Modulation of the neutrophil functional activity by chorionic gonadotropin]

The functions of nonpregnant woman neutrophils in the presence of chorionic gonadotropin (CG) have been studied, as well as possible mechanisms of intracellular hormone signaling. Expression of adhesion molecule CD18 and phagocyte activity of the cells are shown to be inhibited by a high dose of CG (100 IU/ml). Oxygen metabolite production by activated neutrophils decreases almost in a half in the presence of hormone, irrespective of the dose. Synthesis of nitric oxide, an oxidant and effective regulator molecule, is also suppressed by CG. Neutrophil incubation with hormone induces a dose-dependent modulation of intracellular cAMP level. The effect of CG is most strongly expressed for a low hormone dose (10 IU/ml), implying the presence of high affinity hormone-binding structures on the cell membrane. Judging from the correlation of data obtained, the functional effects of CG cannot be attributed to the regulation of adenylatcyclase activity and suggests that this is not the major mechanism of hormone signal transduction. Neutrophil sensitivity to suppressive CG effects is one of the ways of regulating nonspecific defence reactions in different physiological and/or pathological states connected with the presence of this hormone in the organism.     

Dissociation of human choriogonadotropin from the pseudopregnant rat ovary as a complex to a receptor fragment during perifusion and incubation

Pseudopregnant rats were injected intravenously with radioactively-labelled human choriogonadotropin (CG). The animals were killed 2 h after the injection and the ovaries, liver and kidney were subjected to perifusion. Radioactivity was released from the ovaries at an increasing rate during perifusion, mainly in a complex form with a molecular size between human CG and the solubilized receptor-human CG complex. The increase in the rate of radioactivity release was inhibited by N-ethylmaleimide and CuCl2, but not by MgCl2, Trasylol, N alpha-tosyl-L phenylalanine chloromethyl ketone or N alpha-p-tosyl-L-lysine and CuCl2 chloromethyl ketone. Intact hormone dissociation from the complex at pH 3. After perifusion the ovarian tissue radioactivity only as receptor-hormone complexes. Only free radioiodine released from the control tissues, liver and kidney during perifusion. The low molecular weight hormone complex was also released from a homogenate of pseudopregnant rat ovaries prelabelled in vivo with radioactivity-labelled human CG during incubation in a hypotonic medium. The release of this complex was likewise inhibited by alkylating agents and heavy metals, and intact hormone dissociated from the complex at pH 3. A similar human CG complex was released also from purified receptor-human CG complex during incubation with ovarian homogenate, and presence of N-ethylmaleimide or use of heat inactivated ovarian homogenate inhibited this process. The present results indicate that the in vivo bound human CG sheds from the luteal tissue in perifusion and incubation as a low molecular weight complex. This may be a facet in the processing and elimination of occupied LH receptors from the ovary.     

Eleven amino acids (Lys-201 to Lys-211) and 9 amino acids (Gly-222 to Leu-230) in the human thyrotropin receptor are involved in ligand binding.

Our previous studies involving chimeric thyrotropin-lutropin/choriogonadotropin (TSH-LH/CG) receptors suggest that multiple segments spanning the entire extracellular domain of the human TSH receptor contribute to the TSH binding site. Nevertheless, the mid-region (segment C, amino acid residues 171-260) of the receptor extracellular domain is particularly important in TSH binding. In the present studies, we constructed seven new chimeric receptors in order to analyze segment C in further detail. Seven small segments spanning segment C of the TSH receptor were replaced with the counterpart of the rat LH/CG receptor. These mutant receptors were stably introduced into Chinese hamster ovary cells and were tested for hormone binding and cAMP responsiveness to hormone stimulation. The results indicate that 11 amino acids of the TSH receptor (Lys-201 to Lys-211) and the corresponding region of the LH/CG receptor (Thr-202 to Ile-212) are important for specific TSH and human CG binding, respectively. In addition, nine amino acids of the TSH receptor (Gly-222 to Leu-230) are also involved in TSH binding. A further conclusion from these data is that TSH and human CG bind to partially overlapping sites on their respective receptor molecules.     

Effects of chorionic gonadotropin on dynamics of primary immune response]

The effect of chorionic gonadotropin (CG) on primary immune response was estimated according to the level of direct and indirect plaque-forming cells (PFC) on day 5, 8 and 12 after immunization of non-castrated and ovariectomized female mice of CBA strain. It was established, that on the 5th day CG (40-200 IU) did not influence the direct PFC level in ovariectomized animals, but stimulated them in non-ovariectomized mice (40 IU). In ovariectomized animals the selective immunodepressive effect of hormone on the IgG-PFC formation processes has been revealed. The CG effect depended on the time of PFC number examination as well as on the hormone dose. In non-castrated animals, where immunomodulating CG effects are partially mediated by ovarian hormones, the injection of hormone only in the dose of 200 IU significantly lowered the number of IgM and IgG-PFC. It is suggested, that sex steroids on the late stages of PFC formation, when the processes of isotype antibody synthesis switch take place, appear to be synergists of CG immunodepressive effect.     

Effect of chorionic gonadotropin on the cooperative interrelations of splenocytes involved in the primary immune response

The influence of chorionic gonadotropin (CG) on the interaction of spleen T- and B-cells has been studied in adoptive transfer system. It has been established that CG increases the primary immune response in non-ovariectomized mice. This effect reversely depended on the hormone concentration and was independent of prostaglandins (PG). In the experiments on ovariectomized mice the influence of CG was opposite and the immune response was decreased. This action was completely abolished by Voltren-induced blockade of UG-synthetase. In spleen cell culture of female mice CG suppresses the synthesis of interleukin-2 (IL-2). It is suggested that CG may have an independent immunosuppressive action, the mechanism of which consists of disturbed cell-to-cell communications on the level of short-acting mediators of the immunity--PG and IL-2.     

The position of the alpha and beta subunits in a single chain variant of human chorionic gonadotropin affects the heterodimeric interaction of the subunits and receptor-binding epitopes

The glycoprotein hormone family represents a class of heterodimers, which include the placental hormone human chorionic gonadotropin (CG) and the anterior pituitary hormones follitropin, lutropin, and thyrotropin. They are composed of common alpha subunit and a hormone-specific beta subunit. Based on the CG crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, recent observations using single chain glycoprotein hormone analogs, where the beta and alpha subunits are linked (NH(2)-CGbeta-alpha; CGbetaalpha orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for receptor binding/signal transduction. To study the heterodimeric alignment of the two subunit domains in a single chain and its role in the intracellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of alpha was fused to the CGbeta amino terminus (NH(2)-alpha-CGbeta; alphaCGbeta orientation). The secretion rate of alphaCGbeta from transfected Chinese hamster ovary cells was less than that seen for CGbetaalpha. The alphaCGbeta tether was not recognized by dimer-specific monoclonal antibodies and did not bind to lutropin/CG receptor. To define if one or both subunit domains were modified in alphaCGbeta, it was co-transfected with a monomeric alpha or CGbeta gene. In each case, alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes were formed indicating that CG dimer-specific epitopes were established. The alphaCGbeta/alpha complex bound to receptor indicating that the beta domain in the alphaCGbeta tether was still functional. In contrast, no significant receptor binding of alphaCGbeta/CGbeta was observed indicating a major perturbation in the alpha domain. These results suggest that although dimeric-like determinants are present in both alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes, the receptor binding determinants in the alpha domain of the tether are absent. These results show that generating heterodimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are distinct from those associated with intracellular behavior.     

Effects of collagenase on the structure of the lutropin/choriogonadotropin receptor

The structure of the lutropin/choriogonadotropin (LH/CG) receptor of a clonal strain of cultured Leydig tumor cells (designated MA-10) and primary cultures of porcine granulosa cells was studied by cross-linking 125I-labeled derivatives of human CG and ovine LH with bifunctional succinimidyl esters. We show that in both cell types, both subunits of the receptor-bound hormone become cross-linked to a single cellular component of Mr = 106,000, when analyzed in the absence of reducing agents, and of Mr = 83,000 when analyzed in the presence of reducing agents. We also present a detailed investigation on the effects of several collagenase preparations on the structure and some functions of the LH/CG receptor. Our results show that the LH/CG receptor is exquisitively sensitive to degradation by these preparations of collagenase; degradation products can be detected only in the presence of reducing agents; the enzyme(s) responsible for degradation is not collagenase itself, but rather a contaminating enzyme(s), presumably a protease(s); and receptor degradation has little effect on the ability of the cells to bind hormone or to respond with increased steroid biosynthesis. Since normal gonadal cells are usually isolated following dispersion of the tissue with collagenase, our results suggest that these cells are likely to bear a degraded (albeit functional) form of the LH/CG receptor, and thus should not be used in studies dealing with the structure of this receptor.     

The nucleotide sequences of baboon chorionic gonadotropin beta-subunit genes have diverged from the human

The placental glycopeptide hormone chorionic gonadotropin (CG) is involved in establishing and maintaining pregnancy. The hormone consists of two different non-covalently associated subunits termed alpha and beta. In man there are seven closely linked genes coding for beta CG-like peptides, but only three of these appear capable of expression in the placenta. The organization of beta CG-like genes in man and baboon appears to be similar. We demonstrate here that the baboon genome contains multiple copies (at least five) of beta CG-related genes, and that these genes are closely linked in the genome. Nucleotide sequence data from several beta CG cDNA clones indicates that at least two of these beta CG-related genes are expressed in the baboon placenta. Analysis of beta CG sequences from baboons and human subjects demonstrates that these genes have diverged markedly between species. In contrast, these sequences are remarkably homogeneous within their respective genomes. Gene conversion events may be responsible for retaining the high degree of identity among the various beta CG gene family members. Knowledge of beta CG sequences from baboon may lead to development of a long-term antipregnancy vaccine. The ability of CG antibodies to interfere with the maintenance of pregnancy can now be investigated within a homologous system.     

Mutagenesis and gene transfer define site-specific roles of the gonadotropin oligosaccharides

Human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone and thyroid-stimulating hormone are a family of glycoprotein hormones that share a common alpha subunit but differ in their hormone-specific beta subunits. Using site-directed mutagenesis and gene-transfer, we analyzed the role of the N-linked oligosaccharides of alpha and chorionic gonadotropin (CG)beta in the secretion, assembly, and biologic activity of hCG. Absence of carbohydrate at alpha asparagine (Asn) 52 decreased combination with CG beta but did not alter monomer secretion. Absence of the alpha Asn78 oligosaccharide increased the degradation of the alpha subunit, but the presence of CG beta stabilized this alpha mutant in an efficiently formed dimer complex. Alternatively, absence of both alpha oligosaccharides slowed both secretion and dimer formation but allowed an intermediate level of alpha secreted or dimerized compared to the single-site mutants. Analysis of the CG beta glycosylation mutants revealed that absence of the Asn30 oligosaccharide, but not Asn13, slowed secretion but not assembly, whereas absence of both oligosaccharides slowed both secretion and dimer formation. Analysis of the receptor binding of the hCG glycosylation mutants showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on receptor affinity of the derivatives. However, the absence of alpha Asn52, but not the alpha Asn78 or the CG beta carbohydrate units, reduced the steroidogenic effect, unmasked differences in the beta oligosaccharides, and converted the deglycosylated derivatives into antagonists.     

Structural differences in the hinge region of the glycoprotein hormone receptors: evidence from the sulfated tyrosine residues

Tyrosine sulfation is a late posttranslational modification of proteins that takes place in the Golgi network. In the past few years, this process has been identified as an important modulator of protein-protein interactions. Sulfated tyrosine residues have recently been identified in the C-terminal, so-called hinge region of the ectodomain of glycoprotein hormone receptors [TSH, LH/chorionic gonadotropin (CG), and FSH receptors] and were shown to play an important role in the interaction with their natural ligands. The position of two sulfated tyrosine residues in a Y-D/E-Y motif appears perfectly conserved in the alignment of TSH and LH receptors from different species, and site-directed mutagenesis experiments demonstrated that sulfation of the first residue of this motif was responsible for the functional effect on hormone binding. In contrast, the corresponding motif is not conserved in the FSH receptor, in which the first tyrosine residue is missing: the Y-D/E-Y motif is replaced by F(333)DY(335). We extend here our previous observation that, in this case, it is sulfation of the second sole tyrosine residue in the motif that is functionally important. An LH/CG receptor harboring an F(331)DY(333) motif (i.e. displaying decreased sensitivity to human CG) was used as a backbone in which short portions of the FSH receptor were substituted. Segments from the FSH receptor capable of restoring sensitivity to human CG were identified by transfection of the chimeras in COS-7 cells. These experiments identified key amino acid residues in the hinge region of the FSH receptor associated with the functional role of the second sulfated tyrosine residue in a Y-D/E-Y motif, allowing for efficient hormone binding. The experiments represent strong evidence that structural differences in the hinge regions of FSH and LH/CG receptors play a significant role in hormone-receptor-specific recognition.     

Effects of chorionic gonadotropin on peripheral monocytes vary with the phases of the menstrual cycle

The qualitative and quantitative effects of physiological concentrations of chorionic gonadotropin (CG) on monocytes of women vary with the phases of the menstrual cycle. During the follicular phase, the hormone inhibits phagocytosis; stimulates the secretion of interleukin (IL) 6 (IL-6), interferon α (IFN-α), and the protein component of apolipoprotein A1 (APO-A1); and activates myeloperoxidase (MPO). During the luteal phase, CG stimulates phagocytosis and APO-A1 secretion, inhibits MPO, and does not shift the levels of IL-6 and INF-α. Regardless of the menstrual phase, the hormone does not modify the release of elastase or the production of granulocytic colony-stimulating growth factor, IL-1α, and tumor necrosis factor α.     

The significance of chorionic gonadotropin in regulating the antigen-independent differentiation of immunocompetent splenic cells]

The functional activity of fractionated splenocytes were investigated in the syngeneic transfer system. Chorionic gonadotrophin (CG) was injected to ovariectomized and noncastrated lymphocyte donors, the T or B lymphocytes being isolated on a column with a nylon fiber and by means of anti-BaO-serum accordingly. In some series CG was introduced only into recipients before their being irradiated and before syngeneic cells transferring. CG has been stated to be capable of influencing either independently or by means of ovarian hormones splenic B lymphocytes without exhibiting a significant effect on T helper precursors as well as radioresistant cells of the splenic stroma. The character of immunoregulating effect was completely dependent on the presence of ovaries in mice and on the hormone dose.     

Role of cAMP and Neutrophil Cyclooxygenase in Gonadotropin-Dependent Regulation of T Lymphocyte Proliferation

The effect of the main pregnancy hormone, chorionic gonadotropin (CG), on proliferation of peripheral blood mononuclear cells (PBMC) was studied in the presence of autologous neutrophils; also, hormone-dependent regulation of the cAMP levels in T lymphocytes and neutrophils was evaluated. PBMC proliferation in response to a mitogen is suppressed by physiological doses of CG (10, 50, and 100 IU/ml). Autologous neutrophils enhance the suppression induced by the low dose of CG (10 IU/ml), but when cyclooxygenase was inhibited this effect was not observed; this suggests that the anti-proliferative effects of the low dose of CG can be mediated by the products generated by neutrophil cyclooxygenase. The effect of CG was associated with increased cAMP levels in T lymphocytes and neutrophils. Comparison of functional and cAMP-related effects of CG in both cell populations indicates that cAMP is involved in the anti-proliferative effects of CG.     

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.     

Effect of chorionic gonadotropin on the structure and endocrine function of the thymus gland in mice

The thymic secretory function changes in the pubertal female mice under the influence of chorionic gonadotrophin (CG). The latter is activated after administration of hormone in doses, corresponding to its levels in animals in different terms of pregnancy and is depressed with doses, greatly exceeding those levels. The lack of the effect in thymectomized mice testifies to the realization of the modulating CG action at the thymus level. Changes of mass and cellularity of the thymus and spleen reflect dose-dependent alteration of the thymocyte migration rate.     

Partial characterization of chorionic gonadotropin-like binding sites from the bacteria Xanthomonas maltophilia

The gram-negative bacterium, Xanthomonas maltophilia, has low- and high-affinity luteinizing hormone/chorionic gonadotropin (LH/CG)-binding sites, similar to the LH/CG receptor found in mammals. Although the low-affinity site binds both LH and human CG (hCG), the high-affinity site is specific for hCG. In the current investigation, these two binding sites were independently isolated from X. maltophilia for further characterization. To isolate functional binding sites, we developed a solubilization method using the detergent zwittergent 3,14 and high glycerol concentrations that allowed for the maintenance of ligand-binding integrity. Gel filtration experiments established molecular weights of 170 and 11.5 kDa for the two binding sites, which were supported by data from photoaffinity labeling and ultracentrifugation experiments. Gel filtration data also suggested the presence of a third binding site of 5.4 kDa. The 170-kDa site had a binding affinity of Kd = 12 x 10(-6) and bound both LH and hCG. The small molecular weight site had an affinity of Kd = 9.4 x 10(-8) and was CG specific. Collectively, these data demonstrate the presence of multiple hormone binding sites in X. maltophilia that differ in molecular size, binding affinity, and ligand specificity.     

Expression of recombinant human choriogonadotropin in Chinese hamster ovary glycosylation mutants

Human CG, a member of the glycoprotein hormone family that includes LH, FSH, and TSH, is composed of two nonidentical subunits each containing two asparagine linked (N-linked) oligosaccharides. The role of the oligosaccharides in the action of these hormones is unclear. To examine the structure-activity relationships of the glycoprotein hormone oligosaccharides using nonenzymatic and nonchemical methods, we transfected CG subunit genes into mutant cell lines derived from Chinese hamster ovary cells. Two mutant cell lines that synthesize truncated oligosaccharides were used. Cell line 15B, lacking N-acetylglucosaminyltransferase I, synthesizes N-linked carbohydrates containing Man5 oligomannosyl structures, and 1021, defective in transporting CMP-sialic acid into the Golgi, results in sialic-acid deficient glycoproteins. The binding of these derivatives to the LH/CG receptor did not differ significantly from purified CG (CR119), but the ability of the mutant hormones to stimulate cAMP biosynthesis in vitro is reduced compared to wild-type CG or CR119. Since the amino acid sequence of CG from the mutant and wild-type cells is identical, these data indicate that oligosaccharide structures, while not influencing receptor binding, directly affect signal transduction.     

Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity

The lutropin-choriogonadotropin (LH/CG) receptor is a cell surface receptor comprised of two domains of roughly equivalent size. The amino-terminal half of the receptor is relatively hydrophilic and is located extracellularly, whereas the carboxyl-terminal half of the receptor shares amino acid homology with other receptors that couple to G proteins and is similarly thought to span the plasma membrane seven times, ending with a relatively short carboxyl-terminal tail. In order to test the role of the extracellular domain in binding hormone, we constructed a mutated rat luteal LH/CG receptor cDNA (termed pCLHR-D2), which encodes for only the extracellular domain, and used it to transiently transfect human kidney 293 cells. Here we report that the expressed extracellular domain of the LH/CG receptor is capable of binding human CG with a high affinity, comparable with that of the full-length receptor. Thus, not only is the extracellular domain of the glycoprotein hormone receptors involved in binding hormone, but it alone is capable of conferring high affinity binding. Unexpectedly, it was also found that this truncated receptor is not secreted into the culture media but remains trapped within the cells.     

A new subclass of the luteinizing hormone/chorionic gonadotropin receptor lacking exon 10 messenger RNA in the New World monkey (Platyrrhini) lineage

The luteinizing hormone receptor (LHR) plays an essential role as a mediator of LH and CG action during embryonic sexual differentiation and in gametogenesis. In a hypogonadal male patient, we recently demonstrated that a genomic deletion of exon 10, located in the hinge region of the extracellular domain, results in discrimination of LH and hCG action. In the common marmoset (Calltithrix jacchus), exon 10 of the LHR is naturally missing at the mRNA level. In order to investigate whether this is an isolated species-specific phenomenon, we performed a phylogenetic screening, searching for the presence of LHR exon 10 mRNA in a number of primate species representative for the major lineages of primate evolution. The expressed LHR region encompassing exon 10 was amplified from testicular tissue by RT-PCR, cloned, and sequenced. In addition, we performed Southern blot analysis of the LHR of selected New World and Old World primates. The results revealed that exon 10 mRNA is lacking in the complete New World monkey (Platyrrhini) lineage but is present in both more primitive and more advanced primates. However, exon 10 seems to be present at the genomic level, arguing for a splicing failure possibly due to a genomic mutation or the lack of appropriate splicing factors. Considering that, in the human, LH is far less active than hCG on the LHR lacking exon 10, we addressed the question whether the existence of such a receptor has any consequences on the dual hormone LH/CG system present in Platyrrhini. Using primers specific for the known marmoset CG beta cDNA, we amplified the CG beta subunit cDNA from male common marmoset pituitaries by RT-PCR, while LH beta could not be amplified, suggesting a possible physiological role of pituitary CG in this species. In conclusion, we demonstrated for the first time that the LH mRNA without exon10 is the natural wild-type LHR in the Platyrrhini lineage. We propose that this LHR represents a new subclass of receptors that should be named LHR type II. In addition, the high expression of CG beta in the marmoset pituitary suggests a physiological role of CG in the reproductive function of these primates beyond pregnancy.     

beta-arrestin-dependent desensitization of luteinizing hormone/choriogonadotropin receptor is prevented by a synthetic peptide corresponding to the third intracellular loop of the receptor

Desensitization is a ubiquitous response of guanine nucleotide-binding protein-coupled receptors (GPCRs) characterized by the waning of effector activity despite continued presence of agonist. Binding of an arrestin to the activated, often phosphorylated GPCR triggers desensitization. We reported for the luteinizing hormone/choriogonadotropin receptor (LH/CG R) that beta-arrestin tightly bound to porcine ovarian follicular membranes mediates agonist-dependent desensitization of LH/CG R-stimulated adenylyl cyclase (AC) activity (Mukherjee, S., Palczewski, K., Gurevich, V. V., Benovic, J. L., Banga, J. P., and Hunzicker-Dunn, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 493-498). We now show that addition of a synthetic peptide corresponding to the entire third intracellular loop (3i) of the LH/CG R completely and specifically reverses desensitization of AC activity, with an ED50 of 10 microM but does not modulate basal, hCG-stimulated, or forskolin-stimulated AC activities. beta-Arrestin binds selectively to the 3i peptide coupled to activated Sepharose. Desensitization of LH/CG R-stimulated AC activity is rescued when the 3i peptide is preincubated with exogenous beta-arrestin. These results show that endogenous beta-arrestin participates in cell-free desensitization of agonist-dependent LH/CG R-stimulated AC activity in follicular membranes by interacting directly with the 3i loop of the receptor, thereby preventing Gs activation.