The effects of neuraminidase and gangliosides on ovarian LH/hCG receptors during rat development

Ovaries of neonatal rats are not endowed with specific LH/hCG receptors up to 6-8 days of age. Treatment of ovarian membranes of the neonatal rat with neuraminidase results in a specific binding of radioactively labeled hCG, while an increase of hormone binding is observed after neuraminidase treatment of ovarian membranes of the 21-day-old rat. These changes in hormone receptor sites in the ovary are dependent on the neuraminidase concentration used and are due to a receptor with a dissociation constant (KD) of about 10(-9) M. The KD of the receptor in the LH/hCG sensitive ovary without neuraminidase treatment is about 10(-10) M. These results indicate the presence of two different LH/hCG receptors in the ovarian membrane. The unmasking effect of neuraminidase onto LH/hCG receptors indicate that ganglioside-like structures are responsible for the masking of receptors in the neonatal, insensitive rat ovary and also in the 21-day-old sensitive ovary. Ganglioside preparations are able to inhibit the binding, and the fractionation of ovary gangliosides results in a fraction with a rather high inhibition potency of LH/hCG binding to the receptor. It is hypothesized that the masked receptor in the sensitive period represent a store of receptors for the reconstitution of the ovarian cells with active receptors after internalization of the hormone-receptor complex. Thus the masking of the receptors in the early postnatal rat ovary could be a prerequisite for the female differentiation of hypothalamic centers. The observed neuraminidase effect in vitro could reflect a physiologic situation. Neuraminidase was found in the ovary, and during early postnatal development the neuraminidase activity pattern coincides with that of the ovarian LH/hCG receptor changes.     

The Effects of Neuraminidase and Gangliosides on Ovarian LH/hCG Receptors During Rat Development

Ovaries of neonatal rats are not endowed with specific LH/hCG receptors up to 6–8 days of age. Treatment of ovarian membranes of the neonatal rat with neuraminidase results in a specific binding of radioactively labeled hCG, while an increase of hormone binding is observed after neuraminidase treatment of ovarian membranes of the 21-day-old rat. These changes in hormone receptor sites in the ovary are dependent on the neuraminidase concentration used and are due to a receptor with a dissociation constant (K_D) of about 10⁻⁹ M. The K_D of the receptor in the LH/hCG sensitive ovary without neuraminidase treatment is about 10⁻¹⁰ M. These results indicate the presence of two different LH/hCG receptors in the ovarian membrane. The unmasking effect of neuraminidase onto LH/hCG receptors indicate that ganglioside-like structures are responsible for the masking of receptors in the neonatal, insensitive rat ovary and also in the 21-day-old sensitive ovary. Ganglioside preparations are able to inhibit the binding, and the fractionation of ovary gangliosides results in a fraction with a rather high inhibition potency of LH/hCG binding to the receptor. It is hypothesized that the masked receptors in the sensitive period represent a store of receptors for the reconstitution of the ovarian cells with active receptors after internalization of the hormone-receptor complex. Thus the masking of the receptors in the early postnatal rat ovary could be a prerequisite for the female differentiation of hypothalamic centers. The observed neuraminidase effect in vitro could reflect a physiologic situation. Neuraminidase was found in the ovary, and during early postnatal development the neuraminidase activity pattern coincides with that of the ovarian LH/hCG receptor changes.     

Biological function of the LH receptor is associated with slow receptor rotational diffusion

The biological activity of luteinizing hormone (LH) receptors can be affected by modifications to the receptor's amino acid sequence or by binding of hormone antagonists such as deglycosylated hCG. Here we have compared rotational diffusion of LH receptors capable of activating adenylate cyclase with that of non-functional hormone-occupied receptors at 4 degrees C and 37 degrees C using time-resolved phosphorescence anisotropy techniques. Binding of hCG to the rat wild-type receptor expressed on 293 cells (LHR-wt cells) or to the LH receptor on MA-10 cells produces functional receptors which exhibit rotational correlation times longer than 1000 micros. However, modification of the LH receptor by substitution of Lys583-->Arg (LHR-K583R) results in a receptor that is non-functional and which has a significantly shorter rotational correlation time of 130+/-12 micros following binding of hCG. When these receptors are treated with deglycosylated hCG, an inactive form of hCG, the rotational correlation times for the LH receptors on LHR-wt and MA-10 cells are also shorter, namely 64+/-8 and 76+/-14 micros, respectively. Finally, a biologically active truncated form of the rat LH receptor expressed in 293 cells (LHR-t631) has slow rotational diffusion, greater than 1000 micros, when occupied by hCG and a significantly shorter rotational correlation time of 103+/-12 micros when occupied by deglycosylated hCG. The effects of rat LH binding to LH receptors on these various cell lines were similar to those of hCG although the magnitude of the changes in receptor rotational diffusion were less pronounced. We suggest that functional LH receptors are present in membrane complexes that exhibit slow rotational diffusion or are rotationally immobile. Shorter rotational correlation times for non-functional hormone-receptor complexes may reflect the absence of essential interactions between these complexes and other membrane proteins.     

Modulation of LH/hCG receptors and physical state of ovarian membranes in rat pseudopregnancy

Previous investigations have demonstrated that increased ovarian function during pseudopregnancy in the rat may be associated with alterations of the physical state of membranes. Changes in rigidity of membrane lipids were observed during the formation as well as regression of corpora lutea. The effects of cyclooxygenase inhibitors (indomethacin and acetylsalicylic acid (ASA)) and of selected steroids (estradiol, testosterone and dihydrotestosterone) on the functional state of luteinized ovaries were studied. The compounds were administered to the animals in silastic capsules on different days after hCG injection. ASA and indomethacin administration on days 10 and 11 after hCG injection resulted in an increase in the LH/hCG receptor binding activity and rigidity of ovarian membrane lipids, as determined by fluorescence polarization of 1,6-diphenyl-1,3,5 hexatriene (DPH) probe. This effect was apparent within 7 days after indomethacin and ASA treatment. Both estradiol and testosterone significantly increased the ovarian LH/hCG binding activity, however estradiol did not affect the membrane lipid rigidity. Unlike testosterone, the administration of dihydrotestosterone induced a decrease in membrane lipid rigidity and reduced the accessibility of the LH/hCG receptor. Inhibitors of prostaglandin F2alpha (PGF2alpha) synthesis, as the endogenous mediator of luteolysis, were shown to delay the regression of the corpora lutea and to prolong the luteal activity in pseudopregnant rats.     

Application of the immunofluorescence technique and confocal laser scanning microscopy for studying the distribution of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor on rat luteal cells

We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.     

The porcine LH/hCG receptor. Characterization and purification

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.     

Different processing of LH/hCG receptors in cultured rat luteal cells and murine Leydig tumour cells (MLTC-1)

The metabolic fate of LH/hCG receptors after exposure to human chorionic gonadotropin (hCG) was examined in cultured rat luteal cells and murine Leydig tumour cells (MLTC-1). Kinetic studies performed after pulse-labelling of the cells with [125I]hCG indicated that the bound hormone was lost much more rapidly from the tumour cells than from the luteal cells (t1/2 = 4.5 and greater than 12 h, respectively). The tumour cells were also found to internalise and degrade the hormone more effectively than the luteal cells, as measured by disappearance of acid-releasable (i.e. surface-bound) radioactivity from the cells and by the appearance of trichloroacetic acid (TCA)-soluble label in the medium. In MLTC-1 cells, over 80% of the radioactivity released was TCA-soluble at all times examined, whereas in the luteal cells most (65-75%) was TCA-precipitable. Chemical cross-linking and analyses by SDS-PAGE of this material revealed that both cell types also released, in addition to intact hCG, two previously characterized receptor fragment-[125I]hCG complexes (Mr 96,000 and 74,000) (Kellokumpu & Rajaniemi, Endocrinology 116 (1985) 707) into the medium, although their amount was negligible in MLTC-1 cells. Possibly, due to rapid discharge of the ligand from its receptor, no similar complexes could be detected inside the MLTC-1 cells, suggesting that they were released directly from the cell surface. However, the Mr 74,000 complex was observed inside MLTC-1 cells if chloroquine, a lysosomotropic agent, was present during the incubations. This suggests that the internalised receptor also becomes degraded, at least when complexed to hCG. The results thus provide evidence that there exist two different mechanisms for proteolytic processing of LH/hCG receptors in these target cells. In tumour cells, the degradation seems to occur almost exclusively intracellularly, whereas in luteal cells a substantial portion of the receptors is also degraded at the cell surface.     

Effects of truncations of the cytoplasmic tail of the luteinizing hormone/chorionic gonadotropin receptor on receptor-mediated hormone internalization.

The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG-stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.     

Temporal relationship of the prolactin-dependent LH-induced LH receptor to the LH stimulus

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of ¹²⁵l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased ¹²⁵l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.     

Labeling of LH/hCG receptor in rat ovaries.

Human chorionic gonadotropin (hCG) was found to stimulate the incorporation of [¹⁴C] N-acetyl-D-glucosamine in rat ovary in vitro. Subcellular fractionation of the ovarian tissue revealed that the plasma membranes were stimulated maximally to the extent of 200 to 300% by the hormone indicating the stimulation of the synthesis of plasma membrane glycoproteins. In addition, and appreciable amount of the radioactivity was incorporated in the cell surface LH/hCG receptor. The evidence in support of the labeling of the receptor was derived from the behavior of the detergent solubilized receptor on Sepharose 6B column and on hCG-Sepharose affinity adsorbent. The labeled receptor thus purified showed binding affinity for [¹²⁵I] hCG. Thus, the hormone stimulates the synthesis of cell surface glycoproteins as well as the LH/hCG receptor.     

Evidence for the existence of gonadotropin receptors in the nuclei isolated from rat ovary

Specific binding of radiolabeled human chorionic gonadotropin (hCG) to nuclei isolated from pseudopregnant rat ovaries was studied. Incubation of cultured luteal cells or isolated nuclei with fluorescein isothiocyanate conjugated hCG showed concentration of fluorescence in the nuclear region. Isolated nuclei exhibited saturable high affinity binding of radiolabeled hCG with an apparent Kd of 3.42 X 10(-10) M. The binding was inhibited by increasing concentrations of unlabeled hCG. Under dissociating conditions, the bound hCG was dissociated from the nuclei. However, unlike the plasma membranes, the hCG bound to nuclei was not degraded before dissociation. Radiolabeled hCG bound to the nuclei could also be dissociated by brief exposure to MgCl2 or acidic incubation medium. The bound hCG was not extractable with 4M KCl or 2% Triton X-100. The available evidence suggest that nuclear receptors are distinct from plasma membrane receptors for hCG.     

Differences in the characteristics and distribution of rat luteal receptors for native and deglycosylated human choriogonadotropin.

Previous work has suggested that rat luteal cells have two populations of LH/hCG receptors that are located in different parts of the cell membrane. The possibility that these two receptor pools may have functional differences has been investigated through examination of the binding and action of native and deglycosylated hCG to different membrane fractions. Ovaries from eCG/hCG-primed immature female rats were separated into 1,000 x g (heavy) and 20,000 x g (light) particulate fractions. Increasing concentrations of NaCl had a biphasic effect on the binding of native and deglycosylated hCG to both membrane fractions, causing an increase in binding at low concentrations and a decrease in binding at higher concentrations. The binding of deglycosylated hCG to both membrane preparations and the binding of native hCG to light-membrane preparations was maximal at approximately the same NaCl concentration (50-65 mM). This was higher than the concentration of NaCl necessary for maximal binding of native hCG to the heavy-membrane preparation. In addition, maximal native hCG binding to this preparation occurred over a broader NaCl concentration range (15-65 mM). Equilibrium binding experiments showed differences in hCG binding to both fractions. In light membranes there were significantly more receptor sites for deglycosylated hCG (11.2 +/- 4.8 fmol/mg ovary) than for native hCG (4.8 +/- 0.7 fmol/mg ovary), with no significant different in affinity. In contrast, in heavy membranes the affinity for deglycosylated hCG (6.30 +/- 0.19.10(9) M-1), was significantly higher than that for native hCG (2.60 +/- 0.13.10(9) M-1), with no significant differences in receptor number.(ABSTRACT TRUNCATED AT 400 WORDS)     

Porcine follicular fluid containing water-soluble LH/hCG receptor

Porcine follicular fluid has been shown to have a specific water-soluble receptor for human chorionic gonadotropin (hCG). The binding of [125I] hCG to follicular fluid is inhibited by unlabelled hCG, LH but not FSH, ACTH and GH. The binding of hormone to the receptor in follicular fluid is a saturable phenomenon and Scatchard analysis suggested that the receptor has high affinity to hCG with no changes as the follicle enlarges. In contrast, follicular fluid from large follicles (6-12 mm) has higher binding capacity (2.04 +/- 0.12 fmol/mg protein) than follicular fluid isolated from medium (3-5 mm) and small (1-2 mm) follicles (0.60 +/- 0.05 and 0.44 +/- 0.04 fmol/mg protein, respectively). With the aid of affinity chromatography on hCG-CNBr-Sepharose 6-B a homogeneous fraction with Mr about 65,000 as estimated by SDS-PAGE was isolated. Treatment of follicular fluid with several protein-modifying reagents changed interactions of [125I] hCG with both soluble receptor and that bound to granulosa cell membrane in the similar manner. The [125I] hCG binding capacity of follicular fluid represents about 9.5% of the total binding capacity of granulosa cells. Finally, soluble LH/hCG receptor is probably secreted actively by follicular cells into follicular fluid. Dead granulosa cells do not release receptor into follicular fluid or incubation medium.     

Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.     

Restricted lateral diffusion of luteinizing hormone receptors in membrane microdomains

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nm hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homotransfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.     

Effects of cholesteryl esters on the accessibility of LH/hCG receptors and membrane lipid fluidity in rat testes

Incubation of rat testicular membranes with cholesteryl hemisuccinate resulted in an increase in both membrane lipid microviscosity and 125I-labelled hCG specific binding. The purpose of this investigation was to establish which functional groups of cholesteryl hemisuccinate are important for the stimulatory effects. The data obtained showed that only esters of cholesterol with dicarboxylic acids, not those of monocarboxylic acids, increase the accessibility of LH/hCG receptors and membrane rigidity. Experiments with cholesteryl sulfates showed that there are polar groups on C3 carbon of cholesterol having no stimulatory effect on receptors, although an increase in membrane rigidity occurred. The side-chain of cholesterol is important for the stimulatory action. Androstenolone hemisuccinate was ineffective in this respect. On the other hand, partially modified side-chains (hemisuccinates of beta-sitosterol and stigmasterol) did not result in a marked reduction of the stimulatory action. The carboxyl group of cholesteryl hemisuccinate must be 'free': its esterification abolishes the stimulatory effect of cholesteryl hemisuccinate on both the LH/hCG receptor and membrane microviscosity. These results suggest that an intact carboxyl group of ester and the side-chain of cholesterol are indispensable for the stimulatory effect of cholesteryl hemisuccinate on the accessibility of LH/hCG receptors.     

Functional reconstitution of rat ovarian LH/hCG receptor into proteoliposomes

Rat ovarian membrane LH/hCG receptor was solubilized in various detergents and reconstituted into proteoliposomes. Upon removal of sodium cholate by active absorption on Bio-Beads SM-2, the functional interaction between receptor and adenylate cyclase was restored. Adenylate cyclase was stimulated by hCG, HCG+GTP or GppNHp and NaF. Reconstituted proteoliposomes bound more ¹²⁵I-hCG (528 fmol/mg protein) than membrane-bound receptors (384 fmol/mg protein). There was no difference, however, in the relative affinity of reconstituted receptor preparations for hCG.     

Purification and characterization of lutropin receptor from membranes of pig follicular fluid

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).