Ontogenesis, distribution and relative sensitivity of hCG receptors to hCG and LH in goat ovaries.

The binding of [125I]hCG to immature and mature follicles and corpora lutea of goat ovaries has been studied. The hormone is bound maximally by corpora lutea although mature follicles also exhibit some binding. Immature follicles are practically devoid of receptors for this hormone. In the corpus luteum, the receptors for the hormone are present in thecal and luteal cells. Autoradiographic studies show the location of the bound radioactivity grains primarily along the plasma membranes of these cells, although some radioactivity grains were also seen in the cytoplasm of luteal but not thecal cells. On a mole to mole basis, hCG was found to displace [125I]hCG from binding to receptors on corpus luteum better than hLH and oLH.     

HCG-dependent regulation of gonadotropin receptor sites: negative control in testicular Leydig cells.

A single injection of human chorionic gonadotropin (hCG 500 IU) to prepubertal male rats increases plasma testosterone level and decreases hCG receptors in the testicular Leydig cells for more than 120 h. Injected hCG, measured in plasma using a specific radioreceptor assay for gonadotropins, is maximal at 2 h and decreases thereafter with an apparent half-life of 16 h. Plasma testosterone exhibits a rapid increase (30-40 ng/ml) within 1h after hCG injection. A delayed paradoxical increase (20-30 ng/ml) is observed between 48 and 120 h after the injection. The number of hCG binding sites in the isolated Leydig cells membranes decreases to less than 10% of the control value within 10 h and remains almost undetectable until 96 h after hCG injection. Reappearance of the binding sites is observed around 120 h. Similar, but less pronounced effects are found after the injection of 10 IU hCG. Since receptor occupancy cannot explain such a phenomenon, it is concluded that hCG is exerting a negative control on its own receptors in the Leydig cells.     

hCG suppression of LH receptors and responsiveness of testicular tissue to hCG.

A single injection of 75 IU of human chorionic gonadotropin (hCG) into adult male rats caused a dramatic reduction in the concentration of membrane receptors for luteinizing hormone (LH) in the testis. The mean receptor level reached a nadir which was 5--10% of that in the control testes, 3 days after the injection, after which it gradually returned toward normal. This cannot be due to increased competition caused by the injected hCG since no decrease was observed at a time when the circulating levels of hCG were at a maximum (2--24 h after injection). Furthermore, at a time when receptor levels had been maximally reduced, circulating hCG was at or below the level of detection. Reduction in the number of LH binding sites in the testis was associated with a decreased responsiveness of the testicular tissue to hCG as measured by hCG-stimulated testosterone production in vitro. This inhibitory effect of large quantities of LH on its own receptor is suggested as a possible explantation for the previously observed low concentrations of LH receptor in the testis of the testicular feminized male (tfm) rat. This syndrome is characterized by high endogenous levels of plasma LH (Sherins et al., 1971).     

Characterization of gonadotropin-sensitive adenylate cyclase activity in human testis: uncoupling of the receptor-cyclase complex by specific hormonal antagonist

Basal and gonadotropin stimulated adenylate cyclase activity was assessed in testicular tissues obtained from men (20-80 years). A disparity was observed in the gonadotropin responsiveness of the human testicular adenylate cyclase system to hFSH and hCG stimulation. Of the tissues analyzed, 61% were FSH responsive and 22% showed low response to hCG. Forskolin, a diterpene which activates adenylate cyclase by a receptor independent mechanism, stimulated adenylate cyclase activity in the gonadotropin unresponsive tissues. This suggests that the tissue unresponsiveness is due to an uncoupling of the catalytic subunit of the adenylate cyclase. Several functional properties of the FSH responsive human testicular adenylate cyclase were investigated. hFSH and oFSH stimulated the enzyme activity in a concentration dependent manner. However, the hormone (DG-oFSH) in which 80% of the carbohydrate residues had been removed was inactive, despite its good binding ability to the FSH receptor. hFSH stimulated adenylate cyclase activity was inhibited by DG-oFSH but not by DG-hCG (deglycosylated hCG). The data demonstrates the existence of specific FSH and LH(hCG) receptors in human testicular membranes. The FSH receptors in some tissues are coupled to adenylate cyclase. The link between the FSH receptor and adenylate cyclase may be uncoupled in the presence of the deglycosylated form of oFSH resulting in a loss of hormone response.     

Aprotinin antagonizes the binding of human chorionic gonadotropin to its receptor

Aprotinin caused a dose-dependent inhibition of [125I]hCG binding to its receptor in plasma membranes prepared from luteinized rat ovaries. Displacement of [125I]hCG from its receptor by aprotinin was temperature dependent, with an ID50 of 300 microM at 4 C and an ID50 of 3700 microM at 22 C. Equilibrium binding data showed a decrease in the Ka for hCG in the presence of increasing concentrations of aprotinin; aprotinin had no effect on the number of binding sites. The rate of association of hCG with its receptor was markedly reduced in the presence of aprotinin, but aprotinin had little effect on the rate of dissociation. Control experiments established that aprotinin did not inhibit the binding of [125I]hCG to its receptor due to its lectin properties, an ionic effect or some irreversible impairment of the receptor or hormone. Aprotinin did not inhibit hCG binding when the receptor was solubilized out of ovarian membranes with Triton X-100. Aprotinin inhibited the hCG-mediated activation of adenylate cyclase in rat ovarian plasma membranes in a dose-dependent manner. Similarly, aprotinin antagonized the hCG-stimulated biosynthesis of progesterone by dispersed rat luteal cells. The maximal agonistic activity of hCG was attenuated in these two bioassays. The data are compatible with a hypothesis that aprotinin sterically hinders the entry of hCG into its receptor site by binding to a protease in the plasma membrane that is closely associated with the hCG receptor. This proposal is consistent with the emerging concept that integral membrane proteases are modulators of receptor function.     

Receptor binding dependent structural changes in human choriogonadotropin: photochemical inter-subunit crosslinking

Activation of surface receptors is thought to occur in multiple transient steps with conformational adjustments of hormones and receptors beginning from the initial hormone-receptor contact. In this study, we have established a sensitive photochemical crosslinking method to detect structural change of hCG upon receptor binding. hCG consists of an α subunit and a β subunit. Free α subunit was derivatized with photosensitive reagents and reassociated with unmodified β subunit. Reassociated hCG αβ dimer was capable of high affinity receptor binding and activation. The reagents attached to the α subunit were capable of crosslinking the α subunit to the β subunit. However, the extent of inter-subunit cross-linking in solution was two—three fold greater than inter-subunit crosslinking after hCG bound to the receptor. This difference indicates a novel structural change at the subunit interface in response to hCG binding to the receptor. Although highly unlikely, other microenvironmental factors might have interfered with the crosslinking efficiency without impacting the structure of hCG. This study lays the ground work to precisely define the location and nature of the change. Such information will be crucial for the understanding of the molecular mechanism of the hormone-receptor interaction and receptor activation.     

Identification of hormone-binding regions of the luteinizing hormone/human chorionic gonadotropin receptor using synthetic peptides.

A comprehensive series of overlapping synthetic peptides have been used to study the relationship between the primary structure of the ovarian receptor for LH/human CG (hCG) and hormone binding. Twenty-four consecutive, overlap peptides that replicate the entire extracellular domain of the rat luteal receptor have been synthesized by standard solid-phase techniques on an automated synthesizer. Eight additional peptides from the extracellular domain and three peptides replicating the putative extracellular loop regions have also been synthesized. Each peptide was evaluated in RRAs for interaction with hCG by measuring its ability to competitively inhibit binding of 125I-hCG to membrane receptor. Twelve peptides were found to be potent in RRAs and caused a reduction of half-maximal binding of 125I-hCG at concentrations of 10-250 x 10(-6) M. The 12 active peptides (and adjacent inactive peptides) defined at least 4 independent receptor regions that can interact with hormone. One site near the NH2-terminus was localized to receptor residues Arg21-Pro38. Two more sites of hormone interaction were identified by peptides replicating residues Arg102-Thr115 and Tyr253-Phe272. A fourth binding region was identified in the third putative extracellular loop, replicated by rat luteal receptor peptide Lys573-Lys583. The amino acid sequences of the four active rat LH/hCG receptor regions were aligned and compared with published sequences for other glycoprotein hormone receptors. Three regions (Arg102-Thr115, Tyr253-Phe272, and Lys573-Lys583) showed high sequence homology with the human LH/hCG receptor, human TSH receptor, and rat FSH receptor and may represent contact sites for the alpha-subunit of hormone. The other binding region, Arg21-Pro38 had low sequence homology with the other glycoprotein hormone receptors and is postulated to be a binding determinant for beta-hCG/LH. This report demonstrates that synthetic overlap peptides of confirmed sequence can be used to successively identify hormone interaction sites of glycoprotein hormone receptors.     

Involvement of plasma membrane enzymes in the proteolytic cleavage of luteinizing hormone receptor

In vitro degradation of LH receptor after occupancy by hCG was studied. Rat ovarian membranes labeled with [125I]iodo-hCG were incubated at 37 C; as a result, 30-40% of the radioactivity initially bound was rendered soluble in the medium. The molecular complexes in the medium and in incubated membranes solubilized with 1% Triton X-100 were then cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Particulate receptor-[125I]iodo-hCG complex exhibiting an apparent mol wt of 125,000 was cleaved during incubation into two distinct components (mol wt, 96,000 and 74,000) which appeared in the medium. Using tritiated hCG (beta-subunit labeled) instead of radioiodinated hCG (alpha-subunit labeled), these same two components were also observed, indicating that they both contain intact hCG (alpha and beta) as a part of their structure. In addition to the hormone (mol wt, 48,000), these two components contain receptor fragments with mol wt of 64,000 or 38,000, demonstrated directly by labeling the particulate receptor itself with periodate-tritiated borohydride before tagging with unlabeled hCG and in vitro incubation. These receptor fragments were purified from the medium by hCG-directed immunoaffinity chromatography and detached from the hormone by pH treatment. The intact receptor extracted from the membranes with detergent and purified identically in the absence of proteolysis migrated as a 90,000 mol wt polypeptide. These results demonstrate that after hormone occupancy, proteolytic cleavage of the 90,000 mol wt receptor polypeptide occurs at two specific sites. Thiol-blocking agents selectively prevented the appearance of the larger component (hCG coupled to 64,000 mol wt receptor fragment), while metal-chelating agents markedly decreased the appearance of the smaller component (hCG coupled to 38,000 mol wt receptor fragment) in the medium. Identical observations, obtained upon incubation of plasma membranes purified by sucrose density gradient centrifugation, suggest that plasma membrane enzymes are involved.     

Development of heterologous down-regulation of lactogen receptors in the rat testis

Gonadotropin-induced loss (down-regulation) of testicular lactogen receptors was studied in 5–60-day-old rats. An i.m. injection of 600 IU/kg of hCG elicited in 5-day-old animals a 38–56% increase in testicular lactogen binding, measurable between 6 and 72 h after the hormone injection. In contrast, at the age of 60 days the same hCG dose decreased lactogen receptors by 75% at 24 h, which loss recovered in 2–3 days. When the amount of lactogen receptors was measured 24 h after the hCG injection (600 lU/kg) at different ages, the binding increased on average by 50% between ages 5 and 20 days, no effect on binding was seen at day 30, and a loss of binding by 50% was evident in animals of 40 days of age and older. The present results indicate that the plasma-membrane events elicited by gonadotropin binding to neonatal Leydig cells are clearly different from those occurring in the adult, and that down-regulation and functional coupling of LH and lactogen receptors are accompaniments of functional differentiation of the Leydig cell during pubertal maturation.     

The mode of action of LHRH agonists on the rat Leydig cell

The relationship between LHRH agonist-receptor interaction and subsequent stimulation of steroidogenesis has been investigated using dispersed adult rat Leydig cells. Binding of 125I-labelled LHRH agonist to these cells was rapid and was readily and completely reversible, which contrasted with the binding of 125I-labelled hCG which was never completely reversible and which became progressively less reversible with increase in time. Following 125I-LHRH agonist-receptor interaction for 5 min at 21 degrees C, over 80% of the bound hormone dissociated at a fast rate (t 1/2 1.5 min) and the remainder at a slower rate (t 1/2 20 min). Following LHRH agonist-receptor interaction for a longer period (45 min) at 21 degrees C, only the slower of these 2 components of dissociation was evident, although binding was still completely reversible. Evidence was also obtained that following preincubation of Leydig cells for 2 hr at 34 degrees C with a near-saturating concentration (2000 pg/ml) of unlabelled LHRH agonist, all or most of the bound hormone dissociated during subsequent incubation at 21 degrees C. Incubation of Leydig cells at 34 degrees C in the continuous presence of either a low dose (1 pM) of hCG or a high dose (2000 pg/ml) of LHRH agonist resulted in similar percentage stimulation of testosterone secretion with a similar time lag (2 h) before this increase was evident. Preincubation with this dose of hCG for as little as 15 or 30 min still led to increased testosterone secretion following removal of the free hormone. In contrast, preincubation with LHRH agonist for up to 2 h was without subsequent stimulatory effect, presumably due to dissociation of the bound hormone from its receptors. Therefore, for LHRH agonist to stimulate Leydig cell steroidogenesis in vitro, it must be present continuously in the incubation medium, and the implications of this with respect to the secretion and mode of action of 'testicular LHRH' in vivo are discussed.     

Conversion of human choriogonadotropin into a follitropin by protein engineering.

Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the alpha subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their beta subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG beta-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH beta and hFSH beta in receptor recognition and activation. Since the beta subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH beta subunits, coexpressed them with the human alpha subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH beta residues 33-52 for hCG beta residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG beta residues 108-145 or substitution of residues in the "determinant loop" located between hCG beta residues 93 and 100.     

Phosphorylation and glycosylation of the luteinizing hormone receptor.

Purified testicular and ovarian luteinizing hormone/human chorionic gonadotropin (hCG) receptors are phosphorylated at serine and threonine residues by the catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). Occupancy of the receptors by hCG significantly increased the rate but not the extent of phosphorylation. However, prolonged preincubation of receptors with hCG reduced the subsequent rate of receptor phosphorylation. Identical phosphopeptide maps were obtained for the phosphorylated ovarian and testicular receptors. The phosphorylated receptor, like the native receptor, bound to wheat germ lectin and hCG-Sepharose and migrated as a single band of Mr 90,000 (testis) and Mr 85,000 (ovary) on NaDodSO4/PAGE. Neuraminidase treatment of receptors caused reductions of molecular weight to 82,000 (testis) and 77,000 (ovary), and further treatment with O-Glycanase had minimal effect on molecular size. However, deglycosylation with N-Glycosidase and endoglycosidase F produced a single labeled polypeptide of Mr 59,000 for both gonadal receptors. Treatment of native receptors with neuraminidase caused no apparent change in binding of gonadotropin to blotted receptors, whereas deglycosylated receptors showed a major reduction in hormone binding. These results indicate that luteinizing hormone/hCG receptors are sialoglycoproteins with predominantly N-linked glycosyl residues that account for the size difference between testicular and ovarian receptors and that may participate in the interaction with gonadotropin. Receptor occupancy by agonist leads to a conformational change that facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to hCG.     

Testicular receptors of human chorionic gonadotrophin in adult men. Binding and degradation of the hormone

Binding and degradation of human chorionic gonadotrophin (hCG) to testicular tissue obtained by biopsy from 9 men with gonadal disorders were investigated. Vacant hCG receptors were assayed in partially purified testicular homogenates using [125I]hCG (radioiodinated with chloramine T). Degradation of [125I]hCG during exposure to human testicular preparations was measured in terms of the ability of supernatants to specifically bind to rat testicular receptors. Binding of [125]hCG was time and temperature dependent. At 37 degrees C, a maximum was reached at 8 h. It was also found to be a saturable process with respect to homogenate and hormone concentrations. Association constants and number of binding sites determined in 9 men, using Scatchard plot and saturation curve analysis ranged, respectively, from 0.2 to 1.8 x 10(10) M-1 and from 92 to 3427 fmol/g testis or 7 to 380 fmol/mg protein. Degradation of [125I]hCG increased with temperature and time of exposure to human testicular homogenate. It increased also with increasing human testicular homogenate concentration and substrate concentrations. For a similar concentration of [125I]hCG, per cent of degraded hormone ranged from 32 to 57, according to the subjects. These results show that human testicular homogenates are capable of binding and degrading hCG in vitro. Biological and physiological implications of degradation for hormone binding are discussed.     

Changes in ovarian luteinizing hormone and follicle-stimulating hormone receptor content and in gonadotropin-induced ornithine decarboxylase activity during prepubertal and pubertal development of the female rat

Ovarian hCG and FSH receptor content was measured at different postnatal ages (days 4-32), at 4-day intervals, and expressed as counts per min 125I-labeled hormone bound/micrograms DNA. Specific FSH binding was minimal at day 4 and increased to a maximum at day 28, with the greatest rate of increase between day 4 and 16. hCG receptor content increased 6-fold between day 4 and 32, but contrasting with FSH, its greatest rate of increase occurred between day 16 and 28. The effectiveness of in vivo treatment with LH in inducing ovarian ornithine decarboxylase (ODC) activity, measured in 20,000 X g supernatants by the in vitro formation of 14CO2 from labeled ornithine, increased significantly during prepubertal development (days 21-33), paralleling the changes in hCG receptors. Ovarian hCG and FSH receptor content was also measured during the time of puberty. Specific hCG binding increased from anestrus (juvenile 32-day-old animals) to 1300 h of the first proestrus. The levels declined significantly by 1600 h of first proestrus and reached minimal values by the morning of estrus. Specific FSH binding also increased from anestrus to first proestrus, but to a much lesser degree, the levels showing only minor fluctuations during the rest of the cycle. hCG receptors measured in granulosa cells during puberty increased to an even greater extent than in the whole ovary; binding exhibited an 8-fold increase from anestrus to 1300 h of first proestrus and then declined to much lower values on estrus and the first diestrus. The capacity of LH to induce ovarian ODC activity increased markedly between anestrus and first proestrus, declining thereafter, and again paralleled the changes in LH receptor content. Evaluation of the kinetics of binding of hCG and FSH with their respective receptors in ovaries of 28-day-old rats revealed that the binding of hCG reached equilibrium within 4-8 h, whereas the binding of FSH attained equilibrium between 12-24 h. In both cases, however, the labeled hormones were clearly dissociable from their respective receptors by addition of an excess of unlabeled hormone. Moreover, although the ovarian receptor content of both hCG and FSH increased during prepubertal development, the equilibrium association constant of either hormone with its receptor remained unchanged. The results suggest that the infantile increase in FSH receptors may be induced by the high serum FSH levels present at that age and that, as shown by others in hypophysectomized rats, the subsequent increase in hCG receptors is, at least in part, an FSH-dependent phenomenon. The parallel changes in hCG receptors and LH-induced ODC activity during pre- and peripubertal development strongly suggest that an increase in LH binding capacity plays a fundamental role in the process of ovarian growth. It is also suggested that the changes in the steroidogenic ovarian response to hCG previously observed in peripubertal rats are, at least in part, determined by changes in ovarian LH receptor content.     

Osmolytes improve the reconstitution of luteinizing hormone/human chorionic gonadotropin receptors into proteoliposomes.

Rat ovarian membrane luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor was reconstituted into proteoliposomes. The ability of sodium cholate to extract and reconstitute hCG binding activity was dependent on the protein/detergent ratio. Trypsinization of the LH/hCG receptor containing proteoliposomes indicated that approximately 57% of hCG binding sites were oriented extravesicularly. The presence of 20% glycerol or other osmolytes during reconstitution increased the accessibility of LH/hCG receptors but not the activity of adenylate cyclase in proteoliposomes. This beneficial effect was independent of any specific detergent or its presence during detergent solubilization of proteins. Dynamic properties of membranes were monitored by electron spin resonance of 16-, 12-, and 5-doxyl stearic acid. Reconstituted proteoliposomes contain less ordered membrane lipids than do native membranes. Addition of glycerol before reconstitution increased the order of lipid bilayer and shifted it to the physical state of the native membrane. These findings are consistent with the hypothesis that a rise in membrane ordering increases the accessibility of membrane-bound LH/hCG receptors.     

Inhibition of synthesis of luteinizing hormone (LH) receptors by a down-regulating dose of LH

Although LH is known to cause down-regulation of the LH/human CG (hCG) receptor, the mechanisms underlying this process are not well understood. Here we have analyzed the effects of LH on the turnover of LH receptors in cultured granulosa cells. Down-regulation was stimulated in FSH-primed granulosa cells by exposure to LH for 1 h. By 24 h after LH exposure, the LH/hCG receptor content, as measured using [125I]iodo-hCG was 70% less than controls. To determine whether this loss of LH/hCG receptors was due to accelerated receptor degradation, turnover measurements were made using tunicamycin to inhibit LH/hCG receptor synthesis. Under these conditions, there was a small (16%) increase in the rate of degradation of LH/hCG receptors in LH-treated cells compared to controls. By contrast, the rate of LH/hCG receptor synthesis, estimated mathematically, was reduced by 75% in the LH-treated cells. LH treatment had no effect on the incorporation of [35S]methionine into total cellular protein. Withdrawing FSH from the culture medium had a similar effect, slightly enhancing LH/hCG receptor degradation while markedly reducing its synthesis. The role of lysosomes in the degradation of LH/hCG receptors was also investigated. The lysosomotropic amine NH4Cl failed to cause LH/hCG receptors to accumulate, contrary to what would be expected if LH/hCG receptors were degraded lysosomally. In contrast, NH4Cl inhibited the degradation of receptor-bound [125I]iodo-hCG, suggesting that the ligand and the receptor may be handled differently by the granulosa cell. Our results suggest that in the granulosa cell, LH down-regulates its own receptor by inhibiting the synthesis of LH/hCG receptors, possibly by interfering with the ability of FSH to stimulate the synthesis of LH/hCG receptors. Furthermore, they suggest that the degradation of LH/hCG receptors may occur by nonlysosomal mechanisms.     

Luteinizing hormone receptors are self-associated in the plasma membrane

We have evaluated rat LH receptor self-association and lateral dynamics for functional and nonfunctional receptors after binding of hormone. We demonstrate, for the first time, that grouped receptors observed in electron or light microscopy represent actual receptor dimers or oligomers rather than simply the concentration of receptors within membrane microdomains. Fringe fluorescence photobleaching recovery methods showed that, after binding of either LH or human CG (hCG), functional wild-type LH receptors, expressed on 293 cells (LHR-wt cells), have mobilities that are 25% lower than those of nonfunctional LH receptors containing an arginine substitution for lysine at position 583 (LHR-K583R cells). Because lateral diffusion coefficients in two dimensions depend only on the logarithm of the molecular size of the diffusing species, this result implies that functional receptors exist in substantially larger membrane complexes than do nonfunctional receptors. In single-cell measurements of fluorescence energy transfer after hormone binding, functional LH receptors were also characterized by receptor self-aggregation. Values for fluorescence resonant energy transfer efficiency were 13 +/- 2% and 17 +/- 3% between fluorophore-conjugated LH or hCG, respectively, bound to receptors on LHR-wt cells. However, there was little or no energy transfer between receptors on LHR-K583R cells. These results suggest that receptor functionality involves receptor-receptor interactions and that the extent of such receptor self-association depends on whether LH or hCG binds the receptor.     

Nonreceptor binding of human chorionic gonadotropin (hCG): detection of hCG or a related molecule bound to endometrial tissue during pregnancy using labeled monoclonal antibodies that bind to exposed epitopes on the hormone

When hCG adsorbs to surfaces, including membranes from tissues that lack specific hCG receptors, it adsorbs with a particular orientation. Some sites on the alpha- and beta-subunits project away from the surface and can be detected with radiolabeled monoclonal antibodies. Other epitopes, which are located on a region on the hormone that presumably contacts the surface, lose their ability to bind antibody. Using antibodies specific for epitopes on hCG which remain exposed and can be detected when the hormone is adsorbed to rat brain homogenates, we found hCG or closely related substances bound to progestational decidual tissues. Immunologically reactive material adsorbed to the decidual tissue increased and decreased in parallel with the serum levels of hCG throughout pregnancy. Binding of labeled monoclonal antibody to substances similar or identical to hCG in other tissues, including placenta and fetal lung, but not red cells, also was identified. Unlike material adsorbed to decidual tissues, receptor-bound hCG was not recognized by any of our alpha-subunit-specific antibodies. This finding suggests either that the adsorbed hormone has a different orientation than receptor-bound hormone or that the adsorbed hormone has dissociated into subunits. These studies represent the first detection of nonreceptor binding of hCG or related molecules to tissues lacking receptors or presumed not to synthesize the hormone. The role of nonreceptor-bound hCG, if any, is unknown. Other than its effects on stimulation of luteal steroidogenesis during early pregnancy, the role of hCG during most of pregnancy has not been determined. Conceivably, the nonreceptor binding we identified is related to a role for hCG in pregnancy that is not associated with an action on the ovarian LH receptor.