Purification and Partial Characterization ofRat Ovarian Lutropin ...

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc

VOl. 262, No. 16, Issue of June 5 , pp. 7920-7926 1987 Printed in i r S . A .

Purification and Partial Characterization of Rat Ovarian Lutropin Receptor* (Received for publication, January 6, 1987)

Kari P. KeinanenS, Sakari Kellokumpu, M. Kalervo Metsikko, and Hannu J. Rajaniemi From the Biocenter and Department of Anatomy, University of Oulu,Kajaanintie 52 A, SF-90220Oulu,Finland

Lutropin (LH) receptor was solubilized from pseudopregnant rat ovaries and purified by two cycles of affinity chromatography on human choriogonadotropin (hCG)-Affi-Gel 10. The purified receptor preparation contained a single class of high-affinity "'1hCG binding sites with an equilibrium dissociation constant (&) of 5.1 X 10"' M (at 20 "C) and hada specific hormone binding capacityof 7920 pmol/mg of protein. The purified receptor migrated as a single 90-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both nonreducing and reducing conditions. Affinity cross-linking of the purifiedreceptor to 12'I-hCG produced a 130-kDa complex. Hormone-binding ability of the purified 90-kDapolypeptide was demonstrated also by ligand blotting. The purified receptor was electroblotted nitrocellulose onto after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducingconditions followed by incubation with "'I-hCG. Autoradiography revealed labeling of a BO-kDa band. This labeling was displaced by unlabeled hCG and human LH but not by human follitropin or rat prolactin. In addition, LH receptors of bovine corpora lutea andmouse Leydig tumor cells were shown by ligand blotting to contain a 90-kDa hormone binding unit, suggesting that LH receptor structure is well conserved among mammalian species. The purified rat ovarian LH receptor bound to immobilized wheat germ agglutinin, implying that the receptor is a glycoprotein. These results demonstratethat the hormone-binding unit of rat ovarian LH receptor is a 90-kDa membraneglycopolypeptide.

of the components involved with the LH receptor in the first place is needed. In spite of numerous studies using photoaffinity labeling (6-8) or chemical cross-linking techniques(9E ) , no general agreement about the number and size of LH receptor subunits has emerged. The LH receptor is a transmembrane protein (13), which can be solubilized with nonionic detergents, although lability (14) and susceptibility to proteolysis (15) of the solubilized receptor have formed a major obstacle for successful purification of the receptor. Several reports on the purification of the lutropin receptor from various sources have, however, appeared (16-21), but the results are ambiguous regarding the composition and size of the receptor polypeptide(s). Thesereports havelargely lacked firm evidence about theability of the purified polypeptides to specifically bind hormone, thus making it difficult to assess whether the bands seen on stained gels represent real receptor units or contaminatingproteins. This laboratory has previously reported purification of rat ovarian LH receptor as a90-kDa polypeptide, but the purified preparation did not have hormone binding activity (17). In the present study, we have further developed the purification procedure to allow purification of rat ovarian LH receptor to near homogeneity with quantitativeretention of hormone binding activity. The purified receptor migrates as a single 90-kDa band in SDS-PAGE. Furthermore, we demonstrate by a recently developed ligand blotting technique (22) that the purified 90-kDa polypeptide binds hormone with the same specificity as the membrane-bound LH receptor. Thus, our results provide direct evidence that the LH/hCG binding activity resides in a 90-kDa polypeptide. EXPERIMENTALPROCEDURES

The action of the glycoprotein hormones lutropin (LH)' and human choriogonadotropin (hCG) is initiatedby binding to specific high-affinity receptors on the target cell plasma membrane (1).The receptor transmits thehormonal signal to the adenylate cyclase of the plasma membrane by activating a regulatory G.-protein (2, 3). There is recent evidence, however, that second messenger(s) other than cyclic AMP may be involved in the action of LH (4,5).In order to understand the action of LH (and hCG) in molecular terms, purification

* This study was supported by grants from The Academy of Finland, The Cultural Foundation of Finland, and,The Aaltonen Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. The abbreviations used are: LH, lutropin; h, human, CG, choriogonadotropin; FSH, follitropin; TSH, thyrotropin; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; WGA, wheat germ agglutinin.

Materials-Highly purified hCG(12,100 IU/mg) was purchased from Diosynth, Oss, The Netherlands. Human lutropin (hLH), rat prolactin, and human follitropin (hFSH) were supplied by the National Pituitary Agency, Bethesda, MD. Na'T (carrier free)was from Amersham Corp. Human CG was radioiodinated by the chloramineT method (23, 24). The specific activity of the hormone was 40-70 Ci/g as determined by self-displacement assay (25). The biological activity of the radiolabeled hormone (determined as the fraction of radioactivity capable of binding to an excess of membrane-bound receptors) averaged 50%. BSA, hemoglobin, N-ethylmaleimide, PMSF, and WGA were from Sigma. Gelatin was an E. Merck product. WGA was covalently coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia P-L Biochemicals) according to manufacturer's instructions. Acrylamide, Affi-Gel 10, nitrocellulose membrane, and SDS were supplied by Bio-Rad. All other reagents were of reagent grade and purchased from commercial suppliers. Twenty-seven-dayold Wistar rats were rendered pseudopregnant by sequential treatments with pregnant mare serum gonadotropin and hCG as previously described (26). The animals were killed on day 7-9 after the hCG injection, and theovaries were collected, freed from extraneous tissue, frozen in liquid nitrogen, and stored at -80 "C until use. Solubilization of the Receptor-The receptor was purified from batches of 50-100 ovaries. The ovaries were homogenized in PBS (10 mM sodium phosphate, pH 7.4, 140 mM NaCI), 5 mM EDTA, 5 mM

7920

Ovarian

Rat

N-ethylmaleimide, 0.2 mM PMSF (buffer A, 1 m1/100 mg of tissue) in a motor-driven all-glass homogenizer on ice with 100 strokes. The homogenate was centrifuged for 30 min a t 27,000 X g. The resulting membrane pellet was lightly homogenized in buffer A containing 1% (v/v) Triton X-100 and 20% (v/v) glycerol (0.5 m1/100 mg of tissue) and stirred for 30 min on ice. Thereafter the suspension was centrifuged at 100,000 X g for 1 h. The supernatant (solubilized receptor) was diluted to a final detergent concentration of 0.5% Triton X-100 with buffer Acontaining 20%glycerol and used for the affinity purification. Affinity Chromatography Procedures-The preparation of the hCG-Affi-Gel 10 matrix has been described in detail (17). For the affinity purification of the LHreceptor, 0.1 volume of hCG-Affi-Gel 10 gelwas added to solubilized receptor, and the suspension was rotated end-over-end a t 4°C for 16 h. Thereafter the gel was packed into a column (100-ml plastic syringe sealed with nylon mesh), and the flow-through was collected. The gel was subjected to three consecutive washes. The first wash consisted of 50 volumes of buffer A containing 0.5% Triton X-100 and 20% glycerol. The second wash was 50 volumes of buffer A containing 0.1% Triton x-100, 0.5 M NaC1, and 20% glycerol, and thefinal wash was 100 volumes of buffer A containing 0.1% Triton X-100 and 20% glycerol. The receptor was eluted in 2 volumes of 0.1 M sodium acetate, pH 4.0, containing 0.1% Triton X-100 and 20% glycerol. The acid eluate was neutralized immediately by adding 1 M NaOH dropwise. The purified receptor was stored frozen a t -80 "C with no decrease in binding activity during a period of several months. A control purification was performed as described above but in the presence of 0.5 p~ hCG in the affinity chromatography to distinguish between specific interaction with the immobilized hormone and nonspecific adsorption on AffiGel 10 matrix. The first affinity eluate was purified further by a second affinity chromatography step. To 10 ml of pH 4 eluate, 500 p l of hCG-AffiGel 10 was added. The suspension was rotated end-over-end overnight at 4 "C and packed into a column. The gel waswashed with the same relative volumes of buffers as described above, and the receptor was eluted in 1 ml of 0.1 M sodium acetate, pH 4.0, containing 0.1% Triton X-100 and 20% glycerol.The eluate was neutralized as before and characterized further by SDS-PAGE and ligand blotting. Iodination of the Affinity-purified Receptor-The affinity-purified receptor was radioiodinated by the chloramine-?' method (23). Briefly, to 75 ng of purified receptor in 40 p1 of PBS containing 0.1% Triton X-100,lO pl of 0.5 M sodium phosphate, pH7.5,140 mM NaCl, and 5 p l (0.5 mCi) of NalZ51were added followed by 10 p l of chloramine-T solution (5 mg/ml). After 1 min of incubation a t room temperature with constant shaking, 100 p l of sodium metabisulfite solution (1.2mg/ml in PBS) was added. The radioiodinated receptor was separated from free lZ5Iby gel filtration on Sephadex G-50 using PBS containing 0.1% Triton X-100 as eluent. Over 90% of the radioactivity eluting in the void volume was precipitatable by 10% trichloroacetic acid, the rest being probably iodinated lipid and free iodine. The radioiodinated receptor was analyzed electrophoretically on 7.5% SDS-polyacrylamide gels under reducing conditions. The gels were stained with Coomassie Blue, destained, dried, and autoradiographed on Kodak XAR-5 film for 24 h. SDS-PAGE and Silver Staining-SDS-PAGE was performed according to Laemmli (27), using 7.5% acrylamide monomer in the separating gel and 5% acrylamide in the stacking gel. The samples were dialyzed against dilute SDS-sample buffer for 5-6 h at room temperature and concentrated by vacuum centrifugation in a Savant Speedvac apparatus. The samples were incubated for 20 min at 80 "C in the presence or absence of 100 mM dithiothreitol. The gels were silver-stained according to Merrill et al. (28). The mass standards (Sigma) were: myosin heavy chain, 205 kDa; &galactosidase, 116 kDa; phosphorylase b, 94 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; and carbonic anhydrase, 29 kDa. Determination ofBinding Parameters-The hormone binding characteristics of the membrane-bound receptor (homogenate and 27,000 X g pellet) were determined by measuring the binding of a constant amount of "'I-hCG (1-2.7 ng) to ovarian membranes in the presence of increasing amounts of unlabeled hCG (1-100 ng). Membrane samples (20-40 p g of protein) were incubated with the hormone in a final volume of 250 p l of buffer A containing 0.1% BSA for 16 h at room temperature. Thereafter, 1 ml of ice-cold PBS containing 0.1% BSA was added to each tube, and the tubes were centrifuged for 15 min at 13,000 X gin an Eppendorf centrifuge. The pellets were washed twice by resuspending in PBS andcentrifuged as above. The '*'I radioactivity in the washed pellets was counted in an automatic

LH Receptor

7921

y counter (LKB Wallac Ultrogamma). Nonspecific binding was measured in the presence of 2 pg of unlabeled hCG and was subtracted from all tubes. The maximal hormone binding capacity of the receptor preparation was deduced from Scatchard plots (29) of the binding data. The assays were done in quadruplicate. The binding assay for solubilized receptor and purified receptor was performed as follows. Triton extract (10-20pgof protein) or purified receptor (10-20 ng of protein) was incubated in PBS containing 0.1% Triton X-100 and 0.1% BSA with a constant amountof lz5I-hCGand increasing amounts of unlabeled hormone as described above for the membrane-bound receptor. The assay volume was 250 pl. The receptor-bound and free hormone were separated by polyethylene glycol precipitation as described previously (30). Nonspecific binding was measured in the presence of 2 pg of hCG/assay and was generally 1%of thetotal counts added. The binding data were transformed into a Scatchard plot (29) to obtain the equilibrium dissociation constant ( K d ) and maximal binding capacity. All assays were done in quadruplicate. Ligand Blotting-The receptor preparations were subjected to ligand blotting following a procedure previously described (22) with minor modifications. The purified receptor (50 ng) was subjected to SDS-polyacrylamide gel electrophoresis under nonreducing conditions as described above but without prior heating. The protein was transferred electrophoretically from the gel to a nitrocellulose sheet in aBio-Rad Trans-Blot apparatus placed in an ice bath. The transfer was performed using a constant current of 0.1 A for 16-18 h. The nitrocellulose sheet was rinsed in PBS, and the lane containing the molecular weight standards was stained with Amido Black. The sample lanes were incubated in PBS containing 1% hemoglobin, 0.25% gelatin, 0.1% BSA, and 10% glycerol/PBS for 4-6 h at room temperature. The nitrocellulose strips were rinsed briefly in PBS and incubated overnight in 10 mM Tris-HC1, pH 7.4, containing 1%BSA, 10%glycerol, and 200,000 cpm/ml lz5I-hCGin the absence or presence of 4 pg/ml unlabeled hCG, hLH, hFSH, or rat prolactin as indicated in the figure legends. The strips were washed 3-5 times in PBS containing 0.1% Triton X-100 (10-15 min/wash), rinsed in PBS, and dried. The dried strips were subjected to autoradiography on Kodak XAR-5 film for 3-7 days a t -80 "C. Covalent Cross-linking of the Purified Receptor to '25Z-hCG-The receptor preparation was dialyzed against PBS containing 0.1% Triton X-100. Dialyzed receptor (100 p l ) was incubated with 200,000 cpm of '"I-hCG in the absence or presence of 5 pg of unlabeled hCG overnight at room temperature in atotal volume of 120 p l . Thereafter, the hormone-receptor complexes were covalently cross-linked by adding 5 pl of 0.1 M glutaraldehyde to the incubation mixture, and the incubation was continued for 1 h at room temperature (17). The cross-linking was stopped by adding 1 M Tris-HC1, pH 7.5, to a final concentration of 50 mM. The cross-linked products were analyzed by SDS-PAGE on 7.5% acrylamide gels under nonreducing conditions followed by autoradiography. Lectin Affinity Chromatography of the Purified Receptor-The pH 4 eluate was dialyzed against 50 mM Tris-HC1, pH 7.4, containing 0.1% Trixon X-100 and 20% glycerol (buffer B) for 4-5 h a t room temperature. To 4.2 ml of dialyzed receptor preparation, 1ml of moist WGA-Sepharose 4B was added, and thesuspension was rotated endover-end for 16 h, at4 "C. The gel waspacked into a column, and the flow-through was collected. The gel was washed with 50 ml of buffer B, and the receptor was eluted in 3 ml of 0.3 M N-acetylglucosamine in buffer B. The hormone binding activity of the dialyzed receptor, flow-through, and the N-acetylglucosamine eluate were analyzed by incubating 10p l of each solution with 250,000 cpm of lZ5I-hCGin 250 pl of PBS containing 0.1% BSA for 16 h at room temperature. The bound and free hormones were separated by polyethylene glycol precipitation as described above. All assays were performed in duplicate. The nonspecific binding was determined in the presence of 2 pg of unlabeled hCG and was subtracted from the total counts/min to get the specific hormone binding. Comparison of Lutropin Receptors from Different Species by Ligand Blotting-Corpora lutea were excised from bovine ovaries obtained from a local slaughterhouse, frozen in liquid nitrogen, and stored at -80 "C until use. The corpora lutea werehomogenized, and the membrane pellet was prepared and solubilized as described above for the rat ovarian receptor. Mouse Leydig tumor cells (MLTC-1 cells) were grown as described previously (31). Cells from ten 10-cm plates were homogenized,and themembrane-bound receptor was solubilized as described above for the rat LHreceptor. The Triton extracts were incubated overnight with 0.1 volumeof WGA-Sepharose 4B. The gels were packed into columns and washed with 20 volumes of buffer A

Rat Ovarian LH Receptor

7922

containing 0.1% Triton X-100 and 20% glycerol. The glycoproteins were eluted with 0.3 M N-acetylglucosamine in the same buffer. The eluates were dialyzed overnight against Laemmli’s SDS sample buffer (without mercaptoethanol) containing 30% glycerol and subjected to SDS-PAGE and ligand blotting as described above. Miscellaneous-The protein content of the homogenate and membrane preparations was measured by the method of Lowry et at. (32). The protein concentration of the solubilized and purified receptor was determined by the Amido Schwarz binding assay of Schaffner and Weissman (33). Bovine serum albumin was used as a standard in both assays. The results are representative of experiments that were performed 3-6 times with essentially identical results.

40% of this was recovered in the pH4 eluate. The final yield of binding activity averaged 15% of the activity present in the original Triton X-100 extract. The specific activity of the purified receptor was 7920 f 432 pmol/mg protein ( n = 3). This is over 70%of the theoretical maximum, which is 11,100 pmol/mg for a 90-kDa receptor subunit capable of binding one molecule of hCG. Scatchard plots of the binding data reveal a single class of high-affinity binding sites in the Triton X-100 extract (not shown) and in thepurified receptor (Fig. 1).The equilibrium dissociation constants (& at 20 “C) were 5.38 f 3.14 X 10”’ M ( n = 4) and 5.05 f 2.57 X 10”’ M ( n = 3) for the solubilized RESULTS receptor and for the purified receptor, respectively. These Purification of LH Receptor-Rat ovarian LH receptor was data reveal that no notable changes took place in the hormone purified by a procedure consisting of two successive affinity binding characteristics of the receptor in the course of the chromatography steps on hCG-Affi-Gel 10. Table I summa- purification. Characterization of thePurified Receptor-The low pH rizes results from a representative purification from 100 ovaries. The hCG binding activity present inthe pseudopregnant eluates of the first and second affinity chromatography were rat ovarian homogenate was nearly quantitatively (85-95%) analyzed by SDS-PAGE under reducing conditions (Fig. 2). recovered in the 27,000 x g membrane pellet. In preliminary Silver-stained gels revealed only one major band of 90 kDa in experiments, the presence of glycerol and proteinase inhibi- the low pH eluates of the first and second affinity steps (Fig. tors was found necessary for retention of hormone binding 2, c and e). The 90-kDa band was displaced by the presence activity in the solubilization and further purification steps. of an excess of soluble hCG in the first affinity chromatogThe membrane-bound LH receptor was solubilized in 1% raphy (Fig. 2d). The pH 4 eluates of the first and second Triton X-100 in the presence of 20% glycerol, 5 mM EDTA, affinity chromatographies, however, produced additional faint 5 mM N-ethylmaleimide, and 0.2 mM PMSF with a yield of bands corresponding to molecular masses of 58,55,43,42,35, 90-140%of the binding activity present in the membrane and 33 kDa. The relative intensities of these additional bands fraction. The yields exceeding 100% in thesolubilization step varied greatly between different batches of purified receptor, could be due to the presence of receptor-containing inverted and they were also observable in the high-salt wash of the membrane vesicles in the membrane preparation (13), thus affinity gel (Fig. 2b). This makes it unlikely that thefaint 3060-kDa bandsrepresentLH receptor units. The relative giving an underestimation of the particulate receptor. Overnight incubation of the solubilized receptor prepara- amount of the additional bands in the size range of30-60 tion with hCG-Affi-Gel 10 removed85-87% of the hCG kDa was reduced in the pH 4 eluate of the second affinity binding activity. The affinity matrix was washed extensively, chromatography with a concomitant increase in the amount and the pooled wash solutions contained less than 1%of the of the 90-kDa polypeptide and hCG binding activity (Fig. 2e). binding activity present in the detergent extract.LH receptor SDS-PAGE of the iodinated receptor followed by autoradiogwas eluted from the affinity gel by 0.1 M sodium acetate, pH raphy revealed a major band at 90 kDa (Fig. 2g),demonstrat4. The eluate contained routinely 40-60%of the binding ing the high purity of the receptor preparation. SDS-PAGE activity present in the original Triton extract. Whenan excess under nonreducing conditions revealed a broad 90-kDa band of soluble hCG was added to thehCG-Affi-Gel 10 incubation indicating that the 90-kDa species is not produced by reducmixture, only 0.5% of the original binding activity was re- tion from a larger aggregate (Fig. 2f).Thesedatataken covered in the pH 4 eluate. The specific hormone binding together with the high specific activity (7920 pmol/mg) sugactivities of the Triton extract and the pH 4 eluatewere 3.61 gest that the 90-kDa band represents the rat ovarian LH f 1.25 pmol/mg and 5190 f 1356 pmol/mg (n = 5), respec- receptor. The ligand binding ability of the purified 90-kDa polypeptively. The first pH 4 eluate was subjected to a second affinity tide was assessed by affinity cross-linking of radioiodinated chromatography on hCG-Affi-Gel 10. From 65 to 85% of the hCG to the purified receptor. The purified receptor was inbinding activity bound to the affinity matrix, and from 15 to cubated with “‘1-hCG in the presence or absence of unlabeled TABLEI Purification of rat ovarian lutropin receptor Data are from a representative purification from 100 ovaries (7.23 g of tissue). The ovaries were homogenized, and the LH receptor was purified by affinity chromatography on hCG-AffiGel 10 as described under“Experimental Procedures.” ‘%hCG binding Preparation

Proteinb mg

Activity

Specific activity

pmol

pmllmg

Affinity

Yield (Kd)

M (X 10“’)

Purification factor

%

1 100 ND’ 1.32 1000 758 Homogenate 2.05 ND 91.7 917 2.71 338 Membranes 3.68 3.1 109.8 1098 4.86 226 Triton X-100 extract 3143 ND 45.6 456 4150 0.109 1st affinity eluate 5742 4.0 13.7 7580 137 0.018 2nd affinity eluate As determined by saturation analysis of Iz5I-hCGbinding to receptor preparations. As measured by the method of Lowry et al. (32) in the homogenate and membranes and by the method of Schaffner and Weissman (33) in the soluble fractions. e ND, not determined.

7923

Rat Ovarian LH Receptor

a 205-

FIG.1. Scatchard plot and saturation isotherm for specific binding of ”“I-hCG to affinity-purifiedLH receptor. Purified receptor was incubated with a constant amount of T - h C G and increasing amounts of unlabeled hCG as described under “Experimental Procedures.” The data presented are from a representative experiment where each point is the average of triplicate determinations. B,, maximal hormone binding capacity; K d , equilibrium dissociation constant; r, correlation factor of linear regression. hCG, and the hormone-receptor complexes were covalently cross-linked with glutaraldehyde using a protocol developed inthis laboratory (17). The cross-linked complexeswere subjected to SDS-PAGE under nonreducing conditions. Autoradiography of the gels revealed radioactive bands corresponding to molecular sizes of 130, 51, and 36 kDa and one migrating near the dye front (Fig. 3a). Comparison to bands produced by radioiodinated hCG alone under similar conditions reveals that the 51-kDa band is produced by crosslinked hCG and that the 36-kDa band and the radioactivity migrating in the dye front correspond to the hCG-P and hCGa subunits, respectively (Fig.3~). The 130-kDa band was specifically displaced by the presence of an excess of unlabeled hCG in the hormone incubation step (Fig. 36). The faint 94kDa band present in lanes b and c (Fig. 3) is probably due to cross-linked hCG dimers. These facts suggest that the 130kDa band is most likely produced by the covalent crosslinking of hCG to the 90-kDa receptor polypeptide, in agreement with previous observations (17, 19). To further confirm that the major 90-kDa polypeptide is responsible for the hormone binding activity, the purified receptor preparation was subjected to ligand blotting (22). The receptor was subjected to SDS-PAGE under nonreducing conditions and electroblotted onto nitrocellulose. Incubation of the blot with lZ5I-hCGfollowed byautoradiography revealed labeling of a single 90-kDa band (Fig. 4a). The labeling was displaced by unlabeled hCG and hLH (Fig. 4, b and c) but not by heterologous hormones hFSH or rat prolactin (Fig. 4, d and e). Ligand blotting of the original Triton extractproduced the 90-kDa band (Fig. 5a),showing that no gross structural changes in the receptor take place in the course of the purification. A previous study from this laboratory (19) suggested that rat ovarian LH receptor contains sialic acid residues. Therefore, it was of interest to test whether the purified 90-kDa receptor protein interacts with WGA, a lectin that binds to

b

C

d

e

f

g

I

FIG.2. SDS-PAGE of purified receptor preparations.Samples from various stages of purification were subjected to SDS-PAGE on 7.5% acrylamide separating gel and 5% stacking gel as described under “Experimental Procedures.” All samples were reduced prior to electrophoresis with 100 mM dithiothreitol except in lane f. The gels were stained with silver (lanes a-f) or subjected to autoradiography after drying the gel. Lunes: u, Triton X-100extract (10 pg of protein); b, lyophilisate of 5 ml of the high-salt (0.5 M NaCl) wash of the first hCG-Affi-Gel10 chromatography; c, purified receptor after firsthCGAffi-Gel 10 chromatography (500 ng of protein); d , pH 4 eluate from hCG-Affi-Gel 10 chromatography that was performed in the presence of an excess of soluble hCG and corresponding to a similar amount of the original detergent extract as in lane c ; e, purified receptor after the second hCG-Affi-Gel 10 chromatography (500 ng of protein); f, same as e but nonreduced, g, radioiodinated double affinity-purified receptor (l0,OOO cpm). The sizes (in kDa) of mass markers are indicated on theleft. N-acetylglucosamine and sialic acid residues. 70-75% of the binding activity present inthe affinity-purified receptor preparation bound to WGA-Sepharose 4B in an overnight incubation, and 42-53% of the original binding activity was recovered in the N-acetylglucosamine eluate. The lectin chromatography did not, however, increase the electrophoretic purity or specific activity of the receptor (not shown). The purified receptor bound quantitatively to concanavalin ASepharose 4B as well but was difficult to elute from the gel even with 0.5 M specific sugar (not shown). These results suggest that the90-kDa LH receptor is a glycoprotein. Finally, to find out whether there are any major interspecies differences among the LH receptors from various mammalian sources, the LH receptors of bovine corpora lutea and mouse Leydig tumor cells (MLTC-1 cells (31)) wereanalyzed by ligand blotting together with the rat ovarian LH receptor. This comparison was relevant asthe reported molecular weight and subunit composition of purified bovine luteal LH receptor (18)is very different from the rat receptor observed inthis study. Wehavepreviouslyshown that a 90-kDa hormone binding species is detected by ligand blotting in rat ovarian and mouse Leydig cellextracts (22). Although the LH receptor of luteinized rat ovaries could be easily detected when unpurified detergent extracts were analyzed by ligand blotting, preliminary purification of bovine and MLTC-1 receptors by adsorption to WGA-Sepharose and subsequent

Rat OvarianLH Receptor

7924

a

b

a

C

205-

205-

116, 94-

94-

C

d

e

w

66-

6645-

b

45-

I

l

29FIG. 3. Covalent cross-linking of I2%hCG to purified LH receptor. Purified LH receptor was incubated with '=I-hCG in the absence ( l a n e a ) or presence ( l a n e b ) of an excess of unlabeled hCG. The molecular complexes were cross-linked with glutaraldehyde and analyzed by SDS-PAGE undernonreducing conditions. Lane c shows cross-linked products produced by '%I-hCG alone. An autoradiogram of a dried gel is shown. The sizes (in kDa) of the mass markers are indicated on the left.

29FIG. 4. Demonstration of the hormone-binding activity of the purified LH receptor by ligand blotting. Purified receptor after the second hCG-Affi-Gel 10 chromatography was subjected to SDS-PAGE under nonreducing conditions and without prior heating of the samples and electroblotted onto nitrocellulose. The lanes containing receptor were incubated with '=I-hCG in the absence of added hormones ( l a n e a ) or in the presence of 4 pg/ml unlabeled hCG ( l a n e b), hLH ( l a n e c), hFSH ( l a n e d ) , or rat prolactin ( l a n e e). Autoradiograms of the dried nitrocellulose strips areshown. The sizes (in kDa) of mass markers are indicated on the left.

ported by Dias et al. (34) and was later demonstrated with the solubilized LH receptor of Leydig cells (14). Proteinase inhibitors were also found to markedly improve the yield. Previous studies in this laboratory have shown that membrane-bound LH receptor is prone to extensive proteolysis, which is further stimulated by hCG (15,35). elution by N-acetylglucosaminewere found to be necessary. An analysis of the affinity-purified receptor by SDS-polyThis procedure yielded a 3-5-fold enrichment of the specific acrylamide gel electrophoresis under nonreducing conditions binding activity for the bovine luteal and MLTC-1 LH recep- followed by silver staining revealed only one major band of tors. As seen from Fig. 5, rat and bovine corpora lutea and 90 kDa. The receptor migrated as a 90-kDa species under MLTC-1 cells all produce the 90-kDa band in ligandblotting. reducing conditions as well, demonstrating the absence of The labeling of the 90-kDa band is saturable and hormone- disulfide-bonded subunits in the LH receptor. The hormonespecific as it is displacedby an excess of unlabeled hCG (Fig. binding ability of the purified 90-kDa polypeptide apparent, is 5). as ligand blotting revealed saturable and specific binding of lZ5I-hCGto the 90-kDa band. These results indicate that the DISCUSSION 90-kDa polypeptiderepresents the LH receptor. The present This study describes purification of the rat ovarian LH data do not distinguish between the various stoichiometric receptor to near homogeneity in an active hormone-binding alternatives of the native receptor in its membrane environform. The major contributing factors to the retention of ment. Whether the native LH receptor is a monomer or an hormone binding activity were the presence of 20% glycerol oligomer of the 90-kDa polypeptideis presently unknown. It in the solubilizedpreparations and inclusion of the proteinase is also possible that thereceptor contains additional polypepinhibitors N-ethylmaleimide, EDTA, and PMSF to all solu- tides that are not required for specific high-affinity hormone tions used in the purification scheme. The preserving effect binding but areoperative in the transduction of the hormonal of glycerol to solubilized follitropin receptors was first re- signal. If that were the case, the association between the 90-

Rat OvarianLH Receptor

7925

regulating dose of hCG (37). Wimalasena et al. (20) recently reported that LH receptor of porcine corpus luteum consists of 60-65-kDa hormone-binding subunits. Covalent cross-linking studies by Rapoport et al. (8) and by Rebois et al. (9) suggest that rat gonadal LH receptor is an 86- or 100-kDa species,respectively. Finally, our results are in agreement with a recent report demonstrating that a monoclonal antibody that blocks the binding of 'Y-hCG to the receptor in particulate andsolubilized receptor preparations recognizes a 90-kDa protein in Western blots of rat Leydig cells (38). Our results on the structure of the LH receptor disagree with several reports suggesting a hetero-oligomeric structure for the receptor. Bruch et al. (21) recently reported purification of rat ovarian LH receptor as a disulfide-bonded heterotetramer of 320 kDa, which dissociated into 79-, 66-, 55-, and 47-kDa components upon reduction with 8-mercaptoethanol and dithiothreitol. This result is hard to reconcile with ours, as the LH receptor isolated in this study migrates as a 90kDa molecule in SDS-PAGE under nonreducing as well as reducing conditions, indicating that the receptor is a single polypeptide. It should be noted, however, that while the hormone binding activity of the 90-kDa protein purified in this study was substantiated by ligand blotting, the only evidence for the biological activity of the 47-79-kDa proteins was the presence of three faint radioactive bands in the mass range 66-85 kDa after cross-linking radioiodinated hCG to purified receptor. The reason for the discrepancy between our results and those of Bruch et al. (21) is presently unknown. Dattatreyamurty et al. (18)have presented an immunoglobulin-like structure for the bovine LH receptor, consisting of two heavy (85 kDa) and two light chains (38 kDa) stabilized by disulfide bonds. The similarity in molecular size between FIG. 5. Comparison of LH receptors from three different the 85-kDa heavy chain and the 90-kDa receptor characterspecies by ligand blotting. Triton X-100extract of pseudopregnant is interesting, especially asthis study rat ovaries (lanes a and b) and N-acetylglucosamine eluate of WGA- ized inthisstudy affinity chromatography of solubilized mouse Leydig tumor cells demonstrates by ligand blotting that bovine LH receptor (lanes c and d ) or solubilized bovine luteal membranes (lanes e and contains a 90-kDa hormone-binding unit. Studies using chemf ) were subjected to SDS-PAGE under nonreducing conditions and ical cross-linking (10, 11) and photoaffinity labeling (6, 7) electroblotted onto nitrocellulose. The nitrocellulose strips were in- techniques have produced several differently sized radiolacubated with T - h C G in the absence (lanes a, c, and e ) or in the beled complexes on electrophoresis, indicating the existence presence of 4 pg/ml unlabeled hCG. Autoradiograms of dried strips are shown. Sizes (in kDa) of mass markers are indicated on the left. of multiple receptor subunits. Evidence from this laboratory (15,35) andfrom that of Ascoli (12) suggests that endogenous About 50 pg of soluble protein was applied to SDS-PAGE/lane. proteolysis or the use of collagenase in the preparation of cells, respectively,produces receptor-derived proteolytic fragkDa receptor polypeptide and these putative additional sub- ments that could leadto erroneous interpretations of receptor units is not very tight because no other polypeptides are structure. purified in stoichiometric amounts with the 90-kDa polypepThe ligand blotting experiments of this study show the tide. The question of putative additional receptor units can presence of a 90-kDa hormone-binding unit in luteinized rat be resolved bymeasuring the capacity of the purified receptor ovary, bovine corpora lutea, and MLTC-1 cells, a cultured to reconstitute LH-responsive adenylate cyclase in a heterol- Leydig tumor cell line shown to exhibit functional LH recepogous system. Such studies are in progress in this laboratory. tors (31). These results suggest that no major structural Our purification of the 90-kDa LH binding protein from differences exist between LH receptors of different mammapseudopregnant rat ovaries agrees well with many reports lian species. This is hardly unexpected in the light of the suggesting that the gonadal LH receptor hormone-binding central role of the LH receptor in the hormonal control of unit is a single 65-100-kDa polypeptide. Due to anomalous reproduction. migration of glycoproteins in SDS-PAGE (36), the molecular Because of the close structural relationship between the weight estimates for the LH receptor in this study aswell as glycoprotein hormones LH, hCG, FSH, and TSH (39), it in others aresuggestive only. An early report by Dufau et al. would beinteresting to know whether the structuralsimilarity (16) described purification of rat testicular LH receptor, which extends to the receptors as well. Preliminary molecular charbehaved as a 180-kDa molecule in gel filtration and as a 90- acterization of FSH andTSH receptors has been reported but kDa moleculein SDS-PAGE. The authors suggested that the no coherent picture has emerged (40-44). The ligand blotting receptor is a dimer of 90-kDa subunits. A 90-kDa polypeptide approach could possibly be usefulin identifying the hormone was also purified by hCG-affinity chromatography in this binding subunit of these receptors. laboratory from pseudopregnant rat ovaries, although loss of In conclusion, rat ovarian LH receptor has been purified to binding activity was apparent (17). Furthermore, specific near homogeneity. The purified receptor is a 90-kDa glycoantibody prepared against this polypeptide recognized a 90- polypeptide and retains its ability to bind hormone. AvailakDa species in pseudopregnant rat ovarian extracts prepared bility of microgram amounts of pure receptor nowallow from intact rats but notfrom rats that had received a down- further studieson the molecular biologyof LH receptor.

a

205-

11694-

6645-

29-

b

c

d

e

f

7926

Ovarian

Rat

Acknowledgments-The skillful technical assistance of Sirpa Jamsa, Aino Kuha, and Eero Oja is gratefully acknowledged. The MLTC-1 cell line was a generous gift from Dr. V. Rebois (National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD).

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