Amino-terminal leucine-rich repeats in gonadotropin receptors determine hormone selectivity.

Recombinant expression of truncated receptors for luteinizing hormone/chorionic gonadotropin (LH/CG) revealed that the amino-terminal leucine-rich repeats 1-8 of the extracellular receptor domain bind human chorionic gonadotropin (hCG) with an affinity (Kd = 0.72 +/- 0.2 nM) similar to that of the native LH/CG receptor (Kd = 0.48 +/- 0.05 nM). LH/CG receptor leucine-rich repeats 1-8 were used to replace homologous sequences in the closely related receptor for follicle stimulating hormone (FSH). Cells expressing such chimeric LH/CG-FSH receptors bind hCG and show elevated cylic AMP levels when stimulated by hCG but not by recombinant human FSH (rhFSH). Similarly, a chimeric LH/CG receptor in which leucine-rich repeats 1-11 originated from the FSH receptor is activated by rhFSH but not by hCG. For this chimera, no residual [125I] hCG binding was observed in a range of 2 pM to 10 nM. Our results demonstrate that specificity of gonadotropin receptors is determined by a high affinity hormone binding site formed by the amino-terminal leucine-rich receptor repeats.     

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.     

Purification and partial characterization of rat ovarian lutropin receptor

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 125I-hCG binding sites with an equilibrium dissociation constant (Kd) of 5.1 X 10(-10) M (at 20 degrees C) and had a specific hormone binding capacity of 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 purified receptor to 125I-hCG produced a 130-kDa complex. Hormone-binding ability of the purified 90-kDa polypeptide was demonstrated also by ligand blotting. The purified receptor was electroblotted onto nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by incubation with 125I-hCG. Autoradiography revealed labeling of a 90-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 and mouse 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 demonstrate that the hormone-binding unit of rat ovarian LH receptor is a 90-kDa membrane glycopolypeptide.     

Identification of LH/hCG receptors in rabbit uterus

Luteinizing hormone (LH) is believed to act via specific receptors to control gonadal steroidogenesis and reproductive processes. Recently A. J. Ziecik, P. D. Stanchev, and J. E. Tilton (Endocrinology 119:1159, 1986) reported surprisingly that LH/hCG receptors were present in porcine uterus, a tissue not known to be a target for LH action. We report herein the identification of high-affinity LH receptors in the rabbit uterus. Uteri from adult New Zealand white rabbits were homogenized in Tris-HCl, 0.25 M sucrose. After filtration and sequential centrifugation, a partially purified pellet containing receptors was obtained. This preparation was incubated with a trace (1300 cpm) (50 pg) 125I-labeled chorionic gonadotropin and with various unlabeled protein hormones. Receptor bound was separated from free hormone by centrifugation at 1000 g. Affinity was estimated by Woolf plot analysis. Specific binding sites for LH/hCG were identified. The following Kd's were calculated: human LH, 1.6 X 10(-11); hCG, 0.5 X 10(-11); human TSH, 1.3 X 10(-9); and human FSH, 7.85 X 10(-9). The reaction of human FSH and TSH with the receptor is best explained by LH contamination of these hormones. A similar preparation of rat liver showed that no binding sites were present. Rabbit ovarian LH receptors had a Kd slightly higher at 4.1 X 10(-11) than that of the uterine LH receptors. Rabbit ovarian receptors were present at 2.27 X 10(-13) M/mg protein compared to uterine receptors at 4.65 X 10(-15) M/mg protein. We conclude specific- and high-affinity binding sites (receptors) for LH are present in the rabbit uterus. The function of these receptors remains unknown.     

Purification and characterization of Leydig cell luteinizing hormone receptor

We have purified the testicular luteinizing hormone (LH/human choriogonadotropin (hCG)) receptor by sequential affinity chromatography on hCG-Sepharose. The purified LH/hCG receptor was identified as a single protein of Mr = 90,000 +/- 2,000 on sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE), showed high affinity binding for hCG, and a binding capacity of 3.8 nmol/mg of protein. Electrophoretically blotted receptor retained the ability to bind 125I-hCG on nitrocellulose membrane, and the Mr of radioactive band was consistent with that revealed by silver staining. Autoradiography after SDS-PAGE analysis of cross-linked purified receptor-hCG complex showed Mr = 145,000 and Mr = 105,000 bands. These results are consistent with a Mr value for the receptor of 90,000 after accounting for contribution by the intact hormone or its alpha-subunit. Analysis of the free receptor by fast protein liquid chromatography on Superose 12 revealed a single peak of binding activity for 125I-hCG which eluted in the position of Mr = 200,000-240,000 in the presence of Triton X-100. Since a single protein species is observed under reducing or nonreducing conditions in SDS-PAGE, the receptor could exist in the membrane as a dimeric form composed of subunits Mr = 90,000 associated through noncovalent interactions. The pure receptor can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (approximately 0.3 mol of phosphate/mol of receptor). This phosphorylation does not affect the binding characteristics of the receptor. The method described is simple and allows rapid purification of microgram amounts of biological active Leydig cell LH/hCG receptor for structural, functional, and immunological studies.     

Purification, characterization, and amino-terminal sequence of rat ovarian receptor for luteinizing hormone/human choriogonadotropin

The luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor of rat ovary was solubilized with Lubrol PX in the presence of 20% glycerol and protease inhibitors, and purified by one-step affinity chromatography. Purified receptor had a specific hCG binding capacity of 4900 pmol/mg protein, and displayed a single class of high affinity binding sites (Ka = 6.20 X 10(9) M-1). An 11,200-fold purification over the starting crude homogenate was achieved. The purified LH/hCG receptor was identified by sodium dodecyl sulfate-gel electrophoresis and silver staining as a single protein of 92 kDa. The ability of the purified 92-kDa protein to specifically bind hormone was demonstrated by electroblotting onto Immobilon P membrane, incubation with 125I-labeled hCG, and autoradiography of the blot. In addition to a 92-kDa band, ligand blotting also yielded a 170-kDa band representing receptor dimer. Covalent cross-linking of hCG, with isotope in either the alpha- or beta-subunit, to membrane-bound receptor produced complexes that contained a single receptor component of approximately 92 kDa. The cross-linking studies indicated that both subunits interact with receptor and also suggested receptor dimer formation. Following sodium dodecyl sulfate-electrophoresis, purified receptor was electroblotted onto polyethylenimine-treated glass fiber filters for direct microsequencing in a gas-phase sequenator. Eleven cycles of sequence analysis yielded the unique sequence: NH2-Arg-Glu-Leu-Ser-Gly-Ser-Leu-XXX-Pro-Glu-Pro-COOH. These results indicate that the rat ovarian LH/hCG receptor is a protein of 92 kDa which can be easily purified in microgram amounts. This study also describes a relatively simple technique for electroblotting and microsequencing that should be applicable to other membrane-bound hormone receptors.     

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.     

Two distinct forms of receptors for atrial natriuretic factor in bovine adrenocortical cells. Purification, ligand binding, and peptide mapping

The atrial natriuretic factor (ANF) receptor of bovine adrenal cortex was solubilized with Triton X-100 and purified by sequential chromatography on ANF-(99-126)-agarose, GTP-agarose, and wheat germ agglutinin-Sepharose. Two subtypes of ANF receptors were isolated, both of which showed specific ANF binding, whereas one of the ANF receptor subtypes also possessed significant cyclase activity. Both of the receptors showed high capacities (Bmax = 5.7-6.8 nmol/mg of protein) and high affinities (Kd = 54-68 pM) for ANF-(99-126). The cyclase-free receptor had high affinity (Ki = 150-220 pM) to C-terminal truncated ANF analogs, whereas the cyclase-containing receptor had a much weaker affinity (Ki = 10(6)-10(7) pM). When treated with dithiothreitol, the purified cyclase-containing and cyclase-free ANF receptors migrated as a single band at Mr 135,000 and 62,000, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cyclase-free receptor is not a product derived from the cyclase-containing receptor because (i) two proteins with Mr of 135,000 and 62,000 were specifically labeled with 4-azidobenzoyl 125I-ANF-(102-126) in nonsolubilized intact membranes; (ii) the truncated ANF analogs (10(4) pM) prevented the photolabeling of the 62,000-dalton protein but not that of the 135,000-dalton protein; and (iii) two-dimensional peptide mapping showed more than 90% difference between the profiles of the two purified ANF receptor subtypes. This study provides first direct evidence for the existence of two distinct ANF receptors which are different not only in their pharmacological properties but also in their primary structure.     

Isolation and characterization of a gonadotropin receptor binding inhibitor from porcine follicular fluid

The presence of a gonadotropin receptor binding inhibitor in pooled porcine follicular fluid has been demonstrated. Porcine follicular fluid fractionation on DE-32 at near neutral pH, followed by a cation exchange chromatography on SPC-50 and Cibacron blue affinity chromatography, yielded a partially purified gonadotropin receptor binding inhibitor (GI-4). The partially purified GI binding inhibitor inhibited the binding of both 125I labelled hFSH and hCG to rat ovarian receptor preparation. SDS electrophoresis of radioiodinated partially purified GI followed by autoradiography made it possible to identify the binding component as a protein of molecular weight of 80,000. Subjecting 125I labelled GI-4 to chromatography on Sephadex G-100 helped obtain a homogeneous material, GI-5. The 125I labelled GI-5 exhibited in its binding to ovarian membrane preparations characteristics typical of a ligand-receptor interaction such as saturability, sensitivity to reaction conditions as time, ligand and receptor concentrations and finally displaceability by unlabelled inhibitor as well as FSH and hCG in a dose dependent manner. This material could bind ovarian receptors for both FSH and LH, its binding being inhibited by added FSH or hCG in a dose dependent manner.     

The stimulus-secretion coupling of glucose-induced insulin release. Environmental influences on L-glutamine oxidation in pancreatic islets.

Rat ovarian luteinizing hormone/human choriogonadotropin binding sites were labelled with 125I-choriogonadotropin in vivo, and the resulting 125I-choriogonadotropin-receptor complexes were solubilized by Triton X-100 and purified by use of antibodies to choriogonadotropin immobilized to agarose. The purified 125I-choriogonadotropin-receptor complex was treated with glutaraldehyde to crosslink radiolabelled hormone to the receptor. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the crosslinked product revealed a labelled Mr 130 000 major band in addition to the hormone and its alpha-subunit, indicating that a single receptor component was linked to the hormone. Unoccupied binding sites for luteinizing hormone were also solubilized by Triton X-100 from pseudopregnant rat ovaries, and attached to choriogonadotropin-agarose. The agarose gel was washed, and eluted with 0.1 M-sodium acetate, pH 4. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the pH 4 eluate revealed an Mr 90 000 major band which was abolished when ovaries presaturated with choriogonadotropin were used as starting material. These observations suggest that the hormone-binding component of the luteinizing hormone receptor is a polypeptide of Mr 90 000. This polypeptide was isolated and labelled with Na 125I. The labelled polypeptide showed a single band on sucrose density gradient centrifugation and on gel filtration on agarose.     

Characterization of a lipid-associated gonadotropin receptor from the luteinized rat ovary

Gonadotropin receptors which bind luteinizing hormone (lutropin) and human chorionic gonadotropin (hCG) in the ovaries of immature female rats showed a 30-fold increase after treatment of animals with pregnant mare serum gonadotropin (PMSG) and hCG. This marked induction of lutrophin/hCG receptors in the rat ovary was not accompanied by a change in binding affinity for labeled hCG. Such luteinized ovaries have been found consistently to contain a small proportion of soluble receptor sites, which comprised about 5% of the total receptor population. The soluble receptor sites were present in the floating lipid fraction of the 360 000 × g supernatant of homogenate prepared from luteinized ovaries, and could not be detected in similar fractions prepared from interstitial cells or homogenates of the normal rat testis.The physico-chemical properties of the spontaneously soluble ovarian receptors were similar to those derived for detergent-solubilized receptors prepared by extraction of particulate ovarian binding fractions with Triton X-100. The affinity constant to the soluble ovarian receptor sites for [¹²⁵I]hCG was 0.70 · 10¹⁰ M^?1, and that of the receptors solubilized by Triton X-100 was 0.72 · 10¹⁰ M^?1. The sedimentation pattern of the soluble receptors during sucrose density gradient centrifugation showed extensive aggregation into rapidly sedimenting forms. However, centrifugation of the cytosol receptor in the presence of Triton X-100 gave a single 6.5 S component, corresponding to the solubilized receptors previously characterized in detergent extracts of the rat ovary and testis.The pesence of a spontaneously soluble lutropin/hCG receptor in ovarian cytosol fractions suggests that rapid synthesis and assembly of receptors in ovaries of PMSG-hCG-treated rats is accompanied by increased production of cytoplasmic receptor precursors; alternatively, this receptor population may represent a fraction that has been internalized or processed as during receptor turnover in the cell membrane.     

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.     

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.     

Interleukin 1: an inhibitor of luteinizing hormone receptor formation in cultured rat granulosa cells

We have shown that interleukin 1 (IL 1) suppresses follicle-stimulating hormone (FSH)-induced progesterone secretion and 125I-labeled human chorionic gonadotropin (hCG) binding (a measurement of LH receptors) in cultured rat granulosa cells. The present study was designed to examine if the reduction of FSH-stimulated 125I-labeled hCG binding by IL 1 was caused by a decline in the binding capacity or by an alteration in the affinity of the LH receptor and, further, to determine the minimum period of exposure of the granulosa cells to IL 1 necessary to suppress 125I-labeled hCG binding. IL 1 produced a dose-dependent inhibition of 125I-labeled hCG binding in FSH-stimulated granulosa cells. Scatchard analysis revealed that this effect resulted from a reduction of the binding capacity of the LH receptor with no change in affinity. Also, a minimum of 12-24 h of exposure to IL 1 is necessary to significantly inhibit FSH-induced LH receptor formation. These results suggest that IL 1 decreases the number of LH receptors and that protein synthesis may be necessary for IL 1's action. However, a physiological/pathological role for IL 1 in ovarian regulation has yet to be established.     

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.     

Purification and characterization of the rat ovarian receptor for luteinizing hormone. Structural studies of subunit interaction

We have purified the luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor by sequential affinity column on wheat germ lectin-Sepharose and hCG-Sepharose. The method was designed to allow also the purification of lactogen receptor from the initial starting material. The purified LH/hCG receptor retained full binding affinity and was identified as a single protein of Mr = 73,000 +/- 3,000 on sodium dodecyl sulfate-gel electrophoresis. Cross-linking studies performed after binding of hCG to the purified LH/hCG receptor indicated that the hCG alpha-subunit undergoes predominant interaction with the receptor molecule. The influence of the beta-subunit in this interaction seems to occur mainly through its association with the alpha-subunit, presumably by conferring specificity to the alpha-subunit for its hormonal interaction with the receptor. The technique described in this study is simple and allows rapid purification of microgram amounts of biologically active receptor suitable for further molecular characterization, microsequencing, and functional reconstitution studies.     

Purification of follitropin receptor from bovine calf testes

Follitropin (FSH) receptors were solubilized from pure light membranes of bovine calf testis, using an optimum detergent to protein ratio of 0.01. The soluble FSH receptor fraction was gel filtered through Sepharose 6B to isolate an active fraction (6B-Fr-1) which behaved as a complex of FSH receptor and Gs protein. The 6B-Fr-1 was concentrated by ultrafiltration and further purified by sequential Sepharose 4B gel filtration, DEAE-cellulose chromatography (to separate the receptor from Gs protein), and wheat germ lectin affinity chromatography. The purified receptor had an FSH-binding capacity of approximately 3.47 nmol/mg of protein with a Kd of 1.9 X 10(-10) M. Yield was 526 micrograms/11.5 kg tested. Radioiodinated, as well as unlabeled purified FSH receptor, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single major band of Mr approximately 240,000. This band was not affected by 8 M urea treatment prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but treatment with dithiothreitol induced the loss of the 240-kDa band, with appearance of an Mr approximately 60,000 band. The availability of highly purified, stable FSH receptor should allow direct studies on its structure-function relationships.     

Purification and Characterization of Ovarian Lh/Hcg and Prolactin Receptors

Abstract

We have purified the luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor to homogeneity by sequential affinity column on wheat germ lectin-Sepharose and hCG-Sepharose. The method was designed to allow also the purification of lactogen receptor from the initial starting material. Comparable purification of lactogen receptor can be attained using Con A-Sepharose as initial step. The purified LH/hCG receptor was identified as a single protein of Mr=75,000 on SDS gel electrophoresis. The lactogen receptor is composed of two dissimilar active subunits of Mr 88,000 and 40,000, the latter probably being an integral part of the larger form. Comparison of Mr's derived from SDS gels with those from fast performance liquid chromatography suggested that the native LH holoreceptor is present in a dimeric form, while the lactogen receptor seems to be composed of aggregates that could represent dimeric or trimeric forms of holoreceptor Mr 80,000. Cross-linking studies performed after binding of hCG (radiolabeled in the individual subunits) to the purified LH/hCG receptor indicated that the hCG α-subunit undergoes predominant interaction with the receptor molecule. The influence of the β-subunit in this interaction seems to occur mainly through its association with the α-subunit, presumably by conferring specificity to the α-subunit for its interaction with the receptor. The α-subunit, which is identical within species, has an important role in the receptor binding interaction and biological activity of glycoprotein hormones.     

Human chorionic gonadotropin. V. Tissue specificity of binding and partial characterization of soluble human chorionic gonadotropin-receptor complexes.

An in vivo human chorionic gonadotropin (hCG)-receptor complex was solubilized from the subcellular fraction of ovarian and testicular tissues of rats that had been injected with 125-I-labeled hCG. The soluble hCG-receptor complex was partially characterized by Sepharose 6B chromatography in the presence of the nonionic detergent, Emulphogene, and was shown to have a molecular size of about 65 A. By this method it was also shown that the in vivo uptake of radioactivity by rat gonadal tissues represents 125-I-hCG and not the dissociated subunits or degradation products of the hormone. A soluble hCG-receptor complex isolated in vitro in approximately the same yield from both rat testicular and ovarian homogenates was shown to be the same size. The hCG-receptor appears to be specifically located in gonadal tissue; a corresponding hCG-receptor complex was not obtained from liver or kidney that incorporated significant levels of 125-I-hCG administered in vivo. Furthermore, a desialyzed hCG-receptor complex was obtained from rat testis but not liver; desialyzed hCG, like other desialyzed glycoproteins, is nonspecifically bound by rat liver homogenates. The binding of hCG and luteinizing hormone (LH) by rat testis receptor exhibits a high degree of specificity. Other glycoprotein hormones without LH activity, such as follicle-stimulating hormone and thyroid-stimulating hormone, and glycoproteins such as fetuin or alpha1-acid glycoprotein do not bind to the hCG/LH receptors. Desialyzed hCG was 2 times more effective in competing for binding to rat testis receptors than "native" hCG, indicating that caution must be exercised when the radioligand receptor assay is utilized to assay hCG preparations varying in sialic acid content.