43

Molecular and Cellular Endocrinology, 12 (1990) 43-53 Elsevier Scientific Publishers Ireland, Ltd.

MOLCEL

02318

Subunit interaction of human chorionic gonadotropin (hCG) with rat ovarian luteinizing hormone (LH)/CG receptor Ulla E. Petgjij-Repo

and Hannu

J. Rajaniemi

Biocenter and Department of Anatomy, University of Oulu, SF-90220, (Received

Key words: Human

chorionic

gonadotropin;

14 February

Luteinizing

1990; accepted

hormone

receptor;

Ouly Finland

2 May 1990)

Receptor-hormone

interaction;

(Rat ovary)

The subunit interaction of hCG with its rat ovarian LH/CG receptor was studied by cross-linking the solubilized receptor-hormone complex with glutaraldehyde (GA), disuccinimidyl suberate (DSS) or dithiobis(succinimidy1 propionate) (DSP) and analyzing the complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The hormone was labeled either in its a-subunit (‘251-hCG) or in its P-subunit (3H-hCG) or the label (3H) was introduced into the receptor molecule instead of the hormone. All of the labeling procedures led to the detection of only the receptor-( a,P)hCG and receptor-( cu)hCG complexes on the autoradiograms. The sizes of these complexes were 137,000 and 106,000, respectively, under reducing conditions. These results suggest that the receptor binds one hormone molecule, and that hCG interacts with the receptor mainly through its n-subunit. In addition, polyclonal antibodies directed against the LH/CG receptor and the (Y- and P-subunits of hCG were used to detect the non-reduced receptor-(a,P)hCG complex in immunoblotting. As antibodies directed against both the a-subunit and the P-subunit were able to detect the M, 130,000 complex, it is conceivable that both of the subunits are at least partially exposed on the receptor-hormone complex. ‘251-hCG was also cross-linked to the membrane-bound receptor. The membrane-bound complex had an M, of 144,000 under reducing conditions, i.e. approximately 7000 higher than that of the solubilized complex (M, 137,000). This may indicate that the membrane-bound receptor is covalently modified or differs in conformation from the solubilized receptor.

Introduction hCG belongs to the glycoprotein hormone family along with the pituitary hormones LH, follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH) (see Pierce and Par-

Address for correspondence: University of Oulu, Department A, SF-90220 Oulu, Finland.

0303-7207/90/$03.50

Ulla E. Pet;ijl-Repo, M.Sc., of Anatomy, Kajaanintie 52

0 1990 Elsevier Scientific

Publishers

Ireland,

sons, 1981). These four hormones are structurally related heterodimers formed by non-covalent association of two glycosylated subunits, designated (Yand /3. The a-subunit is identical in all four and is highly conserved from species to species. The P-subunits also show considerable homology in structure and confer hormonal specificity. The isolated subunits have little biological activity, if any, and their non-covalent association is necessary for high affinity receptor binding. Among the four glycoprotein hormones, hCG and LH display Ltd.

44

the closest similarity and they also share the same receptor site on gonadal target cells. The fact that the hormone and species specificities of glycoprotein hormones are associated with different subunits has prompted several laboratories to elucidate through which subunit these hormones interact with their corresponding receptor. Various indirect approaches have been employed to address this question. Physicochemical approaches have included modifications of the free subunits or intact hormone molecules by enzymatic or chemical means (see Pierce and Parsons, 1981; Gordon and Ward, 1985) and preparation of synthetic peptide fragments of the subunits (Sluss et al., 1986; Charlesworth et al., 1987; Keutmann et al., 1987, 1988, 1989; Morris et al., 1988) while immunochemical methods have used both polyclonal and monoclonal antibodies directed against the hormone molecule, free subunits or synthetic fragments (Moyle et al., 1982; Dighe and Moudgal, 1983; Milius et al., 1983; Bidart et al., 1987a, b; Hill et al, 1987; AlonsoWhipple et al., 1988; Costagliola et al., 1988; Schneyer et al., 1988; Schwartz et al., 1988). In a third approach, glycoprotein hormones have been cross-linked to their specific receptors (Ji and Ji, 1981; Hwang and Menon, 1984; Ascoli and Segaloff, 1986; Kusuda and Dufau, 1986; Petaja et al., 1987; Roche and Ryan, 1989). Despite intensive research, no general agreement has emerged about the mode of subunit interaction with the receptor and the involvement of both the a- and the P-subunit have been implicated in receptor-glycoprotein hormone interaction. The present work was undertaken to elucidate the subunit interaction of hCG its rat ovarian LH/CG receptor by cross-linking cr-subunitlabeled hCG (iz51-hCG) and P-subunit-labeled hCG ( 3H-hCG) to the rat ovarian LH/CG receptor. To obtain cross-linkages of various lengths, three different cross-linkers were used. Unlabeled hCG was also cross-linked to the 3H-labeled receptor. In addition, polyclonal antibodies directed against the receptor and the (Y- and /?-subunits of hCG were used to detect the cross-linked receptorhCG complex in immunoblotting. The evidence presented indicates that the rat ovarian LH/CG receptor binds hCG in its a,/%dimer form so that the a-subunit is in closer contact with the recep-

tor. Both of the subunits of hCG are, nevertheless, exposed on the stable receptor-hormone complex. Materials and methods Chemicals, hormone preparations and antisera Disuccinimidyl suberate (DSS) and dithiobis(succinimidyl propionate) (DSP) were purchased from Pierce (Rockford, IL, U.S.A.) and iv-ethylmaleimide (NEM) and phenylmethylsulfonyl fluoride (PMSF) from Sigma (St. Louis, MO, U.S.A.). Pregnant mare serum gonadotropin (PMSG, 2820 IU/mg) and hCG (10,900 IU/mg, 12,100 IU/mg) were obtained from Diosynth (Oss, Netherlands) and Na’251 and NaB3H, from Amersham (Bucks, U.K.). Sephadex G-SO and Sepharose 4B were from Pharmacia (Uppsala, Sweden) and the equipment and reagents for electrophoresis and blotting from Bio-Rad (Richmond, CA, U.S.A.). All the other reagents were of analytical grade and obtained from commercial suppliers. hCG (12,100 IU/mg) was radioiodinated with Na’251 by the chloramine-T method (Hunter and Greenwood, 1962) as described by Markkanen et al. (1980). The specific activity of the preparations averaged 60 Ci/g as determined by self-displacement assay (Ketelslegers et al., 1975) and their biological activity (determined as the fraction of the radioactivity capable of binding to an excess of rat ovarian membrane-bound receptors) averaged 50%. 3H-labeled hCG was prepared by a modification of the periodate-NaB3H, method described by Gahmberg and Andersson (1977). Highly purified hCG (12,100 IU/mg; 100 pg) in 10 mM phosphate buffered saline (PBS, pH 7.4, 300 ~1) was oxidized in the dark with 0.7 mM (final concentration) NalO, for 10 min at 20°C. The reaction was quenched by adding 10 mM (final concentration) glucose and dialyzing the preparation against PBS for 20 h at 4 o C. Reduction was performed by incubating the preparation with 4 mCi NaB3H, (stored in 0.01 M NaOH at - 70 o C) for 60 min at 20” C. The labeled hormone was separated by gel filtration using a Sephadex G-50 column and PBS containing 0.1% bovine serum albumin (BSA) as an eluent. The specific activity of the preparations averaged 1 Ci/g and the biological activity was approximately 30%.

45

The antiserum against the a-subunit of hCG was obtained from UCB (Braine L’Alleud, Belgium), and according to the manufacturer its cross-reaction with the a,/%dimer of hCG was 25%. The antiserum against the P-subunit, from Dakopatts (Globstrup, Denmark), recognized the free P-subunit and the cu,/3-dimer of hCG equally well. Antiserum against the LH/CG receptor was prepared by immunizing rabbits with an affinity purified receptor preparation. About 10 pg of purified receptor was suspended in complete Freund’s adjuvant (1 : 1) and injected i.d. at ten sites 4 times at 2-week intervals (Lakkakorpi et al., unpublished data). Animals Immature 27-day-old female Sprague-Dawley rats were rendered pseudopregnant by sequential injections of PMSG (40 IU) and hCG (25 IU) as described by Parlow (1961). The luteinized ovaries were collected 7-9 days after the hCG injection, freed from extraneous tissue, frozen in liquid nitrogen and stored at - 70 o C before use. Ovarian membrane preparation and solubilization of the receptor-hCG complex Crude ovarian membrane particles were prepared as described previously (Petaja et al., 1987). The luteinized ovaries were homogenized in buffer A (10 mM PBS containing 5 mM EDTA, 5 mM NEM and 0.2 mM PMSF; 2 ml/ovary) in a glass homogenizer with 100 strokes, after which the homogenate was centrifuged at 100 X g for 10 min and the supernatant further centrifuged at 27,000 x g for 30 min. The pellet containing the crude membrane particles was suspended in buffer A (0.25 ml/ovary) with mild homogenization and incubated with either ‘251-hCG (3. lo6 cpm/ ovary) or ‘H-hCG (3 . 10e6 cpm/ovary) at 37 o C for 1 h or at 20 o C for 16 h. For control purposes the labeling was also performed in the presence of an excess of unlabeled hCG (20 pg/ovary). In some experiments the crude membrane particles were incubated only with unlabeled hCG (20 pg/ ovary). The unbound hormone was removed by washing twice with buffer A (1.5 ml/ovary). The final pellet was solubilized in buffer A containing 1% Triton X-100 by stirring on ice for 30 min. The suspension was diluted (1 : 1) with buffer A and

centrifuged at 100,000 x g for 60 min to obtain the soluble receptor-hCG complex. All the above steps were performed at 0-4OC. Covalent cross-linking of the receptor-hCG complex The receptor-hCG complex was cross-linked with either 2 mM glutaraldehyde (GA) (Petajij et al., 1987) 1 mM DSS or 1 mM DSP (all final concentrations) using 100 mM GA (in water) and 50 mM DSS or DSP (in dimethyl sulfoxide, prepared just before use). GA cross-linking was continued for 60 min at 20” C and DSS and DSP cross-linking for 15 min at 0 ’ C. The cross-linking reaction was quenched by adding 0.125 volumes of 1 M Tris-HCl (pH 7.4 at 4°C). When the membrane-bound receptor-hCG complex was cross-linked, the crude membrane particles were suspended in buffer A (4 ml/ovary; 0.5 mg of protein/ml) after removing the unbound hormone. The solubilized receptor-hCG complex was crosslinked after diluting the Triton X-100 extract to 1 : 20 (25 pg of protein/ml) with buffer A containing 0.5% Triton X-100. In control experiments, unlabeled hCG (1 pg/ml in PBS) and ‘251-hCG and 3H-hCG (10,000 cpm in PBS) were also cross-linked without attaching them to the receptor. Labeling of the ovarian membranes with periodatetritiated borohydride The labeling procedure was a modification of that of Gahmberg and Andersson (1977) as described previously by Metsikkb (1984). Crude membrane particles were suspended in buffer A (0.3 ml/ovary) and NaIO, (0.1 M) was added to a final concentration of 2 mM. The suspension was incubated in the dark for 10 min at 4O C, after which the excess of periodate was removed by centrifugation at 27,000 x g for 30 min at 4” C. The pellet was washed twice with buffer A (1 ml/ovary) and centrifuged as above. The final pellet was resuspended in 50 mM PBS (pH 8.3,0.3 ml/ovary) and NaB3H, (2.5 mCi/ovary) was added. After incubation (60 min at 20°C) and centrifugation (27,000 X g, 30 min) the pellet was washed twice with buffer A (1 ml/ovary). Unlabeled hCG (20 pg/ovary) was attached to tritiated membrane particles as described above. After solubilization and cross-linking the 3H-LH/CG

46

receptor-hCG complex was purified by immunoaffinity chromatography. In the control experiments, tritiated membrane particles were solubilized with buffer A containing 1% Triton X-100 and 20% glycerol and the tritiated LH/CG receptor was purified essentially as described earlier (Keinanen et al., 1987) using a single affinity chromatography step.

ImmunoafJnity chromatography After cross-linking, the preparations containing the 3H-LH/CG receptor-hCG complex (0.5 mg of protein) were dialyzed against diluted (1 : 4) buffer A containing 0.5% Triton X-100 for 16 h at 4°C and concentrated to one-fourth of their volume (5 ml/ ovary). The concentrated preparations were incubated with 100 ~1 of immunoaffinity matrix by mixing end-over-end for 16 h at 4” C. The immunoaffinity matrix was prepared as described previously (Metsikko and Rajaniemi, 1979) by coupling ammonium sulfate-precipitated y-globulins of hCG antiserum to cyanogen bromideactivated Sepharose 4B. The hCG antiserum has been characterized previously (Kinnunen et al., 1981). After incubation, the matrix was washed twice at 20°C with buffer A containing 0.5% Triton X-100 (4 ml/ovary) followed by buffer A containing 0.1% Triton X-100 and 0.5 M NaCl (2 ml/ovary) and again buffer A containing 0.1% Triton X-100 (2 ml/ovary). The gel matrix was pelleted between the washes by centrifugation at 5000 x g for 15 min. The receptor-hCG complex was eluted by boiling the matrix at 100°C for 5 min in SDS-sample buffer (pH 6.8, 100 #ovary) containing 2% SDS, 125 mM Tris-HCl, 10% glycerol and 0.001% bromophenol blue. In the control experiments, no unlabeled hCG was attached to the membranes, or else the complex was purified in the presence of an excess of unlabeled hCG.

SDS-polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of SDS was performed according to Laemmli (1970), using 4% stacking gels and 7.5% or 10% separating gels. The samples containing the solubilized receptor- ‘251-hCG or receptor-3H-hCG complexes were dialyzed against diluted (1 : 4-

1 : IO) SDS-sample buffer for 20 h at 4” C and concentrated using a Speed-Vat concentrator. Before electrophoresis the samples were diluted with SDS-sample buffer to obtain the same amount of radioactivity into each well and heated at 95°C for 2 min in either the presence or the absence of 2% 2-mercaptoethanol. Molecular weight markers (SDS-6H, SDS-7, Sigma) detected by staining with Coomassie brilliant blue (Fairbanks et al., 1971) were used to calibrate the slab gels. Pre-stained molecular weight markers (SDS-7B, Sigma) were used, when 3H-labeled samples were analyzed. For the detection of radioactivity, the slab gels were dried and exposed at - 70 o C for 2-30 days using Kodak XAR-5 film (Eastman Kodak, Rochester, NY, U.S.A.) with an intensifying screen (Kodak X-Omatic). The gels containing 3H-labeled samples were treated for fluorography (Bonner and Laskey, 1974) before drying.

Immunoblotting The cross-linked receptor-hCG complex (5- 10 pg of protein) was subjected to SDS-PAGE under non-reducing conditions as described above but without prior heating. The proteins were transferred from the gel onto a nitrocellulose sheet electrophoretically in 25 mM Tris, 192 mM glycine using a Mini-Trans-Blot apparatus. The transfer was performed using a constant voltage of 100 V for 1 h. The nitrocellulose sheet was rinsed in PBS, and the lane containing the molecular weight markers stained with 0.02% Ponceau S. The strips containing the sample lanes were incubated in porcine serum for 16 h at 4” C and rinsed briefly with PBS before incubation for 2 h in buffer C (10 mM Tris-HCl, pH 7.4, 1% BSA, 10% glycerol) containing immune serum at the dilutions indicated in the legend for Fig. 6. The strips were washed 6 times in PBS containing 0.05% Triton X-100 (lo-15 min/wash), rinsed in PBS and incubated for 1 h in buffer C containing horseradish peroxidase conjugated to goat antirabbit immunoglobulin G (Bio-Rad, final dilution 1 : 3000). The strips were then washed as described above and incubated in PBS containing 0.03% diaminobenzidine and 0.01% H,O,. All the incubations were performed at 20” C unless otherwise indicated.

a-subunit of hCG, respectively. In the absence of the cross-linker, almost all of the a,/?-dimer of hCG dissociated and the radioactivity appeared in the labeled subunits. Densitometric scanning of the autoradiograms (n = 5) containing the noncross-linked samples showed that in ‘251-hCG 9195% of the radioactivity was in the a-subunit and in 3H-hCG 62-808 was in the P-subunit. The number and intensity of the radioactive bands was similar with all the cross-linkers used, and reduction with 2% 2-mercaptoethanol did not change the appearance of the bands, except when DSP was used. As DSP contains a disulfide bridge, it is a cleavable cross-linker, and thus the a,Sdimer of hCG dissociated under reducing conditions to the same extent as the non-cross-linked hormone.

Protein determination Protein was assayed by the method of Lowry et al. (1951) as modified by Dulley and Grieve (1975). Results

Cross-linking of the a-subunit and P-subunit labeled hCG to the LH/CG receptor Cross-linking of ‘251-hCG (cY-subunit-labeled hormone) and 3H-hCG (/&subunit-labeled hormone) with either GA, DSS or DSP followed by SDS-PAGE under non-reducing conditions produced three bands of M, 49,000, 32,000 and 22,000 on the autoradiograms (Fig. 1). They represent the a,/?-dimer of hCG and the labeled P-subunit and

A ‘*%-hCG 2

1 (a,O)-hCG (fi)_hCG

4

5

6

7

8

Mr

8

M,

‘a

_

3

_.

non- reduced

reduced

6 3H-hCG 1

2

3

4

5

6

7

- 45K - 36~ 1;::

non-reduced

reduced

Fig. 1. Covalent cross-linking of “‘1-hCG (panel A) and ‘H-hCG (panel B). I*’ I-hCG and 3H-hCG were treated with either 2 mM GA (lanes A2, A6, B2, B6) for 60 min at 20 o C or 1 mM DSS (lanes A3, A7, B3, B7) or 1 mM DSP (lanes A4, AS, B4, B8) for 15 min at 0 o C. In the controls (lanes Al, A5, Bl, B5) no cross-linker was added. The samples were analyzed by SDS-PAGE (10% slab gel) under non-reducing or reducing conditions. Numbers on the right denote the molecular weight markers (from the top: ovalbumin, glyceraldehyde-3-phosphate dehydrogenase, carbonic anhydrase, trypsinogen and trypsin inhibitor).

48

In order to determine through which subunit hCG interacts with its receptor, the a-subunitlabeled hCG (‘251-hCG) and the /3-subunit-labeled hCG (3H-hCG) were attached to rat ovarian membranes. The complexes formed were solubilized with Triton X-100 and cross-linked with either GA, DSS or DSP. SDS-PAGE of the solubilized receptor-“‘1-hCG complex under non-reducing conditions gave a band of M, 130,000 on the autoradiograms in addition to the q/3-dimer and the a-subunit of hCG (Fig. 2). The M, 130,000 band represents the receptor-‘251-hCG complex, as it was absent when ‘251-hCG was attached to the membranes in the presence of an excess of unlabeled hCG. A faint, smaller band of M, 103,000 was occasionally detected, which probably contains the a-subunit of hCG bound to the receptor, as some of the bound hormone molecules which are not stably cross-linked dissociate into subunits during SDS-PAGE. This conclusion is supported by the finding that the appearance of the M, 103,000 band was clearly dependent on the concentration of the cross-linker used (data not shown). The relative proportions of the M, 130,000 and 103,000 bands were interdependent. The intensity of the M, 103,000 band decreased with increasing concentrations of the cross-linker, but increased under reducing conditions. Both the M, 130,000 and the 103,000 band seemed to migrate slightly more slowly under reducing conditions, 1

2

3

12345678

Mr - 205K - 116K - 97.4 K - 68K - 45K

unlabeled

hCG

reduction

-

-

-

+

-

-

-

+

-

-

-

-

+

+

+

+

Fig. 2. SDS-PAGE and autoradiography of the solubilized LH/CG receptor- 1251-hCG complex. Crude ovarian membrane particles were incubated with ‘251-hCG in the absence or presence of an excess of unlabeled hCG and solubilized with Triton X-100. The solubilized LH/CG receptor-‘251-hCG complex was treated with either 2 mM GA (lanes 1, 4, 5, 8) for 60 mm at 20 o C or 1 mM DSS (lanes 2,6) or 1 mM DSP (lanes 3,7) for 15 min at 0 o C. Samples were analyzed by SDS-PAGE (7.5% slab gel) under non-reducing or reducing conditions. Numbers on the right denote the molecular weight markers (from the top: myosin, P-galactosidase, phosphorylase h, BSA and ovalbumin).

and their relative mobilities were 137,000 and 106,000. This change in migration can be explained by opening of the internal disulfide bonds and straightening of the molecules (Griffith, 1972). SDS-PAGE of the solubilized receptor- 3H-hCG complex under non-reducing conditions again gave the M, 130,000 band on the autoradiograms in 5

6

7

8

W - 180K - 116K -84~ -58K - 48.5 K

unlabeled hCG reduction Fig. 3. SDS-PAGE and autoradiography LH/CG receptor-3H-hCG complex were the molecular weight markers (from the fumarase). Note that both

-

-

-

-

-

-

of the solubilized LH/CG receptor-3H-hCG complex. Samples containing the solubilized prepared and analyzed by SDS-PAGE as described for Fig. 2. Numbers on the right denote top: a,-macroglobulin, P-galactosidase, fructose-6-phosphate kinase, pyruvate kinase and the a-subunit and the P-subunit of hCG migrate at the dye front in 7.5% gel.

49

addition to the a,&dimer and the CX-and P-subunits of hCG (Fig. 3). Thus, the M, 130,000 band must contain one hormone molecule bound to one M, 90,000 receptor molecule (see Fig. 6). Under reducing conditions the intensity of the M, 137,000 band decreased and a faint M, 106,000 band was occasionally seen, representing the a-subunit of hCG bound to the receptor. No band in the range llO,OOO-120,000, corresponding to the receptor(j3)hCG complex was detected, however, suggesting that hCG can be cross-linked to its receptor mainly through the a-subunit. The non-reduced q/3-dimer of hCG which dissociated from the receptor migrated faster in SDS-PAGE (M, 47,000; Figs. 2 and 3) than the non-reduced free hormone molecule (M, 49,000; Fig. 1). This difference in electrophoretic mobility was clearly dependent on the cross-linker concentration used, and the change was gradual as the concentration increased (data not shown). The change was detected at lower concentrations with DSS and DSP than with GA. No difference in electrophoretic mobility was detected under reducing conditions, however, and the dissociated and free hCG had the same M, of 49,000 (Figs. 1, 2 and 3) regardless of the cross-linking conditions used. These differences in the electrophoretic mobility of the hormone molecule can be explained by differences in internal cross-linking (see Peters and Richards, 1977) which may indicate that the

1

2

3

4

- 205K - 116K

- 97.4K - 66K - 45K

reduction

-

-

+

+

Fig. 4. Covalent cross-linking of the solubilized and the memcomplex. The solubibrane-bound LH/CG receptor- “‘1-hCG lized (lanes 1, 3) and membrane-bound (lanes 2, 4) LH/CG receptor- ‘251-hCG complexes were prepared as indicated in Materials and Methods. The complexes were treated with 2 mM GA for 60 min at 20°C and analyzed by SDS-PAGE (7.5% slab gel) under non-reducing or reducing conditions.

conformation of the bound hCG differs from that of the free hormone. When the hormone was cross-linked to the membrane-bound receptor, the M, of the receptor-hormone complex was approximately 7000 higher than that of the solubilized complex (Fig. 4). This difference was clear when the crosslinked complexes were analyzed side by side on the same gel.

5

6

7

8

9

4 -180 K -116 K -84~ -58~

-48.5 -

reduction

-

-

-

-

-

.,

+

+

+

K

-

Fig. 5. SDS-PAGE and autoradiogra hy of the immunoaffinity-purified 3H-LH/CG receptor-hCG complex. Crude ovarian P membrane particles were labeled with H and incubated with hCG. The ‘H-LH/CG receptor-hCG complex was solubilized with Triton X-100, treated with 2 mM GA (lanes 2, 5) for 60 min at 20° C or 1 mM DSS (lanes 3, 6) or 1 mM DSP (lanes 4, 7) for 15 min at 0 o C and purified by inununoaffinity chromatography. The purified complex was analyzed by SDS-PAGE (7.5% slab gel) under non-reducing or reducing conditions. In the control experiments, no unlabeled hCG was attached to the membranes (lane S), the ‘H-LH/CG receptor-hCG corn lex treated wih 2 mM GA was purified in the presence of an excess of unlabeled hCG (lane 9) or the P H-LH/CG receptor was purified by affinity chromatography (lane 1).

50

Cross-linking of the unlabeled hCG to the “H-LH/ CG receptor In order to eliminate the possibility that the labeling of hCG may somehow influence its interaction with its receptor, another approach was used in which unlabeled hCG was cross-linked to the 3H-LH/CG receptor. Ovarian membranes were labeled by the periodate-Na3BH, method, which introduces the label into the receptor molecule (Metsikko, 1984). Unlabeled hCG was attached to the 3H-labeled membranes and the 3Hreceptor-hCG complex was solubilized, cross-linked with GA, DSS or DSP and purified by immunoaffinity chromatography. SDS-PAGE of the 3H-receptor-hCG complex under non-reducing conditions showed two labeled bands of iV, 130,000 and 90,000 on the autoradiograms (Fig. 5). The smaller band must represent the LH/CG receptor, as it was also observed when the purified 3H-LH/CG receptor was analyzed in the same way. The larger labeled band clearly represents the 3H-receptor-hCG complex. No labeled bands were detected if hCG was not attached to the membranes or if the immunoaffinity purification of the complex was performed in the presence of an excess of hCG. The cross-linking efficiency of GA was somewhat lower than that of DSS and DSP, because the J4, 90,000 band was more intense with GA. A faint band of M, 106,000 appeared upon reduction of the GA-cross-linked complex, representing the receptor-( a)hCG complex. As not all of the DSP-cross-links broke down under reducing conditions, the 106,000 receptor-( cu)hCG complex was occasionally detected with this cross-linker. Detection of the receptor-(a,P)hCG complex by immunoblotting Receptor-hormone interaction was further elucidated using an approach in which polyclonal antibodies were employed to detect the receptorhormone complex in immunoblotting. The non-reduced LH/CG receptor-hCG complex that was cross-linked with GA was transferred to a nitrocellulose sheet, and the sheet incubated with the appropriate antiserum. All of the polyclonal antibodies used - one directed against the purified LH/CG receptor, one against the a-subunit of hCG and one against the /?-subunit of hCG -

were able to detect the M, 130,000 LH/CG receptor-hCG complex (Fig. 6, panel A). As the crosslinking was not complete, the receptor antibodies A 1

2

3

4

5

6

Mr - 205K -116K - 97.4K

:_

I

-66K

:*:

-45K

I AM-1

1

I

antianti(a)-hCG (B)-hCG

B 123456 (a,B)-hCG -

Mr

8 :.(

..‘.

-45K -36K

(l3)-hCG -

:

(a)-hCG -

- 20.1 K anti(a)-hCG

reduction

:R !I

-

-

anti(D)-hCG +

-

-

+

Fig. 6. Panel A: Identification of the LH/CG receptor-hCG complex by immunoblotting. Crude ovarian membrane particles were incubated with unlabeled hCG and solubilized with Triton X-100. The solubilized LH/CG receptor-hCG complex was treated with 2 mM GA for 60 min at 20 o C and subjected to SDS-PAGE (7.5% slab gel) under non-reducing conditions and electroblotted onto nitrocellulose. The nitrocellulose strips were incubated with antibodies to the LH/CG receptor (lane 2; final dilution 1: 300), the ol-subunit of hCG (lane 4: final dilution 1: 200) or the P-subunit of hCG (lane 6; final dilution 1: 500). For control purposes, Triton X-100 extract of the ovarian membranes was analyzed using LH/CG receptor antibodies (lane l), and hCG treated with 2 mM GA was analyzed with (ol)hCG (lane 3) or (/3)hCG (lane 5) antibodies. Panel B: Specificity of the (ol)hCG and (B)hCG antibodies. hCG was treated with 0 mM (lanes 1, 4) or 2 mM (lanes 2, 3, 5, 6) GA at 20 o C for 60 min and subjected to SDS-PAGE (10% slab gel) under non-reducing or reducing conditions and electroblotted onto nitrocellulose. The nitrocellulose strips were incubated with antibodies directed against either the o-subunit (lanes 1-3) or the P-subunit (lanes 4-6) of hCG.

51

also detected the dissociated receptor and the (Yand /3-subunit antibodies the dissociated hormone molecule. The specificity of the LY-and P-subunitdirected antibodies was confirmed, as the former detected only the free a-subunit and the latter the free /3-subunit when the non-cross-linked hormone was analyzed by SDS-PAGE and immunoblotting (Fig. 6, panel B). Unfortunately, the antibodies achieved poor detection of the reduced hormone (P-subunit antibody) or none at all (a-subunit antibody) (Fig. 6, panel B), so they could not be used to detect the receptor-(a)hCG and receptor( p)hCG complexes. Similarly, the LH/CG receptor antibodies were poor at detecting the reduced receptor (data not shown). Discussion The results of the present study suggest that the rat ovarian LH/CG receptor binds one hCG molecule and that the hormone interacts with the receptor mainly through its a-subunit. This is demonstrated by two lines of evidence. Firstly, only the a-subunit could be cross-linked to the ovarian receptor when either the cY-subunit-labeled ‘251-hCG or the /3-subunit-labeled 3H-hCG was used to label the receptor. The receptor-(a)hCG complex was detected even with H-hCG, although the a-subunit contains only about 30% of the label in this hormone preparation. No receptor-( j3)hCG complex was found even though three different cross-linkers were used. Secondly, analysis of the complexes formed after attaching unlabeled hCG to the 3H-receptor by SDS-PAGE revealed only receptor-( a,p)hCG and receptor(a)hCG complexes. Although the a-subunit of hCG seems to play a more important role in receptor-hormone interaction, it is exposed on the receptor-hormone complex and not buried under the P-subunit. This is supported by findings showing that antibodies directed against both the (Ysubunit and the P-subunit were able to detect the receptor-(a,P)hCG complex in immunoblotting. It can be argued that the receptor-P(hCG) complex cannot be detected by a cross-linking approach, because either the P-subunit or the receptor may not contain suitable amino acids for cross-linking close enough to the receptorhormone interface. This possibility was taken into

account, and to obtain cross-linkages of various lengths three cross-linkers were used. GA was particularly useful, as it has been claimed that there is a large number of different polymeric forms simultaneously in the GA solution (see Han et al., 1984). On the other hand, it is very likely that there are suitable functional groups for crosslinkers close to the receptor-hormone interface. The P-subunit of hCG contains four lysine residues (see Pierce and Parsons, 1981) and can readily be cross-linked to the a-subunit (Fig. 1). In addition, the extracellular domain of the rat LH/CG receptor, which is claimed to possess the ligand binding site, contains 17 lysine residues (McFarland et al., 1989). The finding that only the a-subunit of hCG can be cross-linked to its receptor is in good accordance with a recent report by Kusuda and Dufau (1986). In contrast, Ji and Ji (1981) Hwang and Menon (1984), Ascoli and Segaloff (1986) and Roche and Rayan (1989) have reported that both of the subunits can be cross-linked to the receptor. One explanation for the first two disparate reports is that the authors interpret the LH/CG receptor to be an oligomeric complex which causes difficulties in distinguishing the receptor-(o)hCG and receptor-(p)hCG complexes. The reason is more difficult to ascertain in the case of results obtained by Ascoli and Segaloff (1986) and Roche and Rayan (1989). It must be noticed, however, that these authors prepared the P-subunit-labeled hCG by labeling the free P-subunit with radioiodine and combining it with the unlabeled a-subunit, and it is unlikely that the combined hormone was conformationally intact, as the P-subunit is labeled only poorly when the intact hormone is iodinated (Markkanen et al., 1980). The results obtained using polyclonal antibodies directed against the 01- and P-subunits of hCG were somewhat suprising, as both of them were able to detect the receptor-(a,P)hCG complex in immunoblotting. Several other laboratories have shown previously that almost all immunoreactive sites on the a-subunit are blocked upon binding of the hormone to the receptor but only a few on the P-subunit (Moyle et al., 1982; Millius et al., 1983; Bidart et al., 1987a, b). The results of these experiments can be regarded as tentative, because only two commercially available polyclonal antibodies

were used. One reason for the different results may be that the methods used here deviate somewhat from those in previous experiments. Moyle et al. (1982) and Bidart et al. (1987a, b) measured the ability of radiolabeled antibodies to bind to the membrane-bound receptor-hCG complex, and Milius et al. (1983) precipitated the solubilized receptor-hCG complex by a double antibody technique. The close contact between the a-subunit of hCG and the receptor is somewhat perplexing in view of the fact that the P-subunit confers the hormone specificity. It has been shown, however, that the conformation of the cx-subunit changes upon association with the P-subunit (Bewley et al., 1974; Strickland and Puett, 1982; Hojo and Ryan, 1985; Endo et al., 1989). Thus, it is possible that the a-subunit gains such a conformation that it may participate in the formation of the specific high affinity binding. The notion that conformational changes resulting from dimer formation are necessary for high affinity receptor-hormone interaction is also supported by findings showing that it is only at very high concentrations that the free subunits of the glycoprotein hormones (Moudgal and Li, 1982; Charlesworth et al., 1987) and the synthetic peptides corresponding to their internal amino acid sequences (Charlesworth et al., 1987; Keutmann et al., 1987, 1988, 1989; Morris et al., 1988) can compete with the intact hormone molecules for binding to the receptor. The close association of the a-subunit of hCG with the receptor can also be explained by means of the two-step binding model proposed by Milius et al. (1983) for hCG and analogously for all glycoprotein hormones. According to this model, the P-subunit of hCG first recognizes the receptor, forming an initial complex. Then, through a structural transformation in either the receptor or the hormone molecule, a stable complex is formed between the a-subunit and the receptor. The results of the present study support the notion that structural changes occur in the hCG molecule upon binding to the receptor, as the free hCG had a different electrophoretic mobility than the bound, dissociated hormone. Analogously Moyle et al. (1987) detected changes in the availability of antigenic epitopes when hCG was bound to the receptor. The two-step binding model is also sup-

ported by kinetic evidence showing that binding of hCG to testicular membranes changes from ‘loose’ to ‘tight’ with time (Katikineni et al., 1980). Our results also show that the receptor-hCG complex had a molecular weight about 7000 hihger when the complex was cross-linked in the membranes prior to solubilization. This size difference is interesting and has many possible explanations. Firstly, either the receptor or the hormone may have a different conformation when the complex is associated with the plasma membrane. Secondly, some of the available cross-linking sites may be hidden when the cross-linking takes place in the membranes, rendering the complex less compact. Thirdly, the receptor may be covalently modified, such as phosphorylated, in the plasma membrane, and it may remain modified after solubilization, because cross-linking of the membranes may inhibit the protein phosphatases. Phosphorylation of the /3-adrenergic receptor has been found to retard the migration of the receptor molecule slightly in electrophoresis (Stadel et al., 1983). In conclusion, evidence is presented that the rat ovarian LH/CG receptor binds one hormone molecule and that the a-subunit of the hormone plays a significant role in the formation of the high affinity stable complex. The larger size of the membrane-bound receptor-hCG complex may indicate that the receptor is covalently modified in the plasma membrane or has a different conformation than the solubilized receptor. Acknowledgements The skillful technical assistance of Ms. Paula Soininen, Ms. Aino Kuha, Mr. Eero Oja and Ms. Sirkka Martti is gratefully acknowledged. References Alonso-Whipple, C., Couet. M.L., Doss, R.. Koziarz. J.. Ogunro, E.A. and Crowley, Jr., W.F. (1988) Endocrinology 123, 1854-1860. Ascoli, M. and Segaloff, D.L. (1986) J. Biol. Chem. 261. 3807-3815. Bewley, T.A., Sairam, M.R. and Li, C.H. (1974) Arch. Biothem. Biophys. 163, 6255633. Bidart, J.-M., Troalen, F.. Bohuon, C.J., Hennen. G. and Bellet. D.H. (1987a) J. Biol. Chem. 262. 1548315489.

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