Molecular Structure of the Luteinizing Hormone Receptor'

BIOLOGY

OF

REPRODUCTION

40,

1-12

Molecular

J.

HANNU

(1989)

Structure

of the

RAJANIEMI,

ULLA

KARl

Luteinizing

P. KEINANEN,

E. PETAJA-REPO, Biocenter

Hormone

and

Receptor’

SAKARI

M. KALERVO

KELLOKUMPU, METSIKKO

and Department of Anatomy University of Oulu SF-90220

Oulu

Finland INTRODUCTION

terization

A great deal has been learned years about the mechanisms hormone (hCG) target

(LH) exert cells.

binding

to

surface of Markkanen Rajaniemi, mediated

human

chorionic

their endocrine control The action of LH and a common the

1981). activation

evidence,

the past ten luteinizing

the

receptor

however,

that

on

the

1980; Abramowitz 1984). There

the

binding

hCG also activates other plasma membrane enzymes such as polyphosphoinositide-specific pholipase C. This suggests that second

of

recent possible

methodological to purify the

is

the

or

Attempts linking results,

is a single

90 kDa

special

concept

that

glycopolypeptide.

necessary. LH receptor task, however.

high

LH the

to identify techniques however. Ji

have

proved to be and structural

receptors 1984). The stable under

(Pilch and cross-linked denaturing

of their molecular sulfate-polyacrylamide and autoradiography.

the LH receptor have produced and his coworkers

molecular

weight

using crosscontradictory were among

radiolabeled

porcine granulosa cells, suggesting ture for the receptor (Ji and Ji, 1981,

The has Only it

amounts to enable The purpose of progress in the charac-

‘This work was supported by grants from the Academy Cultural Foundation of Finland, and the Emil Aaltonen requests: Hannu Rajaniemi, M.D., Biocenter ment of Anatomy, University of Oulu, Kajaanintie 52 Oulu, Finland.

techniques identification

of membrane Ruoho et al., complexes are

1985;

Nishimura

et

complexes

al.,

composed 28 kDa,

of four subunits 24 kDa, and

of molecular 18 kDa, the

containing subunits

one disulf

each to

copy of ide-linked

that

1986;

They

al., 1986a,b). et a!. have

proposed

The cross-linking suggested that the

techIn a several in

a multimeric 1980, 1981;Ji

1986a,b).

contains four subunits kDa, 58 kDa, and the

with the

the first to use different affinity cross-linking niques to characterize LH receptor structure. long series of investigations, they identified

into the action,

advances have rendered receptor in an active, hormone-

binding form and in sufficient reliable structural characterization. this article is to discuss recent

structure

substantiating

conditions, enabling estimation size by sodium dodecyl gel electrophoresis (SDS-PAGE)

is

LH

characterization of the components involved in

signal transduction pathways molecular characterization of turned out to be a very difficult

receptor

characterization Czech, 1984; radiolabeled

effector phosmessengers

1987; Allen et al., 1988). To gain an insight molecular mechanisms of LH (and hCG) and structural and the other

receptor

Affinity cross-linking highly useful for the

other than cyclic adenosine 3’, 5’-monophosphate may also play a significant role in the action of LH and hCG (Davis et al., 1986a,b, 1987; Dimino et a!.,

purification receptor

LH

LH evidence

IDENTIFICATION OF THE LH RECEPTOR BY AFFINITY CROSS-LINKING AND LIGAND BLOTTING TECHNIQUES

1) (Catt et al., 1976; 1980; Metsikk#{246} and

et al., Gilman,

on

gonadotropin

This results in guanine nucleotideof the adenylate cyclase in the (Dufau 1982;

of

emphasis

upon the gonadal hCG is initiated by

high-affinity

target cell (Fig. and Rajaniemi,

plasma membrane and Birnbaumer, recent

and

during whereby

the

with each

strucet al.,

Shin LH

et

al.,

receptor

is

sizes 34 kDa, holoreceptor

the three largest other (Shin et

experiments rat ovarian

of Menon LH receptor

of molecular sizes 89 46 kDa (Hwang and

kDa, 80 Menon,

1984a,b), while the testicular receptor consists of three subunits of molecular sizes 121 kDa, 83 kDa, and 52 kDa (Zhang and Menon, 1988). The size of

of Finland, Foundation. and DepartA, SF-90220

the 1

holoreceptor

in both

tissues

can

be calculated

to

2 be

RAJANIEMI

approximately

250-280

the subunits were found hormone binding (Hwang

kDa. to and

Interestingly,

participate Menon,

in 1984b;

all specific Zhang

one

other research findings that hormone-binding

90-100 Rajaniemi,

kDa

unit

(Rebois 1982,

groups, support

1984;

et

including ours, have the existence of only of

al.,

a molecular 1981;

Rebois,

size

of

Metsikk#{246} and 1982;

Ascoli

and

Segaloff, 1986; Petaja et al., 1987). This concept is also supported by a recent study in which a monoclonal antibody that inhibited hCG binding to the membrane receptor recognized only a 90 kDa polypeptide in a detergent extract membranes (Podesta et al., 1986). The reasons for the contradictory by affinity cross-linking techniques

of

rat

Leydig

cell

results obtained with regard to the

FIG 1. Electron microscopic autoradiograph demonstrating luteal cells. The autoradiograph is prepared from pseudopregnant The figure shows a capillary in the center and four luteal cells

AL.

molecular

size

receptor linking

and Menon, 1988). Rapoport et al. (1984) showed, however, that of the multiple labeled species in the rat ovary, only one, a 86 kDa component, exhibits the saturable hormone binding expected of the LH receptor. Using affinity cross-linking techniques, several reported

ET

to identify certain

and

subunit

are unknown methods, which receptors intrinsic

composition

at present. have been for

small

difficulties

of

ligands,

when

the

LH

Affinity crossused successfully are

used

with

subject

to

glycopro-

tein hormones. The large size of the ligand may cause cross-linking of the receptor-bound hormone to any nearby nonreceptor protein. In addition, the dimeric nature of the hormone makes it difficult to resolve whether the labeled complexes contain both subunits or only putative the

the a- or receptor.

cells

(Ascoli

and

(Kellokumpu and rise to multiple cross-linking

j3-subunit Proteolysis Segaloff,

the localization rat ovarian tissue around it (X 12,000).

1984;

or

to the of

the

tissue

may also components

give in

experiments.

characterization (Fernandez-Pol, al.,

linked preparation

1986)

Rajaniemi, 1985a,b) hormone-binding

The ligand-blotting contributed to successful

et

covalently during

technique identification

of numerous 1982; Gershoni Eidne

of the luteinizing 2 h after the iv.

et

hormone injection

a!.,

1985;

has

substantially and structural

membrane et al., Mitani

receptor in the plasma of ‘25l-human chorionic

receptors 1983; Kroon and

Dufau,

membrane gonadotropin.

of

1986),

and

has

identification

been

of

(1987a,b)

the

in our

the spontaneous electroblotted SDS-PAGE.

STRUCTURE

OF

employed

for

successfully

LH

receptor

laboratory.

by

The

Kein#{224}nen et a!.

method

is based

renaturation of receptor onto a nitrocellulose Alternatively,

some

may retain their hormone in the presence of SDS,

receptor

binding although

A single 90 kDa hCG-binding strated with this technique Leydig tumor cells (MLTC-1),

on

molecules sheet after molecules

conformation this seems

specificity

polypeptide was demonin the rat ovary, mouse and human and bovine

characteristic

of the

(Fig. 3). On the basis of these posed that the 90 kDa polypeptide receptor or its hormone-binding this

90

kDa

and no

human major

binding

species

and bovine structural

corpora differences

LH receptor

rat

ovary,

3

receptors

in

hardly

different

unexpected

possibility

in

that obtained

or

could

explain

PURIFICATION

of the

LH

form

is a prerequisite

tion.

Although

some

et can

a!.,

1981; et

be

al.,

Wimalasena 1983),

solubilized

interorgan the

for

most

and

of

mamthe

(testis

vs.

contradictory techniques.

in a biologically

its ultimate have receptor Dufau,

experiments

quantitatively

is

role

LH RECEPTOR

receptor

LH

This

control of also excludes

investigators

of water-soluble

central

cross-linking

OF THE

active existence

of the

the hormonal This result

by different

Solubilization

species.

light

interspecies

differences

results

mammalian in the

the LH receptor malian reproduction.

sena

results, we have prorepresents the LH unit. The presence of

in the

RECEPTOR

ovary)

even unlikely.

corpora lutea (Fig. 2) (Kein#{228}nen et a!., 1987a,b; Kein#{228}nen and Rajaniemi, 1988). The binding of hCG to this polypeptide exhibited the saturability and hormone

LH/hCG

only

purificareported

species

the (Khan

1982;

Wimala-

indicate

that

it

in detergents.

MLTC-1,

lutea suggest also exist between

that LH

12345 kDa

1234.5678 kDa 116205-

94--

116-

66-

‘-i1#{149} 66-

45-. 45-

FIG.

2.

Identification

of

the

luteinizing

hormone

receptors

from

four mammalian species. Triton X-100 extract of pseudopregnant rat ovarian membranes (Lanes 1 and 2), and N-acetylglucosamine eluate of wheat germ agglutinin affinity chromatography of solubilized mouse Leydig tumor cells (Lanes 3 and 4) or solubilized bovine luteal membranes (Lanes 5 and 6) and Triton X-100 extract of human luteal membranes (Lanes 7 and 8) were subjected to SDS-PAGE under nonreducing conditions and electroblotted onto a nitrocellulose sheet. The nitrocellulose strips were incubated with ‘‘I-human chorionic gonadotropin (hCG) in the absence (Lanes 1, 3, 5, and 7) or presence of an excess of unlabeled hCG (Lanes 2, 4, 6. and 8).

FIG. 3. Saturability and hormone specificity of the ‘“I-human chorionic gonadotropin (hCG) binding to protein blots from pseudopregnant rat ovary. Solubilized membrane proteins were separated by SDS-PAGE under nonreducing conditions and electroblotted onto a nitrocellulose sheet. The nitrocellulose strips were incubated with ‘251-hCG in the absence (Lane 1) or presence of unlabeled hCG (101W ml) (Lane 2), human luteinizing hormone (10 lU/mi) (Lane 3), rat follicle-stimulating hormone (2 sg/ml) (Lane 4) or rat prolactin (2 p5/ ml) (Lane 5).

RAJANIEMI

4 Thus, the membrane Singer

LH receptor protein in

and

can the

Nicholson

solubilized X-100 and

be regarded operational

(1972).

the rat testicular demonstrated that

Dufau

properties analyses suggested

asymmetric molecule mass of 190-200 kDa

with (Dufau

Rebois

1973;

Catt

ity b)

et a!., et al.,

Its

in

a

et

raphy

a!.

(1973)

lability

(Dufau

and obstacle

and

et al.,

and

in

sufficient amounts for detailed structural and functional analyses. An important methodological advance in the solubilization of gonadotropin receptors was the finding by Dias et al. (1981) that glycerol

recently,

but

the

garding reports

its size generally

results

have

ability on thus making

whether the polypeptide SDS gels represented real nating proteins. In one

1. Purification The solubilized

of rat receptor

was

luteinizing purified by

re-

activity

factors binding

contributing activity were

cal 90

purified to assess

Preparation

Homogenate Membranes Triton X.100 extract 1st affinity eluate 2nd affinity eluate

the

aAs

determined

bAs

measured

soluble CND,

758 338 226

1000 917 1098 456 137

0.109 0.018 by by

fractions. not determined.

the the

saturation method

analysis of Lowry

of “5l-hCG et al. (1951)

analyses

(n

on

=

1987a).

The

major

the retention of hormonepresence of 20% glycerol

revealed

in

inclusion of the ethylenedi-

that

contained

Data are from hCG-Affi-Gei

to receptor

which

only

is over

calculated capable of the compared

ovarian second

the

purified

a single

class

70%

of

the

of

specific with

purification by KeinInen

binding hormone

±

theoreti-

to be 11000 pmol/mg of binding one molecule

homogenate, affinity step

a representative 10 as described

for

activity binding

a of

was to

and the receptor yield averaged 15%. When the

from 100 ovaries et al. (1987a).

(7.23

g of

bindin? activity

4150 7580

homogenate

4),

Enrichment 6000-fold

1.32 2.71 4.86

binding

to the

preparation

the total after the

Specific (pmol/mg)

in the

have improved the of the LH receptor to retention of its hormone-

(EDTA), and phenylmethylsul(PMSF) in all solutions in the purificaTable 1 summarizes the results of a purification from 100 ovaries. Equilib-

maximum, kDa receptor

hCG. over

‘251-hCG Activity (pmol)

a preliminary

we

(Kein#{228}nen et al.,

binding

protein

hormone (LH) receptor. affinity chromatography

proteinb (mg)

only

laboratory reported from rat ovaries using and elution of the

high-affinity binding sites for hCG, with a Kd of 5.1 2.6 X i0#{176} M (n = 3). The specific binding capacity of the purified receptor was 7920 ± 432 pmol/mg

bands seen in the stained receptor units or contamiearly paper, Dufau et al.

ovarian

binding

receptor

the

gave

of the LH chromatog-

receptor at low pH (Metsikk#{246} and Rajaniemi, Metsikk#{246}, 1984). The purified receptor prepacontained only one major 90 kDa glycopolybut its hormone-binding activity was substan-

rium

The earlier of any specific

the part of it difficult

but

purification affinity

Later, our of the LH receptor chromatography

aminetetraacetate fonyl fluoride tion procedure. representative

of the appeared

conflicting

and subunit composition. lacked firm evidence

hormone-binding polypeptides,

TABLE tissue).

been

hCG-Affi-Gel

using

the solubilized preparations and the proteinase inhibitors N-ethylmaleimide,

effectively stabilizes the solubilized calf testicular follicule-stimulating hormone (FSH) receptor. Later, Ascoli (1983) showed a similar stabilizing effect of glycerol on the LH receptor as solubilized from cultured mouse Leydig tumor cells. Numerous reports describing purification LH receptor from various sources have

15,000-fold testes

rat

tially reduced. Recently, procedure to allow purification near homogeneity with full

susceptibil-

form

on

bound 1982; ration peptide

Rajaniemi, 1985a, to its successful

hormone-binding

described from

characterization. purification hCG affinity

in this solubilized of the detergentthat it is a highly

1983)

(Kellokumpu a major

(1975) receptor

an apparent molecular et al., 1973;Metsikk#{246},

1976;Ascoli,

to proteolysis have formed

purification

1987).

AL.

as an integral definition of

LH receptor in Triton it retained its character-

istic hormone binding form. Hydrodynamic solubilized LH receptor

1984;

ET

Affin ity (KD) M (X 10-10)

Yield

NDC ND 3.1 ND

100 91.7 109.8 45.6

4.0

Purification factor

%

1 2.05 3.68 3143 5742

13.7

preparations. and

membranes

and

by

the

method

of Schaffner

and

Weissman

(1973)

in

OF LH/hCG

STRUCTURE

purified SDS-PAGE

receptor under

preparation was either nonreducing

conditions, only one appeared, demonstrating receptor and that it linked subunits (Fig.

subjected to or reducing

major 90 kDa polypeptide the high purity of the does not contain covalently 4). The 90 kDa polypeptide

RECEPTOR

The

slight

difference

size of 90 to different lous

5 between

kDa obtained electrophoretic

(1987)

et

al.,

have

properties 1980).

Interestingly,

recently

reported

showed specific hormone binding in ligand blotting (Fig. 5) and formed a 130 kDa complex with radio-

identical to those obtained basis of affinity purification

labeled

the rat testicular polypeptide.

LH

The papers cited male LH receptors

above infer are composed

purified receptor. does since

hormone,

indicating

90 kDa Moreover,

conclusively

polypeptide it is conceivable

not contain no other

that

represents that the

any nonhormone-binding polypeptides were

the

the LH receptor

subunits, copurified in

stoichiometric amounts relative to the 90 kDa species. These results agree well with recent reports by Wimalasena et al. (1985) and Kusada and Dufau (1986, 1988) that purified porcine and rat luteal LH receptors have the form of single polypeptides with molecular masses of 68 kDa and 73 kDa, respectively.

kDa

glycopolypeptide.

LH-receptor Dattatreyamurty

al.

1

molecular

may be assigned and/or anomaof

glycoproteins Minegishi

results

et

at.

essentially

by use, concluding on the and ligand blotting that receptor

is

a single

that of

Totally

structure et

the

different

have (1983)

90

kDa

both female and a single 68-90 views

on

also been reported. purified the bovine

luteal LH receptor using conventional protein cal separation methods and found the preparations to consist of a highly aggregated

12345 kDa

and

in our study conditions

electrophoretic

(Leach

these

chemipurified protein

234

kDa ‘

205-

116-

11694-

.,

66-’.

11 45-

FIG.

4. SDS-PAGE

66-

-.

45-,

of

the

purified

rat

ovarian

luteinizing

hormone

(LH) receptor preparations. Reduced samples from various stages of purification were subjected to SDS-PAGE on 7.5% acrylamide separating gel and 5% stacking gel, and the gels were stained with silver. Lane 1, Triton X-100 extract; Lane 2, purified LH receptor after first human chorionic gonadotropin (hCG)-Affi-Gel 10 chromatography; Lane 3, purified LH receptor after second hCG-Affi-Gel 10 chromatography; Lane 4, control purification in which the LH receptors were attached to the affinity column in the presence of an excess of soluble hCG; Lane 5, mass standards.

5. Demonstration of the hormone-binding activity of the puriovarian luteimzing hormone receptor by ligand blotting. Triton x-ioo extract (Lane 1) and three purified receptor preparations (Lanes 2, 3, and 4) after the second human chorionic gonadotropin (hCG)-AffiGel 10 chromatography were subjected to SDS-PAGE under nonreducing conditions and electroblorted onto nitroceliulose. The nitrocellulose strips were incubated with ‘251-hCG. fied

FIG. rat

6 that

RAJANIEMI

produced

a 280

denaturation. dissociated

Upon first to

kDa

species

in SDS-PAGE

reduction, a 120-140

the kDa

confined molecular

only mass

to the 120 forms (Saxena

sizes binding

receptor,

consisting

light

38

Bruch

kDa

et

receptor obtained

al.

of

chains (1986)

from a 268

dissociated

four

may

be

interchange, and 80 kDa polypeptide

that

a more however, an

which in

and

to

lead

structure.

The

by most recent been assigned

erroneous bands

molecular

caused

size

the same molecular by

disulfide

in primary purification al., 1982), research subunits

produced

in the

receptor receptor

stained

our

type

experiment

of glycosidic

purified

(Kein#{228}nen,

iodinated

glycopeptidase between the

rat

F(PNGase proximal

linkage

ovarian

1988),

(N-linked

LH

in glycoproteins (Tarentino in a decrease in the molecular from 90 kDa to about 62

whereas

neuraminidase

shift from contrast, minidase, (0-linked) the size receptor receptor

90 kDa treatment

of

treatment

treatment

of

receptors

F), which cleaves N-acetyl-glucosamine

paragine resulted receptor

caused

with the bond and as-

et

al., size

kDa only

1985), of the (Fig. 6), a slight

to about 79 kDa (Fig. with endo-a-N-acetylgalactosa-

7).

In

which cleaves the serine-threonine-linked oligosaccharide side chains, did not affect of the intact or neuraminidase-treated iodo(Fig. 7). These results suggest that the LH contains mainly N-linked oligosaccharide

bone Kusuda

of which the size

the receptor is and Dufau (1988)

carry terminal sialic acid of the polypeptide backapproximately have reported

62 kDa. somewhat

group to be are

SDS

careful controls or direct demonstrations hormone-binding ability of these polypeptides. thus possible that some of the proposed polypeptides may also represent nonspecific

In

cultures of one but a

at of

receptor

preparations subunits

the

side chains, some residues, and that

1985a,b) may components of

and

or 0-linked), have not been elucidated until very recently (Kusada and Dufau, 1988; Kein#{228}nen, 1988).

a single Biosyn-

results preparation

interpretations

purified to LH

LH

ovaries, and upon reduction

receptor is in fact and Bahl, 1987).

and Rajaniemi, hormone-binding

two

bonds.

recent report, that the high

subsequent experiment by the same allowed three additional receptor identified. The reasons for these contradictory present unknown. Proteolysis during (Kellokumpu to multiple

and of the

the

thetic labeling of the LH receptor of rat Leydig cells resulted in the 79 kDa polypeptide (Aubry et

the tissue give rise

kDa

disulfide rat

artifact

the (Sojar

suggested bovine LH

purification

subunits In

85 by

pseudopregnant kDa protein,

to

species

heavy

stabilized reported

range 46-79 kD. laboratory suggests, mass

two

of 85 was

kDa dimer and higher et a!., 1986). Based on

these results, Dattatreyamurty et al. (1983) an immunoglobulin-like structure for the

AL.

receptor,

upon

280 kDa species species and then

to two dissimilar subunits with molecular kDa and 38 kDa. Specific hormone

ET

1

2

3

4

kDa 205-

gels

have without of

the It is receptor contam-

inants. GLYCOSYLATION

The evident bound lectin

OF THE

LH RECEPTOR

45-

glycoprotein nature of the LH receptor is from numerous reports. The receptor is to and specifically eluted from wheat germ and

concanavalin

A (MetsikkO

and

Rajaniemi,

1982; Kusuda and Dufau, 1986; Keinanen et al., 1987a), and may be chemically labeled using carbohydrate-specific labeling techniques (Metsikk#{246}, 1984). The contribution of oligosaccharide side chains to the size and hormone-binding properties of the LH

FIG. 6. Effect of glucopeptidase (PNGase) F on the electrophoretic mobility of the ‘251-Iabeled rat ovarian luteinizing hormone receptor. lodoreceptor was incubated at 30#{176}C either alone (Lane 1) for 4 h or with PNGase F (4 U/mi) for 1 (Lane 2), 2 (Lane 3), or 4 h (Lane 4). The products were precipitated by 10% trichioroacetic acid and subjected to SDS-PAGE and autoradiography.

STRUCTURE

similar

results,

showing

N-linked oligosaccharide tion of the purified

that

enzymatic

OF

removal

of

side chains caused a reducreceptor from 80 kDa to 54

LH

LH/hCG

RECEPTOR

yet

been

7 obtained

deglycosylated a hormonal

to

the

receptor

question

is still

of

capable

whether

the

of transmitting

signal.

kDa. The seem

glycan to be

binding. PNGase 62

moiety necessary

the for

We have found F-treated rat ovarian

kDa

binding

polypeptide

complete agreement the iodoreceptor rium-binding tions

of

in

with (Fig.

analysis the

the receptor compared also agree receptors,

amount

does not hormone

(Kein#{228}nen, membranes in

ligand

1988) that produce a blotting,

in

the results obtained with 8). In addition, an equilib-

showed or

LH receptor high-affinity

no

significant

hormone-binding

alteraaffinity

TRANSMEMBRANE

To

gain

membrane

an

insight

signaling

upon its membrane recently, only a few membrane

of

1985a,b).

in the PNGase F-treated membranes as with untreated membranes. These results with those obtained with i3-adrenergic showing that enzymatic deglycosylation

approach bearing

by

OF THE

receptor To [125

extensive Rajaniemi, portion of out-oriented

into

the

the

LH

mechanism receptor,

of

of

any

(Kellokumpu

address

this

question,

trans-

information

topography is needed. reports existed concerning

topography

activating

(Cervantes-Olivier et at., 1985; Stiles, 1985) or inhibition of receptor glycosylation with tunicamycin (George et a!., 1986) have no effects on the ligandbinding characteristics of the receptor. No answer has

TOPOGRAPHY

LH RECEPTOR

adenylate and we

Until the cyclase-

Rajaniemi, adopted

an

in which rat I] -hCG-labeled

ovarian receptors

membrane vesicles were subjected to

chymotrypsin 1986). This

treatment removes

(Keinanen and the extracellular

the occupied vesicles but

receptor only the

in the right-sidecytoplasmic exten-

123 1

2

3

4

kDa

kDa

205116-

1166645.-.

45-

FIG. 7. Effect of neuraminidase and endo-o-N-acetyigalactosaminidase on the electrophoretic mobility of the ‘251-labeled rat ovarian iuteinizing hormone receptor. lodoreceptor was incubated for 3 h at 30#{176}Calone (Lane 1), with neuraminidase (50 mU/mI) (Lane 2), with neuraminidase (50 mU/mi) plus endo-a-N-acetylgalactosaminidase (25 U/mI) (Lane 3) or with endo--N-acetylgalactosaminidase (25 U/mI) (Lane 4). The reaction products were analysed by SDS-PAGE and autoradiography after precipitation by 10% trichloroacetic acid.

FIG. 8. Detection of the rat ovarian luteinizing hormone (LH) receptor by ligand blotting after glycopeptidase (PNGase) F treatment of the membranes. Membranes were incubated in the presence or absence of PNGase F (10 U/mI) for 2 h at 30#{176}C.After solubilizauon, the digesteed receptor was subjected to SDS-PAGE under nonreducing conditions and electroblotted onto nitrocellulose. The niuocelluose strips were incubated with ‘251-human chorionic gonadotropin. Lanes I and 3, untreated control membranes; Lane 2, PNGase F-treated membranes.

RAJANIEMI

8

ET

Sian ifl the inside-out-oriented vesicles, the truncated receptor being retained in the membranes. Extensive chymotrypsin treatment released 70-75% of the radioactivity initially bound to the ovarian membrane

ment.

vesicles in a 1-h-incubation. the water-soluble radioactivity three complexes with molecular

the of

kDa,

and

tory

have

61 kDa.

Previous

demonstrated

SDS-PAGE analysis released pointed masses of 96 kDa,

experiments that

endogenous

and trypsin treatment of ovarian in the release of similar complexes, kDa

and

74 kDa

and 38 kDa (Kellokumpu suggest molecule oriented

complexes

and

can

be

released

1

by

proteolysis results the 96

to the

64 kDa

linked 1985a,b).

part of 90 kDa)

labora-

membranes and that

correspond

receptor fragments and Rajaniemi,

that a substantial (64 kDa out of

in our

of to 74

to intact hCG These results the LH receptor is extracellularly

proteolytic

AL.

Further

increase membrane

the

chymotrypsin

release vesicles,

hormone-occupied

treatment

of radioactivity indicating that

from the 25-30%

receptors

protected

are

enzyme and probably exposed toward the inside-out vesicles. When the

chymotrypsin-resistant

radioactivity

and subjected to SDS-PAGE, observed (Fig. 9), whereas produced

a

130

kDa

for

the

laboratory et al.,

finding

was

a 110 kDa untreated

complex,

other findings in our Rajaniemi, 1982; Peta:ja explanation

did

cited

in

ovarian of the from

the interior solubilized cross-linked complex was membranes

agreement

(Metsikko 1987). The here

not

is that

with and salient while

most of the LH receptors reside in the right-side-out vesicles, some 25-30% are situated in the inside-out vesicles and that the decrease in size of the hormone-

treat-

1234

2

kDa

kDa 205-

116-’ 94-

94-:.,

66-

00

45-

FIG. 9. Detection of the cross-linked membrane-retained proteolytic products of ‘251-human chorionic gonadotropin (hCG)-receptor complex by SDS-PAGE and autoradiography. Rat ovarian membrane vesicles, labeled with 125l-hCG in vivo, were subjected to chymotrypsin (1 mg/mI) treatment for 60 mm at 37#{176}C.The membrane-retained proteolytic products of the ‘“l-hCG-receptor complex were solubilized and subjected to SDS-PAGE after cross-linking with glutaraldehyde. Lanes I, membrane-bound hormone receptor complex from control incubation (enzyme omitted); Lane 2, membrane-retained proteolytic products of hormone receptor complex produced by chymotrypsin.

FIG. 10. Detection of the membrane-retained proteolytic products of the free luteinizing hormone (LH) receptor by ligand blotting. Rat ovarian membrane vesicles bearing free LH receptor sites were subjected to an identical incubation as in Figure 9. The chymotrypsin-treated membranes were solubilized, subjected to SDS-PAGE under nonreducing conditions, and electrobiotted onto nitrocellulose. The nitroceilulose strips were incubated with ‘“I-human chorionic gonadotropin (hCG) in the absence (Lanes I, 2) or presence (Lanes 3. 4) of an excess of unlabeled hCG. Lanes I and 3, intact receptor; Lanes 2 and 4, chymotrypsin-treated receptor.

STRUCTURE

receptor

complex

by

about

20

kDa

is due

OF

to proteo-

lytic cleavage of the cytoplasmic domain of the receptor in these inside-out vesicles. This concept was supported by the finding that enrichment of the inside-out doubled

vesicles by the fraction

chymotrypsin

digestion.

ovarian membrane with chymotrypsin retained

that

bearing analysis

cated in 70 kDa

the inside-out-oriented binding polypeptide,

receptor

took

similar

the

conditions

with results suggests that

form (Fig.

molecule complex.

to

al., hCG

ligand

free

a 90 This

kDa

lo-

produced a the intact species

is in good

affinity change

and Taken

blotting

receptors

vesicles whereas

of 10).

obtained by the molecular

the receptor moiety of the

the

under

agreement

cross-linking and in fact occurs in

not in together,

the hormone these results

support the transmembrane nature of the LH receptor. Moreover, if the receptor traverses the lipid bilayer only once, then the 20 kDa domain released by chymotrypsin digestion will be entirely exposed intracellularly, so that it must thus represent the cytoplasmic

domain

of the

INTERACTION

LH receptor.

OF hCG SUBUNITS

WITH

THE

LH RECEPTOR

questions are through

1987)

antibodies

recognize

whereas generally suggests tact

ties, but

whereas can be

intact dimeric receptor-binding

gonadotropins and biological

the separate reassociated with

biological activity Parson, 1981). On demonstrated that

subunits are full recovery

(Catt et al., the other hand, the dissociated

1976; recent subunits

have

with

intrinsic activity, although very limited, as shown by the ability of the purified a-subunits of LH and hCG (Moudgal and Li, 1982) and synthetic peptides corresponding a-subunit (Charlesworth

to internal (Keutmann et al.,

et 1987)

amino

acid

sequences

(Keutmann

al., 1987) and a-subunit of hCG to compete with

et al.,

1987).

the

a-subunit

of

complex,

the

receptor

and

thus

beyond

the

reach.

subunit (Petaja

([‘251]-hCG) or et al., 1987).

receptor receptor PAGE

complex (130 complex (105 and autoradiography,

in its a-subunit Only the intact

of

we hCG, its a-

([3H1-hCG) hormone-

kDa) and the a-subunit kDa) were found in SDSthe latter being particu-

larly distinct after reduction. This suggests that the LH receptor binds one hormone molecule and that the major interaction of hCG with the receptor occurs through its a-subunit (Fig. 11) (Petaj#{228}et al., 1987). Essentially by Kusuda and

identical results Dufau (1986).

contact receptor

between the demonstrated

plexing common

as it is well known to all the glycoprotein

have been reported The apparent close

a-subunit of hCG and in these is somewhat that the hormones

the

a-subunit and that

LH peris it

123456

kDa

kDa

-205 130105

116

-94

-

-66

(M ()

-

-

h CO h O

-

______

-45

()-hCO

in the

the intact hormone for receptor binding. In addition, the synthetic a-subunit peptides are reported to have weak steroidogenic activity, although at micromolar concentrations

against

hormone-receptor

the specific antibodies. To address this question, have carried out affinity cross-linking tests with which carries the radioactive label either in

the activi-

and has some

activation of hCG interacts

the antibodies against the a-subunit do not recognize the receptor-bound hCG. This that the a-subunit is in more intimate con-

inactive of their

Pierce research have

directed the

Gonadotropins, including hCG, are composed of two dissimilar subunits, of which the a-subunit is responsible for species specificity and the a-subunit for hormone specificity. According to numerous reports, only characteristic

regarding the which subunit

with its gonadal receptor and what is the valency of the receptor. A number of polyclonal (Milius et al., 1983) and monoclonal (Moyle et al., 1982; Bidart et

of

by

of

Interesting receptor

treatment

free receptor sites of the membrane-

components

proteolysis

the

9

RECEPTOR

chromatography resistant

Identical

vesicles and

receptor

showed

lectin-affinity of radioactivity

LH/hCG

FIG. 11. Interaction of the human chorionic gonadotropin (hCG) subunits with rat ovarian luteinizing hormone (LH) receptor. Membranes were incubated with ‘I-hCG (a-subunit-labeled) (Lanes 1-3) or ‘HhCG (-subunit-iabeled) (Lanes 4-6) in the absence (Lanes 1, 2, 4, 5) or presence (Lanes 3, 6) of an excess of unlabeled hCG and solubiized in 1% Triton X-100. Samples were subjected to SDS-PAGE both under nonreducing (Lanes 1, 3, 4, 6) and reducing (Lanes 3, 5) conditions after cross-linking with glutaraldehyde. In the 7.5% separating gel, the a- and a-subunits of hCG migrated at the dye front.

10

RAJANIEMI

is the

a-subunit

specificity.

that

To

should

be

this

remembered

hypothesized of all other two tor,

is responsible

explain

for

apparent that

the

Milius

et

al.

it (1983)

(and analogously takes place

steps. First, the a-subunit recognizes the forming an initial transient complex.

through

a structural

is formed

transformation,

between

the

a stable

a-subunit

and

in

the

DOMAIN

receptor.

1987).

model

binding

evidence show that

membranes with

two-step

changes

is also

offered by hCG binding from

90 kO

supported

Katikineni to rat

a “loose”

EXT RACELLUL AR

complex

at least in the hormone as evidenced by changes epitopes (Moyle et at.,

This

N-Linked Oligosaccharide Side Chains

recepThen,

Structural changes take place upon binding to the receptor, in the availability of antigenic by kinetic (1980) to

AL.

hormone

discrepancy,

that the binding of hCG glycoprotein hormones)

ET

et al. testicular

to a “tight”

form TRANSMEMBRANE

PLASMA

time.

DOMAiN CONCLUSIONS

Advances

in

conformation

of

AND

FUTURE

stabilizing the

PROSPECTS

the

receptor

INTRACELLULAR

hormone-binding

by

means

of

glycerol

and in identification of the receptor have rendered it possible to purify

by ligand blotting LH receptors in an

active,

and

hormone-binding

form

amounts to enable reliable structural and production of antibodies. The

in

sufficient

characterization available results

from our laboratory are summarized in Figure 12, which proposes a schematic structure for the LH receptor. The receptor consists of a single 90 kDa transmembrane glycopolypeptide with a 64 kDa extracellular hormone-binding domain and a 20 kDa cytoplasmic domain. It contains several N-linked oligosaccharide side chains that do not participate in high-affinity hormone binding, and its polypeptide backbone has a molecular mass of 62 kDa. The receptor interaction its

binds one of hCG

a-subunit.

molecule with the

Structural

of hCG, receptor conservation

receptor in different mammalian by ligand-blotting data on the human. As regards the proposed of

the

receptor,

absence of amino that the receptor brane

more

The LH hypothetical tions. determine plasma

than receptor and

it is worth

cannot

model involves

of

the

LH

species is suggested rat, mouse, cow, and membrane insertion

emphasizing

acid sequence polypeptide once

and the major occurs through

data, the may span

that

in the

possibility the mem-

DOMAiN

FIG. 12. Proposed izing hormone receptor. illustrates the 62 kDa

structure and membrane insertion of the luteinThe curved line spanning the plasma membrane polypeptide backbone of the receptor.

Kusuda and Dufau (1988) imply that the LH receptor tends to dimerize in purified form, and the same authors suggest that its functional form may in fact be a homodimer of the 90 kDa species. This result needs to be confirmed by other techniques, however, e.g., The question should of the tute ous

by a target-size of putative

be resolved, purified 90

LH-responsive system. Future

elucidating

gram

amounts

to look

the

into

be excluded. proposed here many unresolved

adenylate work the

1985). units

the capacity to reconsti-

cyclase in a heterologshould also be aimed

molecular

receptor Finally,

of pure

further

(Venter, receptor

e.g., by measuring kDa receptor species

in detail

the coupling of effector enzymes.

analysis additional

events

with plasma the availability

receptor

now

its molecular

makes

involved

at in

membrane of microit possible

biology.

ACKNOWLEDGMENTS

is highly ques-

Investigations are particularly needed to the functional form of the receptor in the membrane. The results published recently by

The Eero

Oja,

skillful and

technical Sirkka

Martti

assistance

of

is gratefully

Sirpa

JmsI,

Paula

Soininen,

acknowledged.

REFERENCES Abramowitz gonadotropin

J,

Birnbaumer interaction

L, 1982. with rabbit

Temporal lutes]

characterisiecs receptors and

of activa-

STRUCTURE

OF

don of adenylyl cyclase: comparison to the mode of action of catecholamine receptors. Endocrinology 111:970-76 Allen RB, Su HC, Snitzer J, Dimino J, 1988. Rapid decreases in phosphatidylinositol in isolated lutes] plasma membranes after stimulation by luteinizing hormone. Biol Reprod 38:79-83 Ascoli M, 1983. An improved method for the solubilization of stable gonadotropin receptors. Endocrinology 113:2129-34 Ascoli M, Segaloff DL, 1986. Effects of collagenase on the structure of the lutropin/choriogonadotropin receptor. J Biol Chem 261: 3807-15 Aubry M, Collu R, Ducharme JR, Crine P, 1982. Biosynthesis of a putative gonadotropin receptor component by rat Leydig cells: isolation of a radiolabeled acidic protein of 79,000 molecular weight by affinity chromatography. Endocrinology 11:2129-3 1 Bidart J-M, Troalen F, Salesse R, Bousfield GR, Bohuon CJ, Bellet DH, 1987. Topographic antigenic determinants recognized by monocloncal antibodies on human choriogonadotropin (3-subunit. J Biol Chem 262:8551-56 Bruch RC, Thotakura NR, Bahl OP, 1986. The rat ovarian lutropin receptor. Purification, hormone binding properties, and subunit composition. j Biol Chem 261:9450-60 Catt KJ, Ketelslegers JM, Dufau ML, 1976. Receptors for gonadotropic hormones. In: Blecher M (ed.), Methods in Receptor Research, Part I. New York: Marcel Dekker, pp. 175-250 Cervantes-Olivier P, Durieu-Trautmann 0, Delavier-Klutchko C, Strosberg AD, 1985. The oligosaccharide moeity of the jl,-adrenergic receptor from turkey erythrocytes has a biantennary, N-acetyllactomsamine-containing structure. Biochemistry 24: 3 765-70 Charlesworth MC, McCormick DJ, Madden B, Ryan Ri, 1987. Inhibition of human choriogonadotropin binding to receptor by human choriogonadotropin a peptides. J Biol Chem 262: 13409-16 Dattatreyamurty B, Rathnam P, Saxena BB, 1983. Isolation of the luteinizing hormone-chorionic gonadotropin receptor in high yield from bovine corpora lutea. Molecular assembly and oligomeric structure. J Biol Chem 258:3140-58 Davis JS, Weakiand LL, Farese RV, West LA, 1987. Luteinizing hormone increases inositol trisphosphate and cytosolic free Ca2 in isolated bovine lutes] cells. J Biol Chem 262 :8515-21 Davis JS, Weakland LL, West LA, Farese RV, 1986a. Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells Biochem J 238:597-604 Davis

JS, West LA, Weakland LL, Farese RV, 1986b. Human chorionic gonadotropin activates the inositol 1, 4, 5-trisphosphate-Ca’#{176} intracellular signalling system in bovine lutes] cells. FEBS Lett 208: 287-91 Dias JA, Huston JS, Reichert LE Jr, 1981. Effect of the structure-stabilizing agent glycerol on detergent-solubilized follicle-stimulating hormone receptors from calf testis. Endocrinology 109:736-41 Dimino MJ, Snitzer J, Brown KM, 1987. Inositol phosphate accumulation in ovarian granulosa cells after stimulation by luteinizing hormone. Biol Reprod 37:1129-34 Dufau ML, Baukal AJ, Catt KJ, 1980. Hormone-induced guanyl nucleotide binding and activation of adenylate cyciase in the Leydig cell. Proc NatI Acad Sci USA 77:5837-41 Dufau ML, Ryan DW, Baukal AJ, Catt KJ, 1975. Gonadotropin receptors. Solubilization and purification by affinity chromatography. I Biol Chem 250:4822-24 Eidne KA, Hendricks DT, Millar RP, 1985. Demonstration of a 60 K molecular weight luteinizing hormone-releasing hormone receptor in solubilized adrenal membranes by a Iigand-immunoblotting technique. Endocrinology 116:1792-95 Fernandez-Pol IA, 1982. lmmunoautoradiographic detection of epidermal growth factor receptors after electrophoretic transfer from gels to diazo-paper. FEBS Lett 143:86-92 George ST, Ruoho AE, Malbon CC, 1986. N-giycosylation in expression and function of (3-adrenergic receptors. J Biol Chem 261: 165 59-64 Gershoni JM, Hawrot E, Lentz TL, 1983. Binding of a-bungarotoxin to isolated a subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots. Proc Nati Acad Sd USA 80:4973-77 Gilman AG, 1984. G proteins and dual control of adenylate cyclase. Cell 36:577-79

LH/hCG

RECEPTOR

J,

Hwang ture

Chem Hwang

J,

11

Menon KIM, of gonadotropin 259:1978-85 Menon KMJ,

1984a.

Characterization receptor in luteinizied

1984b.

Spatial

of

the rat

relationships

subunit ovary. of

the

J

strucBiol

human

chorionic gonadotropin (hCG) subunits in the assembly of the hCG-receptor complex in the luteinized rat ovary. Proc NatI Acad Sci USA 81:4667-71 Ji I, Bock JH, Ji TH, 1985. Composition and peptide maps of crosslinked human choriogonadotropin-receptor complexes on porcine granulosa cells. J Biol Chem 260:12815-21 Jil, Ji TFl, 1980. Macromolecular photoaffinity labeling of the lutropin receptor on granulosa cells. Proc NatI Acad Sci USA 77:7167-70 Ji I, Ji TH, 1981. Both a and (3 subunits of human choriogonadotropin photoaffinity label the hormone receptor. Proc NatI Acad Sci USA 78:5465-69 Ji I, Yoo BY, Kaltenbach C, Ji TH, 1981. Structure of the lutropin receptor on granulosa cells. Photoaffinity labeling with the a subunit in human choriogonadotropin. J Biol Chem 256: 10853-58 Katikineni M, Davies TF, Huhteniemi IT, Catt KJ, 1980. Luteinizing hormone-receptor interaction in the testis: progressive decrease in reversibility of the hormone-receptor complex. Endocrinology 107:1980-88 Kein#{228}nen KP, 1988. Effect of deglycosylation on the structure and hormone binding activity of lutropin receptor. Biochem J (in press) Keinanen KP, Kellokumpu S, Metsikko MK, Rajaniemi JH, 1987a. Purification and partial characterization of rat ovarian lutropin receptor. J Biol Chem 262:7920-26 Keinanen KP, Kellokumpu S, Rajaniemi H, 1987b. Visualization of the rat ovarian lutropin receptor by ligand blotting. Mol Cell Endocr 49:33-38 Kein#{225}nen KP Rajaniemi HJ, 1986. Rat ovarian lutropin receptor is a transmembrane protein. Evidence for an Mr2OOOO cytoplasmic domain. Biochem J 239:83-87 Kein#{228}nen KP, Rajaniemi HJ, 1988. Solubilization of luteinizing hormone receptor from human corpora lutea in a stable form and identification of the hormone binding unit by ligand blotting. J Clin Endocr Metab 67:228-3 3 Kellokumpu S, Rajaniemi H, 1985a. Involvement of plasma membrane enzymes in the proteolytic cleavage of luteinizing hormone receptor. Endocrinology 116:707-14 Kellokumpu S, Rajaniemi H, 1985b. Hormone binding modified endogenous proteolysis of LH/hCG receptors in rat ovarian plasma membranes. Mol Cell Endocr 42:157-62 Keutmann HT, Charlesworth MC, Mason KA, Ostrea T, Johnson L, Ryan RJ, 1987. A receptor-binding region in human choriogonadotropin/lutropin (3 subunit. Proc NazI Acad Sci USA 84: 2038 -42 Khan

FS, Rathnam P, Saxena BB, 1981. Purification and properties of human chorionic gonadotropin/lutropin receptor from plasma membrane and soluble fractions of bovine corpora lutea. BiochemJ 197:7-22 Kroon PA, Thompson GM, Chao Y-S, 1984. A comparison of the low-density-lipoprotein receptor from bovine adrenal cortex, rabbit and rat liver and adrenal glands by lipoprotein blotting. BiochemJ 223:329-35 S, Sufau ML, 1986. Purification and characterization of the rat ovarian receptor for luteinizing hormone. Structural studies of subunit interaction. I Biol Chem 261:16161-68 Kusda S, Dufau ML, 1988. Characterization of ovarian gonadotropin receptor. Monomer and associated form of the receptor. J Biol Chem 263:3046-49 Leach BS, Collawn JF Jr, Fish WW, 1980. Behavior of glycopolypeptides with empirical molecular weight estimation methods. 1. In sodium dodecyl sulfate. Biochemistry 19:5734-41 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ, 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265 -75 Kusda

Markkanen SO, Rajaniemi H, 1980. Role of internalization and degradation in the removal of receptor-bound human chorionic gonadotropin in rat lutes] cells in vivo. Endocrinology 109:1399-1403 Metsikk#{246} MK,

1984.

Covalent

labelling

of

the

lutropin

binding

site.

12

RAJANIEMI Evidence 2 19:583-91

for

a single

Mr

90,000

sialoglycopolypeptide.

Biochem

ET

J

Metsikk#{246} MK, Rajaniemi H, 1981. Antibodies to purified luteinizing hormone receptor localize the receptor at the lutes] cell surface. Endocrinology 109:1399-1403 Metsikko MK, Rajaniemi HJ, 1982. The hormone-binding unit of luteinizing hormone receptor. Biochem J 208:309-16 Metsikk#{246} MK, Rajaniemi Hi, 1984. lmmunoprecipitation of the lutropin receptor. Loss of receptor molecules during down-regulation. Biochem J 224:467-71 RP, Midgley AR Jr, Birken S, 1983. Preferential masking by the receptor of immunoreactive sites on the a-subunit of human chorionic gonadotropin. Proc Nat] Acad Sci USA 80:7375-79 Minegishi T, Kusuda S, Dufau ML, 1987. Purification and characterization of Leydig cell luteinizing hormone receptor. J Biol Chem 262: 17138-43 Mitani M, Dufau ML, 1986. Purification and characterization of prolacdii receptors from rat ovary. J Biol Chem 261:1309-15 Moudgal NR, Ki CH, 1982. (3 subunits of human choriogonadotropin and ovine lutropin are biologically active. Proc Nat] Acad Sci USA 79:2500-03 Moyle WR, Ehrlich PH, Canfield RE, 1982. Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor. Proc Nat] Acad Sci USA 79:2245 -49 Moyle WR, Pressey A, Dean-Emig D, Anderson DM, Demeter M, 1987. Detection of conformations] changes in human chorionic gonadotropin upon binding to rat gonads] receptors. J Biol Chem 262: 16920-27 Nishimura R, Raymond MJ, Ji I, Rebois RV, ii TH, 1986. PhotoMilius

Petaj

affinity labeling and deglycosylated 83 :63 27-3 1 U, Kellokumpu

of

the gonadotropin choriogonadotropin.

5, Keinanen

K,

receptor Proc Metsikko

with Nat]

native, asialo, Acad Sci USA

K, Rajaniemi

H, 1987.

Covalent cross-linking of radiolabeled human chorionic gonadotropin to rat ovarian luteinizing hormone receptor with glutaraldehyde. J Rec Research 7:809-27 Pierce JG, Parsons TF, 1981. Glycoprotein hormones: structure and function. Ann Rev Biochem 50:465-95 Pilch PF, Czech MP, 1984. Affinity cross-linking of peptide hormones and their receptors. In Venter JC, Harrison LC (eds.), Receptor Biochemistry and Methodology. New York: Alan R Liss, pp. 161-75 Podesta EJ, Solano AR, Sanchez ML, 1986. Luteinizing hormone triggers two opposite regulatory pathways through an initial common event, receptor aggregation. Endocrinology 119: 989-97 Rapoport B, Hazum E, Zor U, 1984. Photoaffinity labeling of human chorionic gonadotropin-binding sites in rat ovarian plasma mem-

AL. branes.

J

Biol Chem

259:4267-71

Rebois Ky, 1982. Establishment of gonadotropin-responsive murine Leydig tumor cell line. J Cell Biol 94:70-76 Rebois RV, Bradley RM, Titlow CC, 1987. Hydrodynamic properties of the gonadotropin receptor from a murine Leydig tumor cell line are altered by desensitization. Biochemistry. 26:6422-28 Rebois Ky, Omedeo-Sale F, Brady RO, Fishman PF, 1981. Covalent crosslinking of human chorionic gonadotropin to its receptor in rat testes. Proc Nat] Acad Sci USA 78:2086-89 Ruoho AE, Rashidbaigi A, Roeder PE, 1984. Approaches to the identification of receptors utilizing photoaffinity labeling. In: Venter JC, Harrison LC (eds.), Receptor Biochemistry and Methodology. New York: Alan R Liss, pp. 109-18 Saxena BB, Dattatreyaznurry B, Ota H, Milkov V, Kathnasn P. 1986. Subunits of luteinizing hormone-human chorionic gonadotropin receptor from bovine corpora lutea. Biochemistry 25:7943-50 Schaffner W, Weissman C, 1973. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56: 502-14 Shin J Jil, Ji TH, 1986a. Disulfides of the lutropin receptor. J Biol Chem 261:7501 -06 Shin J, Ji I, Ji TH, 1986b. Structural homologies in the lutropin/human choriogonadotropin receptor and the follitropin receptor on porcine granulosa cells. Biochemistry 25:3410-15 Singer SJ, Nicholson GL, 1972. The fluid mosaic model of the structure of cell membranes. Science 175:720-31 Sojar HT, Bahi OP, 1987. Role of sulhydryl groups in rat ovarian lutropin receptor aggregation. I Cell Biol 105:314a Stiles GL, 1985. Deglycosylated mammalian fi2-adrenergic receptors: effect on radioligand binding and peptide mapping. Arch Biochem Biophys 237:65-71 Tarentino AL, Gomez CM, Plummer TH Jr. 1985. Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F. Biochemistry 24:4665-71 Venter JC, 1985. Size of neurotransmitter receptors as determined by radiation inactivation/target size analysis. In: Conn PM (ed), The Receptors, Vol. 2. New York: Academic Press, pp. 245-80 Wimalasena J, Dufau ML, 1982. Water-soluble gonadotropin receptors of the rat ovary. Endocrinology 110:1004-12 Wimalasena J, Moore P, Wiebe JP, Abel J Jr, Chen TT, 1985. The porcine LI-I/hCG receptor. Characterization and purification. J Biol Chem 260:10689-97 Wimalasena J, Schwab 5, Dufau ML, 1983. Soluble luteinizing hormone! human chorionic gonadotropin receptors of the rat ovary: comparative studies of waterand detergent-soluble receptors. Endocrinology 113:618-24 zhang Q-Y, Menon KMJ, 1988. Characterization of rat Leydig cell gonadotropin receptor structure by affinity cross-linking. J Biol Chem 263:1002-07