

Vol. 64, No. 4, 1975

labeled

were

as described

BIOCHEMICAL

extracted

with

in Materials

control

and experimental

smaller

in size

than

increased

incorporation

The ratio

of the

hormone-receptor ed the complex

0.25%

Emulphogene,

and Methods.

experimental of the

elution

to the

complex

(Fig.

presumably

formed

Fig.

RESEARCH COMMUNICATIONS

and applied 1 shows the

label

sample in the

bed volume, lA),

suggesting

(Figs. ovaries Ve/Vt, that

due to an appreciable

to a Sepharose elution

The 14 C - and 3H-peaks

samples. the

AND BIOPHYSICAL

profiles

in the

1A and lB),

control indicating

from the hCG treated was 0.62, the

same as that

radioactive amount

peaks

of circulating

6B column of the were an animals. of the represent, hCG in

TUBE NUMBER

Fig. 1. Elution profile of the solubilized fraction of 10,000 g pellet of The ovaries from control and hormone treated rat ovaries on Sepharose 6B column. immature rats were incubated with [3H]leucine and N-acetyl-l-[14C1-D-glucosamine. The membrane rich pellet was solubilized with 0.25% Emulphogene and the 144,000 g supernatant was applied to a Sepharose 6B column. See Materials and Methods for details. 14 A. Elution pattern of the C-labeled solubilized membrane fraction from Dotted line the control (-0-O-O) and the hormone treated (O-0-0) ovaries. shows the elution profile of the receptor-[la511hCG complex and [125IlhCG. B. Fractionation of 3H-labeled solubilized membrane fraction from control (-0-O-O) and hormone treated (O-0-0) ovaries.

1202

Vol. 64, No. 4, 1975

the serum. 14

This

C - labeled

the

of the possible after

solubilized peak

by the

In order following

from

clearance

rat

to further

experiment

ovaries

these

last

the

receptor

bindinq

assay

ascertain

was carried

activity

shown by the

C125~1 hCG.

In contrast,

a much greater

from circulation with

since PMSG.

was chromatographed showed (Fig.

whether out:

with

showed

injection

ovaries

RESEARCH COMMUNICATIONS

binding

animals

of the hormone

from

soluble

by the poor

the hCG-primed

one week of the

containing

AND BIOPHYSICAL

evidenced

superovulated

receptor

fractions

assayed

further

ovaries

l4 C -labeled

sacrificed

is

BIOCHEMICAL

an appreciable

binding the

because

animals

were

Furthermore,

when the

on Sepharose

6B, the

binding

activity

when

2A). the LH/hCG receptor

The superovulated

ovaries

was labeled, were

the

incubated

TUBE NUMBER Fig. 2. A. Elution profile of the solubilized membrane fraction from C14ClD-glucosamine and c3~1 leucine incorporated superovulated ovaries 6B column. Solid bar indicates the specific binding with [125IlhCG. binding was determined in the presence of excess of unlabeled hC!G (10 B. Elution of 3H- and 14C-radioactivities on Sepharose 6B as in except the 3H-labeled membrane preparation was incubated with unlabeled (100 IU/ml) prior to solubilization. See text for details.

1203

N-acetyl-lon Sepharose Non-specific IV/ml). Fig. 2A, hCG

Vol. 64, No. 4, 1975

separately

C3~1 leucine

with

hCG (100 IV/ml)

separately

and mixed

for

evidence

in support

of the

When the pooled the

(20-40%

of the

total

readily

by 2M guanidine

show any binding receptor

into

activity, Emulphogene

polyethylene glycol

precipitate

column. of the

significant

was held

hydrochloride

on the

lipids

binding

(12% W/V).

To further

eluted complex.

All

Therefore,

is

eluate

However,

with

this

when

to

0.5%

radioactivity

the

eluate,

complex,

the

by

the polyethylene

to a Sepharose

of 0.61, that

failed

was precipitated

and applied

believed

eluted

the binding

50% of the

Ve/Vt

6B column

of the

for

complex

volume, it

the

was performed

characterize

an elution

additional

of the label

dissociation

, only

in 0.25% mulphogene

with

same

of the radioactivity

(4).

elution

assay

2B

and at the

Sepharose

, necessary

[ 125 11 hCG - [14C1 receptor

of the

The complex

amount

hydrochloride for

Fig.

peak provides

from the

adsorbent.

of the

was used

soluble

in the

due to either

by guanidine

16 hrs)

C-peak

in 2 hr at O", although

or the removal

6B.

solubilized

was labeled.

adsorbent,

was dissolved

hCG-receptor

the receptor

[ 125 11 hCG, possibly

glycol

shift

affinity

When the amount

This

of the

the receptor

4O for

was recovered.

that

ahead

14

with

were

on Sepharose

containing

the receptor

(at

significant

fact

incubated

The l4 C- and 3H-pellets

fraction

subunits

from

peak

and subsequently

was then

to chromatography

complex.

counts)

with

g pellet

60 min at 37O. prior

as the hormone-receptor

D-glucosamine

10,000

of 3 H-radioactive

position

with

labeled

together

shows the appearance

was treated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and N-acetyl-l-C14Cl

The II34 leucine

homogenized. unlabeled

BIOCHEMICAL

6B

characteristic LH/hCG receptor

was labeled.

that

Thus,

the

it

possible

is

above evidence to label

N-acetylglucosamine. not only studies

its

turn

over

from

several

the LH/hCG receptor

Availability

purification

on the

derived

and synthesis

of experimentation

by the incorporation

of the labeled

and physico-chemical

lines

receptor

would

characterization,

indicates

of the certainly but

also

facilitate permit

of the receptor.

Acknowledgement: This (AM-10273)

work

was supported

and the

Population

by research Council

grants of New York

1204

from U.S. (M-74-21).

Public

Health

[ 14c1

Service

Vol. 64, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES

1. Cuatrecasas, P. (1974) Ann. Rev. Biochem., 2, 169-214. 2. Pandian, M.R., Gupta, P.D., Talwar, G.P. and Avrameas, S. (1975) Acta. Endocrinologica, 78, 781-790. 3. Gospodorowicz, D. (1973) J. Biol. Chem., 248, 5042-5049. 4. Haour, F. and Saxena, B.B. (1974) J. Biol. Chem. 2, 2195-2205. 5. Bellisario, R. and Bahl, O.P. (1975) J. Biol. Chem., in press. 6. Cuatrecasas, P. (1974) Biochem. Phann., 23, 2353-2361. 7. Lee, C.Y. and Ryan, R.J. (1972) Proc. Natl. Acad. Sci., U.S.A., 69, 3520-3523. 8. Zeleznik, A.J., Midgley, A.R., Jr.and Reichert, L.E., Jr. (1974) Endocrinology 95, 818-825. 9. PanaT=, M.R. and Talwar, G.P. (1971) J. Exp. Med., 134, 1095-1113. 10. Dufau, M.L., Charreu, E.H., Ryan, D., and Catt, K.J. (1974) FEBS Letters 2, 149-153. 11. Cuatrecasas, P., Wilcheck, M. and Anfinsen, C.B. (1968) Proc. Natl. Acad. Sci., U.S.A., 61, 636-643. 12. Bahl, O.P., Pandian, M.R., Moyle, W.R. and Kobayashi, Y. (1975) Advances in Fertility Regulation Through Basic Research, (eds. W.A. Sadler and S. Segal), Plenum Press, New York, in press. 13. Coleman, R., Michell, R.H., Finean, J.B. and Hawthorne, J.N. (1967) Biochem. Biophys. Acta., 135, 573-579. 14. Ray, T.K. (1970) Bio&. Biophys. Acta., 196, l-9. 15. Rao, Ch.V. (1974) J. Biol. Chem., 249, 2864-2872.

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