Proc. Nati. Acad. Sci. USAVol. 83, pp. 6327-6331, September 1986Biochemistry

Photoaffinity labeling of the gonadotropin receptor with native,asialo, and deglycosylated choriogonadotropin

(human choriogonadotropin receptor structure/granulosa cells/cross-linking/peptdde mapping)

RYUICHIRo NISHIMURA*, MATTHEW J. RAYMOND*, INHAE JI*, R. VICTOR REBOISt, AND TAE H. Ji*t*Department of Biochemistry, University of Wyoming, Laramie, WY 82071; and tMembrane Biochemistry Section, Developmental and Metabolic NeurologyBranch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892

Communicated by Andrew A. Benson, May 12, 1986

ABSTRACT Human choriogonadotropin (hCG) is aheterodimeric hormone composed of an a and a (I subunit.hCG and its asialo (ashCG) and deglycosylated (dghCG) formsvary in their ability to stimulate hormone responsive adenylatecyclase. ashCG is a partial agonist, and dghCG is an antago-nist. Photoactivatable moieties were coupled to hCG, ashCG,and dghCG, and the derivatives were radioiodinated. Com-petitive binding studies indicate that al of the derivatives hada similar affinity for the gonadotropin receptor on porcinegranulosa cell membranes. Radiolabeled derivatives were usedto photoaffmity label the gonadotropin receptor. Radiolabeledcomplexes were separated by NaDodSO4/PAGE. Al of thederivatives produced similar autoradiographic patterns, ex-cept that dghCG produced an additional 48-kDa complex. Toinvestigate the structure of the complexes further, peptidemapping of proteolytic digests was used. All, except for the48-kDa complex, generated similar peptide maps indicating arelationship between those complexes in which the smallercomponents are part of the larger. The 48-kDa complexcontained both subunits of 40-kDa dghCG. Therefore, thiscomplex is expected to contain an additional component of 8kDa. The complex was generated whether the hormone-recep-tor complex was photoaffinity labeled on cells, on isolatedmembranes, or after solubilizing in detergent. Formation wasblocked by excess hCG and did not occur in the absence of UVirradiation. We conclude that the hCG derivatives are able tophotoaffinity label the hCG receptor but that the dghCGderivative can photoaffiity label an additional component thatwas not observed when derivatives ofhCG or ashCG were usedto label the receptor.

Pituitary lutropin and placental human choriogonadotropin(hCG) are heterodimeric glycoproteins that recognize thesame receptor and elicit similar physiological responses. Theamino acid sequence of the a and ,B subunits have beenestablished, and the carbohydrate sequences have beendetermined (1). Removal of terminal sialic acid residues doesnot interfere with receptor binding but results in a loss of invivo, but not in vitro, biological activity (2, 3). It has beenreported that chemically deglycosylated hCG (dghCG) is anantagonist, failing to activate adenylate cyclase but bindingwith affinity similar to that of hCG (4-6). Studies of thisnature indicate that the carbohydrate moieties are not im-portant for binding of the hormone but play a role inactivation of the adenylate cyclase system. To investigatedifferences in the way hCG, the asialo derivative of hCG(ashCG), and dghCG interact with the receptor, we havemade photoactivable derivatives and used them to label thegonadotropin receptor of porcine granulosa cells.

EXPERIMENTAL PROCEDURESPreparation and Characterization of Hormone Derivatives.

hCG, batch CR-123, was supplied by the Center for Popu-lation Research (National Institutes of Health and HumanDevelopment). The biological potency was 12,780 units/mg.ashCG and dghCG were prepared as described (5, 7). TheN-hydroxysuccinimide ester of 4-azidobenzoylglycine(ABG) was synthesized, and hormones (hCG, ashCG, anddghCG) were derivatized with the reagent and radioiodinatedfollowing reported procedures (8). Competitive binding stud-ies were done as reported (9), except that the competitorswere either unlabeled hCG or the corresponding unlabelednonderivatized hormone analogue. ABG-hormone deriva-tives were also tested for their ability to stimulate adenylatecyclase. Murine Leydig tumor cell membranes were prepared(10), and adenylate cyclase activity (11) was assayed as de-scribed.

Cell Preparation. Porcine ovaries, rich with large folliclesbut free of corpora lutea, were selected and rinsed withMedium 199. Follicles were excised (9), and granulosa cellswere collected in 80 ml of Medium 199 containing 1 mMphenylmethylsulfonyl fluoride, 20 uM tosyl-L-lysine chloro-methyl ketone, 5 mM 6-amino caproic acid, 2mM EDTA, 100,um benzamidine, leupeptin at 5 ,ug/ml, and pepstatin at 7,ug/ml. This combination represents inhibitors for all fourmajor classes of proteases (serine, thiol, metallo-dependent,and carboxyl). These inhibitors were included in all buffersexcept that 6-amino caproic acid was deleted when sampleswere cross-linked and that all inhibitors were omitted forsamples to be digested. It is important to prevent aggregationof cells by occasional, gentle mixing and by maintaining thecell concentration below 3 x 106 cells per ml and temperaturebelow 4°C. Cells were collected by centrifugation at 600 x gfor 10 min and rinsed twice in 15 ml of Medium 199. Alloperations were carried out on ice or at 4°C.Membrane Preparation. Washed granulosa cells were in-

cubated at 4°C for 15 min in 25 mM Tris HCl, pH 7.4/1 mMCaCl2 containing the protease inhibitors and homogenized ina Dounce homogenizer with a pestle B. The supernatant,after centrifugation at 1500 x g for 10 min, was centrifugedagain at 45,000 x g for 30 min. The pellets were resuspendedin 0.17 M NaCl/1.5 mM MgCl2/10 mM Tris HCl, pH 7.4, andused as crude membranes. The protease inhibitors werealways included unless otherwise indicated.

Photoaffinity Labeling of the Gonadotropin Receptor.Washed granulosa cells (7 x 106 cells per sample) wereincubated in 400 Al of 0. 15 M NaCl/10 mM Na2HPO4, pH 7.4(PBS), with 125I-labeled hCG-ABG for 60 min at 37°C, washedtwice, and irradiated with a shortwave UV lamp. The cell

Abbreviations: ABG, 4-azidobenzoylglycine; ashCG, asialo humanchoriogonadotropin; dghCG, deglycosylated human choriogonado-tropin; hCG, human choriogonadotropin; SES, bis[2-(suc-cinimidooxycarbonyloxy)ethyl] sulfone.1To whom reprint requests should be addressed.

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pellet was suspended in 20-60 ,ul of the solubilizing solutioncontaining 3% (wt/vol) NaDodSO4, 5% (vol/vol) glycerol,and 100 mM dithiothreitol. After boiling for 2 min, thesuspension was centrifuged in a Beckman Ti50.2 rotor at40,000 rpm for 60 min at 20°C. The supernatant was collectedwithout disturbing the pellet and electrophoresed usually on9-12% gradient gels containing 6 M urea (12). Photoaffinitylabeling of the soluble receptor was carried out after deter-gent extraction of hormone-treated cells in a solution con-taining 0.5% Triton X-100, 10mM NaCl, 1 mM MgCl2, 10mMTris-HCl (pH 7.5), and the protease inhibitors. Solubilizationfor 5 min on ice was followed by centrifugation at 3000 x gfor 10 min, then the supernatant was centrifuged in aBeckman Ti5O.2 rotor at 40,000 rpm for 60 min at 4°C. Thesupernatant was collected and photoaffinity labeled, and theresulting complexes subjected to NaDodSO4/PAGE (12).

Proteolytic Digestion. Cross-linked samples were resolvedon a first-dimensional gel. Gel lanes were excised andincubated in 50 ml of 125 mM Tris'HCl, pH 6.8/0.1%NaDodSO4 for 40 min at 25°C. Following equilibration, thegel lanes were rinsed with distilled water and mounted on thetop of fresh slab gels. A 4% stacking gel was cast up andaround the first-dimension gel lane and overlaid with 0.8 mlof 0.5% NaDodSO4/8% (vol/vol) glycerol/pyronin Y/prote-ases. The gels were initially electrophoresed at 50V for 30-60min until the dye front reached three-quarters of the stackinggel. Electrophoresis was temporarily stopped for a 60-minincubation at 25°C and then resumed at 100 V.

'°°r

Sal.F

E

_O

E

E

C

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201

Table 1. Association constants for hCG, ashCG, and dghCG

Hormone Associationderivative constant, nM-1

hCG 2.6ashCG 3.2dghCG 3.8

Values for association constants were determined in competitivebinding studies. Displacement of ABG- and '25I-labeled hormonederivatives from receptors on granulosa membranes by theirunlabeled and nonderivatized counterparts was measured. Associ-ation constants were obtained by linear regression analysis ofScatchard plots generated from the data in Fig. 1.

Cross-Linking and Cleaving. Washed granulosa cells, 7 x107 cells per 400 dul of Medium 199, were incubated with"25I-labeled dghCG for 60 min with constant, gentle shakingat 37TC. Borosilicate glass tubes (12 x 75 mm) have shown theleast nonspecific binding, compared to soft glass and variousplastic tubes. Critical factors for maintaining a good cellsuspension and hormone binding are the proper number ofcells, the volume of medium, and the size of tubes. Afterincubation, cells were rinsed twice in 1 ml ofMedium 199 andonce in 1 ml of PBS. The cell pellet was resuspended in 400A.l ofPBS containing an alkaline cleavable heterobifunctionalreagent bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone(SES), incubated for 15 min at 250C, rinsed once in 50 mMglycine in PBS, solubilized, and electrophoresed.To cleave cross-linked complexes resolved on first-dimen-

sion slab gels, gel lanes were cut and incubated for 90 min at370C in the cleaving solution containing 0.1% NaDodSO4, 10mM dithiothreitol, and 100 mM Na3PO4 (pH 11.5). Thetreated gel lanes were mounted on the top of fresh slab gelsand subjected to a second electrophoresis.

RESULTSCompetitive binding of the ABG- and 1251-labeled derivativesof hCG, ashCG, and dghCG to their receptors in granulosamembranes was evaluated in the presence of increasingconcentrations of hCG (Fig. 1) or in the presence of theirrespective unlabeled nonderivatized analogue (Fig. 1). The

_jequilibrium association constants for ashCG and dghCGbinding to granulosa cells were slightly higher than that ofhCG (Table 1). These values also agree with those reportedfor binding of gonadotropins to their receptors in othersystems (13). Stimulation of adenylate cyclase by hCG andashCG was also similar, but dghCG did not show anysignificant stimulation of the enzyme (Table 2), similar to thefindings of others (4-6).The a subunits of 125I-labeled ashCG-ABG and 125I-labeled

hCG-ABG were the primary sites for radioiodination, where-as in 125I-labeled dghCG-ABG the 13 subunit was radioiodi-nated in addition to the a subunit (Fig. 2). The electrophoreticmobility of the subunits and the dimers increased in the orderof hCG, ashCG, and dghCG, as expected, with removal of

I Table 2. Activation of adenylate cyclase by hCG, ashCG,100 1000 and dghCG

Untreated hormone, ng

FIG. 1. Competitive binding ofradiolabeled hCG derivatives withtheir unlabeled counterparts and with native hCG. Displacement of1251I-labeled hCG-ABG (e), -ashCG-ABG (o), or -dghCG-ABG (c&)from their receptors on granulosa cells was determined. The com-

petitors were either unlabeled hCG (Upper) or the correspondingunlabeled and nonderivatized native, asialo, or deglycosylated hor-mone (Lower). The data are expressed as a percent of the maximum125I-labeled hormone derivative bound. Nonspecific binding wasdetermined in the presence of 5 ug of untreated hormones and wasless than 10% of the total binding.

Adenylate cyclase activity,pmol per 10 min

Hormone per mg of protein

None 36 ± 2hCG 350 ± 30ashCG 190 ± 10dghCG 69 ± 10

Adenylate cyclase activity on murine Leydig tumor cells wasmeasured in the presence and absence of 100 nM hormones asdescribed (6). Data are expressed as the mean ± SD.

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Proc. Natl. Acad. Sci. USA 83 (1986) 6329

hCG ashCG dghCG

UV - + - + - +

- _ *Ww

4El-0N

hCG ashCG dghCG_ + - + - +

A-w-LR.0.

_ _

FIG. 2. Photoaffinity labeling of the gonadotropin receptor with hCG, ashCG, and dghCG. ABG- and I"I-labeled hormone derivatives wereseparated on NaDodSO4/PAGE without being exposed to UV light or after photolysis as indicated. The derivatives were free in solution (Left),bound to cells (Center), or membranes of granulosa cells (Right). The relative positions of corresponding bands are indicated by arrows. Thedata represent a typical experiment. The experiment was repeated 15 times. 48k, 48-kDa complex. +, Exposed to UV-irradiation. -, Notirradiated.

sialic acids and other carbohydrates. Upon photolysis of freehormone derivatives, the subunit bands diminished while theintensity of the a-,S dimer band increased. Whenhormone-receptor complexes on cells were UV-irradiated,several bands (A, B, C, and D) of slower electrophoreticmobilities appeared in addition to the ao-3 dimer band (Fig. 2Center). Similar results were obtained when isolated mem-branes were used (Fig. 2 Right). The positions of these bandsvaried depending upon the hormone derivative used tophotoaffinity label the hormone receptor. Correspondingbands are indicated by the arrows in Fig. 2. The differencesin the band positions are most likely due to variations in theelectrophoretic mobility of the hormone derivatives. Theseresults suggest that the hormone derivatives photoaffinitylabel the same components of the hormone receptor. ThedghCG derivative, however, photoaffinity labeled one addi-tional band of 48 kDa. Although it is a minor band, itconsistently appeared in samples labeled with the dghCGderivative. This band was absent when 251I-labeled dghCG-ABG alone was extensively photolyzed or when cells wereincubated with hormone derivative but not photolyzed.Addition of an excess amount of native dghCG duringincubation of cells with the 125I-labeled dghCG-ABG prevent-ed formation of the 48-kDa band. The appearance ofthe band

was dependent on the length of UV irradiation (Fig. 3). The48-kDa band was also produced when the hormone-receptorcomplex was photolyzed on intact cells or after solubilizationwith Triton X-100 (data not shown). To determine theapparent molecular sizes of the complex, cross-linked sam-ples were electrophoresed on gels ofthe following acrylamideconcentrations: 7, 8, 9, 11, 6-12, 7-12, 8-12, 9-12, and10-12%. Although the molecular size estimates varied de-pending on the gradient and concentration of acrylamide, theaverage value was 48 kDa.

Photoaffinity labeled hormone-receptor complexes re-solved in one dimension by NaDodSO4/PAGE were subject-ed to proteolytic digestion, and the products were separatedin a second dimension ofNaDodSO4/PAGE (Fig. 4). Peptidemaps of complexes A-D were similar. The smallest radiola-beled peptide generated by protease treatment of thesecomplexes (indicated by the arrows) had the same electro-phoretic mobility but was different from the smallest peptideproduced from the 48-kDa complex (Fig. 4, arrowheads).This was true for both V8 protease and papain digestion.To investigate the hormone subunit composition of the

48-kDa complex, we cross-linked 125I-labeled dghCG to itsreceptor on granulosa cells with SES. NaDodSO4/PAGErevealed that the cross-linker produced a 48-kDa band just as

hCG-R0 10 30 60 120

4W.1_.r_ _

_

_ _ 6wPIb

goOD

ashCG-R

0 10 30 60 120

daf

dghCG-R0 10 30 60 120

..i_i

4*-48k

FIG. 3. Time course of photoaffinity labeling of the gonadotropin receptor with hCG (hCG-R), ashCG (ashCG-R), and dghCG (dghCG-R).The indicated ABG- and '2"I-labeled hormone derivatives were bound to the gonadotropin receptors on granulosa cells and photolyzed for 0-120sec as indicated above each lane. The data are typical, and the experiment was repeated eight times.

Ca C

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--48k

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1 St A CD CIOtt tt

-ac .C',awN

t 0-$t t tt f s t ft f t i 8 kt

tc>4 * ,.... z~~~~~~~~-... !;A lk:

A

c

C,' 4..I

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FIG. 4. Peptide map of the gonadotropin receptor photoaffinity labeled with hCG (hCG-R), ashCG (ashCG-R), and dghCG (dghCG-R).Granulosa cells were photoaffinity labeled with the indicated ABG- and "'I-labeled hormone derivative and separated by NaDodSO4/PAGE.The samples were then subjected to proteolytic digestion with V8 protease or papain before separating by a second NaDodSO4/PAGE. Theposition of the smallest radiolabeled peptide fragment is designated by the arrows. Arrowheads indicate the peptide fragments appearing onlywith the 48-kDa complex (48k). The experiment was repeated five times, and the data presented here are representative of a typical experiment.

the photoaffinity labeling procedure had. The cross-link waspartially cleaved, and the products were separated in asecond dimension by NaDodSO4/PAGE (Fig. 5). By thistechnique we were able to identify a component with thesame mobility as the a3 dimer ofdghCG (Fig. 5). In addition,the a subunit could be identified as a product ofcleavage. Thesample photoaffinity labeled with the dghCG derivativeshowed a faint additional band slightly above band A (Figs.2-4). The identity of this complex is unknown.

DISCUSSIONInvestigations have made it clear that dghCG binds specifi-cally and with high affinity to the gonadotropin receptor;however, it does not activate adenylate cyclase (4-6). It isapparent that the sugar moieties are not important for thebinding activity but are necessary for the biological activity.ashCG also binds with high affinity to the gonadotropinreceptor. ashCG, which lacks only the sialic acid residues, isalso an agonist, although it is less potent than native hCG(14). Since dghCG is an antagonist, its binding to thegonadotropin receptor may not disturb the configuration ofthe unstimulated (basal) state of the hormone-responsive

1 St

48KAe

ag3

FIG. 5. Cross-linked dghCG-receptor complex separated byNaDodSO4/PAGE. "'II-labeled dghCG was cross-linked to its re-ceptor on granulosa cells with SES, and the complexes wereseparated by NaDodSO4/PAGE. The cross-links were then partiallycleaved, and the products were separated by a second NaDodSO4/PAGE. 48k, 48-kDa complex.

adenylate cyclase system. By photoaffinity labeling thereceptor with photoactivatable, radiolabeled hormone deriv-atives, we hoped to discover (i) if there is some portion of thereceptor that can be labeled with hCG and ashCG but not withdghCG and, therefore, that may be implicated in activation ofthe adenylate cyclase system; and/or (ii) if some componentassociated with the receptor in the basal state, but not theactivated state, could be photoaffinity labeled with dghCGbut not with the agonists hCG or ashCG.We have found that a number of high molecular weight

complexes were generated by photoaffinity labeling of thegonadotropin receptor wih ABG- and '25I-labeled deriva-tives. In this report, we have designated them as complexesA-D for the purposes of identification. This suggests aheterooligomeric structure for the receptor, which agreeswith previous reports (12, 15). Some investigators haveclaimed that the heterooligomeric nature of the receptor isdue to proteolytic degradation (16) and that in the absence ofproteolysis the receptor is a single polypeptide chain. This issimilar to the results of others that indicate that thegonadotropin receptor is a polypeptide with a molecular sizeof -90 kDa (10, 17, 18). During this investigation we havebeen careful to avoid proteolysis by including proteaseinhibitors throughout the procedures. We cannot, however,completely eliminate the possibility of proteolytic degrada-tion particularly before hormone binding.

In addition to components A-D, a 48-kDa complex wasgenerated when 1251-labeled dghCG-ABG was used tophotoaffinity label the gonadotropin receptor. Proteaseswere used to generate peptide maps of the complexes tounderstand their relationship to one another. V8- and papain-generated peptide maps of photoaffinity-labeled complexesA-D were similar to each other. This was true regardless ofwhich hormone derivative was used to form the complexes.Thus there is an apparent relationship between these com-ponents such that each successively smaller component iscontained within the larger ones. However, the peptide mapof the 48-kDa complex was different from that of the otherphotoaffinity-labeled complexes.The molecular size of the 48-kDa complex was similar to

that of the native hCG. We considered the possibility that thedghCG preparation was contaminated with hCG. We haveinvestigated this possibility by extensively cross-linking thedghCG preparation and have failed to generate a complex of

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Proc. Natl. Acad. Sci. USA 83 (1986) 6331

the same electrophoretic mobility. Thus the 48-kDa complexis likely to represent a complex composed of dghCG (or oneof its subunits) and a component of the cell membrane.The 48-kDa component may represent a receptor-hormone

complex that contains only the dghCG /8 subunit. The (3subunit of dghCG is unique in that it was radioiodinated,whereas (3 subunits of hCG and ashCG were virtuallyunlabeled (Figs. 2 and 3). Thus it could be argued that the48-kDa complex was present in all of the photoaffinity-labeled samples but appeared as a radioactive band only inthe samples labeled by the dghCG derivative. This can bepossible only if the 48-kDa complex is composed of the 3subunit but not the a subunit. To determine which hormonesubunits were contained in the 48-kDa complex, we firstcross-linked 251I-labeled dghCG to its receptor and thenlocated the complex on NaDodSO4/PAGE. Under the propercleaving conditions some, but not all, of the cross-links canbe broken with the result that a radiolabeled componenthaving the same electrophoretic mobility as the ,a-3 dimer ofdghCG appeared. In addition, if the 3 subunit were the onlycomponent of dghCG in the complex, it should be apparentin the cleaved products. This is not the case, and in fact thea subunit was detected. This result supports the hypothesisthat the 48-kDa complex contains both subunits of dghCGand thus represents a complex not previously observed whenhCG or ashCG is used in photoaffmity-labeling (or cross-linking) experiments. Therefore, we estimate that the 48-kDacomplex contains an 8-kDa component in addition to the a-/3dimer of 125I-labeled dghCG-ABG. A more direct way to testthe hypothesis is to photoaffinity label the receptor with hCGthat has the radioiodinated P subunit. Although the (3subunitin whole hCG is resistant to radioiodination, it can beradioiodinated after its dissociation from the a subunit. Sucha radiolabeled 3 subunit can be recombined with an unlabeleda subunit. Results of such experiments (19) were inconsistentwith the hypothesis, and no radioactive complex equivalentto the 48-kDa complex was produced.The 8-kDa component may represent a component of the

hCG receptor. It is also possible that it represents somecomponent closely associated with the receptor but not a partof the receptor itself. This component may become dissoci-ated from the receptor upon activation and hence it cannot belabeled with the agonists hCG or ashCG. If the 8-kDacomponent is not a part of the receptor, then it mustnecessarily be associated with it since photoaffinity labelingof the detergent solubilized dghCG-receptor complex alsoproduced the 48-kDa complex. A potential candidate thatwould fit the criterion set forth for the 8-kDa component is they subunit of the regulatory guanine nucleotide (G) bindingprotein of the adenylate cyclase system (20, 21). The dghCGappears to have a direct access to the 8-kDa component. If

this 8-kDa component is the y subunit (20), our resultsuggests an intriguing possibility of the hormone's directcontact with the y subunit either at the cell surface and/orinside of cells, its cell surface exposure and the transmem-brane nature of the regulatory subunit. In view of thepotential regulatory interactions of hormone receptors withthe ,Band y complex (20, 21) and the absence of a transmem-brane domain in the y subunit (22), it is possible that the /3subunit could be a transmembrane polypeptide and respon-sible for anchoring the regulatory protein to cell membranes.

This work was supported by Grant HD-18702 from the NationalInstitutes of Health. T.H.J. is the recipient of American CancerSociety Faculty Research Award FRA-262.

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