Biologic effect of a hybrid preparation of human chorionic gonadotropin in human subjects

Alpha and beta-hCG subunits were recombined generating a hybrid hCG preparation (AB1ER-CR-2XY) which met the required specifications of a pharmaceutical product. The biologic activity contained in each vial of AB1ER-CR-2XY was equivalent to 10 000IU of hCG-IS. This preparation was given as a single dose of 10 000IU by the i.m. route to four female subjects presenting unexplained infertility. The hCG hybrid was demonstrated to effect gonadal stimulation in humans.     

Effect of a partially desialylated human chorionic gonadotropin preparation (Desialo hCG) on ovarian function

Six patients with unexplained infertility were administered a partially desialylated hCG preparation (Desialo hCG). 5000 and 10000 IU of this preparation were given as a single IM dose, following the administration of hMG-Pergonal. The circulatory half-life (t 1/2) of Desialo hCG was found to be 42.6 hours, which is similar to the t 1/2 of 35.6 hours previously reported for a hybrid hCG preparation. The t 1/2 values were shorter than the t 1/2 of 65.3 hours estimated for commercial hCG administered by the same route. In spite of the similarities in t 1/2 values between the two preparations, serum progesterone levels were significantly increased 48 to 96 hours after the administration of Desialo hCG, and only 8 to 10 hours after the administration of the hCG hybrid. The difference in response may reflect differences in receptor occupancy at the ovarian level. Large scale testing of preparations with half-lives approaching the t 1/2 of the physiological hormone, hLH, should be carried out in order to determine if the risk of overstimulation and/or multiple pregnancies could be reduced thus, allowing for a safer therapeutic modality in patients pre-treated with hMG.     

Human chorionic gonadotropin-ricin A chain hybrid protein: a hormone analog for the study of signal transduction

We have prepared a disulfide cross-linked hybrid consisting of human chorionic gonadotropin and the toxic ricin A chain, utilizing N-succinimidyl-3-(2-pyridyldithio)-propionate. The purified hybrid exhibited cytotoxicity toward rat testis Leydig cells due to inhibition of protein synthesis within the cells, although it inhibited protein synthesis in a cell-free system of a rabbit reticulocyte lysate only after treatment of the hCG-A hybrid with dithiothreitol. On the other hand, pretreatment of the hCG-A hybrid with dithiothreitol completely abolished its cytotoxicity. The hCG-A hybrid stimulated the testosterone production by rat testis Leydig cells to a similar degree to hCG. The hCG-A hybrid could elicit full testosterone production in the presence of about 1/3-1/2 less accumulated cAMP compared with in the case of hCG stimulation. The steroidogenetic effect of the hCG-A hybrid was largely inhibited by anti-hCG antibody but not by anti-ricin A chain antibody. These results suggest that the hCG-A hybrid first binds to Leydig cells via an hCG receptor and then is internalized into the cells, and finally the inhibition of cellular protein synthesis occurs on the release of the ricin A chain from the hybrid. Furthermore, it was suggested that hCG may stimulate testosterone production via another second messenger system as well as via the cAMP system, since the hCG-A hybrid stimulated full testosterone production without maximal cAMP accumulation.     

Purification and characterization of monoclonal antibodies against the free α-subunit of human chorionic gonadotrophin

Monoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free alpha-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-alpha hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free alpha-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free alpha-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary hormones either as free subunits or intact molecules as follows: alpha hCG 100%; intact hCG 1.8%; beta hCG 0.14%; alpha FSH 24.5%; intact FSH 0.8%; beta FSH 0.09%; alpha LH 20.5%; intact LH 0.9%; beta LH 0.08%; alpha TSH 50.5%; intact TSH 3.7%; beta TSH 0.07%; The affinity constant (K) of this Mab with respect to free alpha-subunit of hCG was found to be 1.5 x 10(7) I/mol as determined by the simple antibody dilution analysis method.     

Necessity for two human chromosomes for human chorionic gonadotropin production in human-mouse hybrids

Through a series of human-mouse hybrids we have identified that two human chromosomes, 10 and 18, must be present for production of the pregnancy protein hormone human chorionic gonadotropin (hCG). Human choriocarcinoma cells producing hCG were hybridized to mouse cells. From 49 independent clones three hybrid clones continued to produce whole hCG. Chromosomal analysis was done on the 3 producer clones and 5 nonproducer clones. The additional 41 nonproducer clones were genetically characterized by isozymes. Only when chromosomes 10 and 18 were present in a clone would the whole hCG molecule be produced. Clones with only 10 or only 18 did not produce hormone. Nine subclones of a producer clone confirmed this observation. Three subclones retaining both 10 and 18 continued to produce hCG. This study demonstrated the need to use cellular chromosome data and population enzyme data to identify two chromosomes necessary for hCG production in heterogeneous human-mouse hybrids.     

Production of monoclonal antibodies to human chorionic gonadotropin

Production of monoclonal antibodies against human chorionic gonadotropin (hCG) has been studied using hCG as an immunogen. Spleen cells of BALB/c mice immunized with hCG were fused with NS-1 mouse myeloma cells. This study reports the successful isolation of a hybrid clone secreting a monoclonal antibody specific for hCG. By using PEG 4,000 as a fusion agent, the fusion rates were between 42.0 and 50.2%. In total 842 hybridomas were produced. Among them, 403 hybridomas had hCG antibody production. After cloning twice by limiting dilution and alternately screening by enzyme immunoassay and by radioimmunoassay, there were 39 cell lines having specific antibody production. Among them, the No. 57-42-2 had the highest reactivity. By Ouchterlony test, the monoclonal antibody was shown to be IgG1. The affinity constant of the antibody to hCG was 0.6 x 10(9) 1/mole. In radioimmunoassay, the cross reactivity of the antibody to human luteinizing hormone (LH) and human follicle-stimulating hormone (FSH) was 1.5% and 0.7%, respectively. There was no cross reaction with human thyrotropin-stimulating hormone (TSH).     

The use of drugs to dissect the pathway for secretion of the glycoprotein hormone chorionic gonadotropin by cultured human trophoblastic cells

Agents that affect intracellular cation and pH gradients and inhibit energy production have been tested for their ability to modulate the processing and secretion of the free alpha subunit and the alpha beta dimer of human chorionic gonadotropin (hCG) by cultured human trophoblastic cells (JAR). Incubation of JAR cells with monensin or nigericin, monovalent cation ionophores that produce equilibration of Na+ and K+ across cellular membranes, dicyclohexylcarbodiimide, an agent that inhibits intracellular membrane ATPases, and methylamine, which neutralizes intracellular pH gradients, produced similar effects on hCG processing and secretion. All these agents inhibited the processing of the asparagine-linked oligosaccharide chains of free alpha subunit and the alpha and beta subunits contained in the hCG dimer. Moreover, after treatment of JAR cells with these agents, there was an intracellular accumulation of precursor forms and an inhibition of secretion of "mature" forms of hCG. Monensin affected the processing and secretion of hCG subunits differently at different concentrations. At 5 X 10(-7) M, monensin inhibited the processing of the asparagine-linked oligosaccharides of hCG without altering the rate-limiting step in the secretory pathway or blocking hCG secretion. The intracellular hCG subunit precursors in both control and monensin-treated cells contained a similar array of high mannose oligosaccharides, predominantly of the Man8GlcNAc2 and Man9GlcNAc2 types. However, monensin-treated cells secreted hCG subunits that contained endo H-sensitive oligosaccharides of the high mannose (mostly Man5GlcNAc2) and hybrid types rather than the endo H-resistant complex chains synthesized by control cells. Nevertheless, a full complement of serine-linked oligosaccharides was added to the hCG-beta subunit in monensin-treated cells. These results indicate that the intracellular movement of hCG from the rough endoplasmic reticulum to the cell surface was not inhibited by monensin at a concentration that impaired Golgi-localized steps in the processing of asparagine-linked oligosaccharides. At 5 X 10(-6) M, monensin significantly inhibited secretion of hCG and created a new rate-limiting step in the processing pathway. hCG subunits bearing Man5GlcNAc2 units accumulated intracellularly, suggesting that the equilibration of intracellular Na+/K+ pools blocked oligosaccharide processing at an intra-Golgi point, perhaps by inhibiting movement of the glycoprotein hormone from the "cis" to the "trans" Golgi compartment. Since the other drugs mentioned above produced similar effects on hCG processing and secretion, it appears that maintenance of intracellular cation and pH gradients is necessary for the intra-Golgi transport of glycoprotein hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.     

Role of glucose in mouse preimplantation embryo development

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca × C57BL/6) were cultured from the one-and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D+L-lactate, in the presence and absence of 1 mM glucose (M16+G and M16-G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16-G were transferred to M16+G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16+G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16-G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 ± 2.5 [28] vs. 105 ± 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16-G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16-G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16+G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage. The results show that mouse preimplantation embryos from F1 hybrid mice (CBA/Ca × C57BL/6) need only be exposed to glucose for less than 24 h between 22 and 94 h post hCG in order to develop from the morula to the blastocyst stage in vitro. However, the exposure time needs to be increased to between 24 and 72 h in order that blastocyst cell numbers reach control levels. The importance of glucose before the morula stage may relate to the need to synthesize glycogen for later use. If the obligatory requirement for glucose is fulfilled, embryos are able to utilize pyruvate in the absence of glucose at the later stages of development. These results show that the mouse preimplantation embryo can, to some extent, adapt metabolically to changes in its external environment. © 1995 Wiley-Liss, Inc.     

Inhibition of adenylyl cyclase activity in rat corpora luteal tissue by glycopeptides of human chorionic gonadotropin and the alpha-subunit of human chorionic gonadotropin

Indirect evidence has indicated that the carbohydrate moieties of the glycoprotein hormones are involved in the activation of the receptor-adenylyl cyclase system of reproductive tissues. In the present study, we have isolated the glycopeptides (GP) from human chorionic gonadotropin (hCG), the alpha-subunit of hCG, fetuin, and bovine gamma-globulin (b gamma G). These along with a number of synthetic oligosaccharides were tested for their ability to inhibit adenylyl cyclase (AC). There was less than 0.001% cross-reactivity of the GP from hCG, hCG alpha, fetuin, and b gamma G when tested in a double-antibody hCG radioimmunoassay or rat corpora lutea radioreceptor assay. The GP of fetuin, b gamma G, and the synthetic oligosaccharides did not inhibit AC activity of 2000 g corpora lutea membranes when coincubated with 100 ng of hCG/mL (ED50). However, when the GP of hCG and hCG alpha were included with intact hCG, there was a dose-related inhibition. Inhibition of cyclase activity was enhanced when the hCG GP were desialylated. This occurred without a change in the lag time of hCG activation which was calculated to be 1-1.5 min. Changing the concentration of ATP and Mg2+ did not affect the inhibitory effects of the hCG alpha GP on hCG-stimulated AC activity. Inhibition by hCG GP followed uncompetitive kinetics. The inhibition by the GP of hCG seems to be restricted to the LH/hCG-stimulatable AC system because the same dosage of hCG GP which inhibited the rat luteal AC system did not have any effect on the rat hepatocyte AC system when coincubated with glucagon or on NaF-stimulated activity in luteal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of conformational changes in human chorionic gonadotropin upon binding to rat gonadal receptors

After binding to rat testicular or ovarian luteinizing hormone (LH) receptors, human chorionic gonadotropin (hCG) and mammalian LH can be detected with monoclonal antibodies directed against a conserved epitope on the beta subunit of the hormones. Two such anti-hCG/anti-LH monoclonal antibodies, known as B105 and B110, compete with one another for binding to this epitope region on free and receptor-bound hormone. By comparing the affinities of B105 and B110 for these two forms of hCG, we have detected apparent changes in the structure of the hormone which develop subsequent to receptor binding. Whereas the affinity of B105 for receptor-bound hCG is approximately 10-fold lower than that for free hCG, the affinity of B110 for receptor-bound hCG is nearly 20-fold greater than that for free hCG. Both B105.hCG and B110.hCG complexes bind to the receptor; however, they have approximately 25 and 50% lower affinity than hCG. Thus, although B110 binds better to the form of hCG which is bound to receptors, binding of B110 to hCG does not appear to induce a conformational change in the hormone which facilitates hormone-receptor binding. Consequently, both B105 and B110 partially inhibit binding of hCG to its receptors. Fab fragments of B105 and B110 are as effective as intact B105 and B110 in inhibiting the binding of labeled B105 and B110 to hCG-receptor complexes, suggesting that circular complexes which might be formed by the interaction of divalent antibody, two molecules of hCG, and two membrane-bound receptors or one divalent receptor are not contributing to the affinity of the antibodies for receptor-bound hCG. Alternatively, formation of circular complexes can explain an increase in apparent affinity of B105 for ovine or bovine LH-receptor complexes. Data obtained with B105 suggest either that the structure of the epitope is altered following binding or that a portion of the epitope is partially obscured when hCG binds to the receptor. In contrast, the data obtained using B110 are not explained by models in which steric factors reduce the affinity of the antibody for the hormone-receptor complex. Therefore, as a minimal explanation for these observations, we postulate that the conformation of the B105/B110 epitope region is altered following binding of the hormone to receptors. The nature of the conformational change and its relationship to LH/hCG action is unknown.     

Localization of residues that confer antibody binding specificity using human chorionic gonadotropin/luteinizing hormone beta subunit chimeras and mutants.

The glycoprotein hormones are a family of conserved heterodimeric proteins which share a common alpha subunit but differ in their hormone-specific beta subunits. We used chimeras of human chorionic gonadotropin (hCG) and luteinizing hormone (hLH) beta subunits to identify residues which enable monoclonal antibodies (mAb) to distinguish the two hormones. The LH beta-CG beta chimeras appeared to fold similar to hCG beta, since they combined with hCG alpha and, depending on their sequences, were recognized by hCG-selective mAbs. Amino acid residues Arg8-Arg10,Gly47-Ala51, and Gln89-Leu92 form a major epitope region and appear to be adjacent to each other on the surface of hCG beta. Gly47-Ala51 and Gln89-Leu92 are recognized by dimer-specific mAbs while Arg8-Arg10 is recognized by mAbs which have highest affinity for the free beta subunit. These observations suggest that the conformation of this region of the beta subunit changes when the alpha and beta subunits combine. Residues which are C-terminal of Asp112 form a second epitope domain. mAbs to the third domain distinguish hCG beta and hLH beta by the presence of Asn77 in hCG beta and can be detected after hCG binds to receptors. These findings were used to develop a model of hCG beta which predicts the locations of these residues and their positions relative to the alpha subunit and receptor interfaces.     

Regulation of progesterone secretion in human syncytiotrophoblast in culture by human chorionic gonadotropin.

In the present investigation we sought to define the specific sites in the pathway of placental progesterone biosynthesis that underlie the action of human chorionic gonadotropin (hCG). When the cells were challenged with dibutryl cAMP (dbcAMP), forskolin or isobutylmethylxanthine, they produced significantly higher amounts of progesterone which in the presence of the hCG antibody was reduced to the level of the control set of cells. Trophoblast cells cultured in serum free medium with 25-hydroxycholesterol (25-OHC) produced increased amounts of progesterone. In the presence of hCG antibody at a concentration which neutralized the secreted hCG, the steroid production was completely blocked, even when the 25-OHC was added to the medium. Also, direct quantitation of the cytochrome P450 SCC enzyme in the absence of hCG indicated a significant decrease. The exogenous addition of low density lipoproteins (LDL) increased the progesterone secretion by the trophoblast cells in culture. Neutralization of hCG by the antibodies, however, drastically reduced the LDL induced progesterone secretion, which was restored by the addition of dbcAMP to the medium. Based on these findings, we suggest a stimulatory effect of hCG on normal trophoblast cells at the level of LDL utilization and cytochrome P450 SCC enzyme. Since dbcAMP could mimic these actions of hCG, the data suggest a possible autocrine/paracrine role of hCG on the trophoblast cells. An additive effect of hCG and cAMP on progesterone secretion observed in our studies, indicate that apart from hCG, adenylate cyclase activity may also be regulated by other factors.     

Construction, purification and characterization of a recombinant baculovirus containing the gene for alpha subunit of human chorionic gonadotropin.

A cDNA encoding the alpha subunit of human chorionic gonadotropin, a placental glycoprotein hormone, was cloned downstream to the viral polyhedrin gene promoter of Autographa california nuclear polyhedrosis virus and the recombinant transfer vector was used to co-transfect Spodoptera frugiperda cells growing in culture. Recombinant baculovirus carrying the alpha hCG gene was detected and isolated after dot hybridization using supernatant from co-transfected cells. Recombinant vAc alpha hCG having a replacement of the viral polyhedrin gene, which is hyper-transcribed very late in the infection cycle, with the alpha hCG cDNA was purified after a single round of plaque purification. Insect cell culture infected with vAc alpha hCG, secreted high levels of hCG which was biologically active.     

Thyroidal responses to human chorionic gonadotropin in the chick and rat

The thyrotropic activity of human chorionic gonadotropin (hCG) has been examined in the chick and the rat. Uptake of 32PO4 by chick thyroid increased significantly with injection of bovine thyrotropin (bTSH) with a maximum response at 2.4 mU per chick. On the other hand, no significant stimulation of 32PO4 uptake was detected with injection of graded doses of highly purified hCG up to 0.25 mg per chick. 1 mg of partially purified hCG, equivalent in biological potency to the maximum dose of highly purified hCG used in the chick, did induce a significant increase in 32PO4 uptake. In rats, highly purified hCG stimulated a very significant release (p less than 0.001) of 125I from the thyroid and partially purified hCG had a thyrotropic activity equivalent to 0.42 microU bTSH/U hCG, identical to the value we reported in mice, 0.42 microU bTSH/U hCG. The duration of hCG action on thyroidal release of 125I in the rat was longer than that for bTSH, as it is in the mouse. hCG also induced a significant rise in the serum level of triiodothyronine in rats. We conclude that pure hCG is a weak thyrotropic substance in the rat but not in the chick. These results and other evidence suggest an inhibitory role for the densely glycosylated 30 amino acid residue C-terminal extension on the beta-subunit of hCG which limits, by steric hindrance, the interaction of the TSH-like hCG 'core' with thyrotropin receptors.     

N-linked oligosaccharides on free alpha interfere with its ability to combine with human chorionic gonadotropin-beta subunit.

The purpose of the study was to examine the role of N-linked oligosaccharides in preventing combination of free alpha molecules with human chorionic gonadotropin (hCG)-beta subunit to form the intact hormone, hCG. Culture media from JEG cells incubated in the presence or absence of Swainsonine were filtered on Sephadex G-100, and free alpha was identified by radioimmunoassay. Swainsonine interferes with glycosylation by inhibiting alpha-mannosidase II, resulting in formation of hybrid structures. Approximately 50% of the free alpha molecules from Swainsonine-treated cells (Swainsonine pool 2) had an apparent molecular size that was smaller than that of free alpha from control cells. The oligosaccharides of control alpha molecules were resistant to endo-beta-N-acetylglucosaminidase H treatment. In contrast, virtually all of the Swainsonine free alpha molecules contained endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides. Swainsonine free alpha and control free alpha molecules were incubated with an excess of hCG-beta subunit, followed by chromatography on Sephadex G-100. Each fraction was assayed by radioimmunoassay for intact hCG and for alpha. Less than 10% of control free alpha molecules combined with hCG-beta. In contrast, 54% of Swainsonine alpha pool 2 and 40% of Swainsonine alpha pool 1 combined with beta to form hCG. Thus, modulation of N-linked oligosaccharide processing converted free alpha molecules to forms that can combine with hCG-beta. These results indicate that the inability of a substantial portion of control free alpha molecules to combine with hCG-beta is due to the presence of N-linked oligosaccharide structures that interfere with combination.     

Upregulation of placental indoleamine 2,3-dioxygenase by human chorionic gonadotropin

We tested the hypothesis that hCG can upregulate human trophoblast indoleamine 2, 3-dioxygenase (INDO), which catalyzes the breakdown of tryptophan in villous circulation. The results revealed that it can. Treatment of human trophoblasts with hCG resulted in a time and dose dependent increase in INDO mRNA and protein levels and its enzyme activity. The hCG effect was hormone specific and required the dimer conformation of hCG. The hCG effect required its receptors and was mediated by a cAMP dependent, but protein kinase A independent, mitogen-activated protein kinase 3/1 (MAPK3/1) signaling mechanism. In summary, the present data demonstrate a novel hCG effect on human placental INDO, which probably plays a key role at maternal fetal interface in preventing fetal rejection.     

The beta-subunit of human choriogonadotropin interacts with the exodomain of the luteinizing hormone/choriogonadotropin receptor and changes its interaction with the alpha-subunit.

Human CG (hCG) consists of a common alpha-subunit and a hormone-specific beta-subunit. Similarly, its receptor is also composed of two domains, an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain). hCG initially binds the exodomain of the receptor after which the resulting hCG/exodomain complex is thought to interact with the endodomain. This secondary interaction is considered responsible for signal generation. Despite the importance, it is unclear which hormone subunit interacts with the exodomain or the endodomain. As a step to determine the mechanisms of the initial and secondary interactions and signal generation, we investigated the interaction of the hormone-specific beta-subunit in hCG with the receptor's exodomain. A photoactivable hCG derivative consisting of the wild-type alpha-subunit and a photoactivable beta-subunit derivative was prepared and used to label the exodomain. The analysis and immunoprecipitation of photoaffinity labeled exodomain demonstrate that the beta-subunit in hCG makes the direct contact with the exodomain.