Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain.

Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.

Luteinizing hormone/human chorionic gonadotropin receptors in breast cancer.

Recent studies have suggested that human choriogonadotropin (hCG), in addition to its function in regulating steroidogenesis, may also play a role as a growth factor. Immunocytochemistry using two different monoclonal antibodies (LHR29 and LHR1055) raised against the human luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor allowed us to detect this receptor in breast cancer cell lines (T47D, MCF7, and ZR75) in individual cancer biopsies and in benign breast lesions. The receptor was also present in epithelial cells of normal human and sow breast. In the latter, its concentration increased after ovulation. The presence of LH/hCG receptor mRNA was confirmed by reverse transcription-PCR using primers extending over exons 2-4, 5-11, and 9-11. The proportion of LH/hCG-receptor positive cells and the intensity of the immunolabeling varied in individual biopsies, but there was no obvious correlation with the histological type of the cancer. These results are compatible with previous studies suggesting that during pregnancy, hCG is involved in the differentiation of breast glandular epithelium and that this hormone may play an inhibitory role in mammary carcinogenesis and in the growth of breast tumors.

Internalization and recycling pathways of the thyrotropin receptor.

Scant information is available to date on the intracellular trafficking of the TSH receptor. In the present study we have used stably transfected L cells that express the TSH receptor, 225I-labeled TSH, and antireceptor antibodies as well as gold-conjugated antireceptor monoclonal antibodies and hormone. The latter allowed us to study, by electron microscopy, the cellular distribution and endocytosis of TSH receptor. The receptor was initially localized on the plasmalemma proper and in clathrin-coated pits but was excluded from smooth vesicles open to the cell surface. It was internalized through clathrin-coated vesicles. Constitutive endocytosis represented 10% of cell surface receptor molecules. Endocytosis was increased 3-fold by incubation with hormone. The majority of internalized receptor molecules (90%) was recycled to the cell surface, whereas the hormone was degraded in lysosomes. This recycling of receptor was inhibited by administration of monensin. Electron microscopic and confocal microscopic studies were repeated in primary cultures of human thyroid cells and showed a distribution, and endocytosis pathways, very similar to those observed in transfected L cells. A previous study has shown the LH receptor to be endocytosed in high proportion and to be degraded in lysosomes. Confocal microscopy and colocalization studies with transferrin receptor confirmed that the highly homologous LH and TSH receptors exhibit, when expressed in the same cells, very different cellular trafficking properties. The use of LH/TSH receptor chimeras showed that transmembrane-intracellular domains contain information orienting the protein toward recycling or degradative pathways. The extracellular domain seems to play a role in the extent of intemalization. These observations should now allow the identification of the molecular signals involved.

Comparison of immunocytochemical and molecular features with the phenotype in a case of incomplete male pseudohermaphroditism associated with a mutation of the luteinizing hormone receptor.

We report the case of an infant who presented at birth with a hypoplastic phallus associated with hypospadias. Low testosterone production, normal serum levels of steroid precursors, and increased LH in response to LH-releasing hormone supported a defect in Leydig cell differentiation or function. Conventional microscopic study of the testes showed fibroblastic cells in the interstitium. However immunocytochemical analysis using anti-LH receptor and anti-P450c17 antibodies demonstrated that about one third of these cells were Leydig cells or precursors of Leydig cells. No histological feature could distinguish the latter cells from fibroblasts. A homozygous substitution of cysteine 133 for arginine was found in the extracellular domain of the receptor. This is the first naturally occurring missense mutation found in the extracellular domain of the LH receptor. COS-7 cells transfected with the mutant receptor exhibited a marked impairment of hCG binding, whereas some cAMP production could be observed at high hCG concentrations. We propose that the partial impairment of LH receptor function, as reflected by the presence of Leydig cells, was responsible for the incomplete male pseudohermaphroditism observed in our patient.

The postmenopausal ovary is not a major androgen-producing gland.

It is currently believed that the postmenopausal ovary remains a gonadotropin-driven, androgen-producing gland. However, the adrenal contribution to circulating androgen levels may explain some conflicting results previously reported. In addition, the steroidogenic potential and gonadotropin responsiveness of the postmenopausal ovary have not been recently reassessed. Plasma T, bioavailable T, free T, androstenedione (Adione), and dehydroepiandrosterone sulfate levels were measured in postmenopausal or ovariectomized women with complete adrenal insufficiency, compared with women with intact adrenals. A stimulation human chorionic gonadotropin test (on d 0, 3, and 6) was performed in postmenopausal women with adrenal insufficiency. Dexamethasone was administered for 4 d in postmenopausal women with intact adrenals. Intraovarian T and androstenedione were also measured in homogenates of ovarian tissue from postmenopausal women. Immunocytochemistry was performed on postmenopausal ovaries and premenopausal controls to detect the presence of steroidogenic enzymes (P-450 aromatase, P-450 SCC, 3beta HSD, and P-450 C17) and gonadotropin receptors. Plasma androgen levels were below or close to the limit of the assay in all women with adrenal insufficiency. They were similar in postmenopausal and oophorectomized women with normal adrenals. No hormonal changes were observed after human chorionic gonadotropin injections in women with adrenal insufficiency. In contrast, a dramatic decrease of all steroids was observed after dexamethasone administration in postmenopausal women with intact adrenals. Intraovarian T and androstenedione levels were negligible in postmenopausal ovarian tissue. P-450 aromatase was absent from the 17 ovaries studied, and the enzymes for androgen biosynthesis were either absent (n = 13) or present in very low amounts (n = 4). In all the postmenopausal ovaries, FSH and LH receptors were completely absent. In the absence of adrenal steroids, postmenopausal women have no circulating androgens. This result is consistent with the immunocytochemical studies showing the almost constantly absent steroidogenic enzymes and LH receptors in the postmenopausal ovary. Thus, the climacteric ovary is not a critical source of androgens. The arrest of androgen secretion after menopause may impact significantly on women's health.

The LH/CG and FSH receptors: different molecular forms and intracellular traffic.

Monoclonal antibodies have been raised against the LH/CG receptor [1] and have allowed to perform immunochemical studies of the receptor in target cells. Three different forms of the LH/CG receptor are physiologically expressed: a mature approximately 85 kDa transmembrane species corresponding to the full length receptor, a approximately 68 kDa high mannose containing species corresponding to a precursor which accumulates inside the cells, and truncated soluble approximately 45-48 kDa molecular weight species corresponding to the variant messanger RNAs generated by alternative splicing. Monoclonal antibodies against the human FSH receptor were also prepared. They allow to observe the existence of two forms of the FSH receptor in the ovaries: a major approximately 87 kDa species corresponding to the mature receptor and a minor approximately 81 kDa species corresponding to a high mannose rich precursor. No variant forms of the receptor corresponding to alternative mRNA transcripts were detected. The transport of hCG was examined in rat testicular microvasculature by electron microscopy and by analyzing the transfer of radiolabeled hormone and antireceptor antibodies. LH/CG receptors were present in endothelial cells and were involved in hormone transcytosis through these cells. Immunocytochemical experiments have shown that the FSH receptor has a polarized expression in the Sertoli cells of the testes whereas the LH/Cg receptor is spread on the surface of thecal granulosa and luteal cells in the ovary and Leydig cells in the testes. To study the mechanism of this polarization FSH, LH and TSH receptors were expressed in polarized MDCK cells. The mechanism of basolateral localization and of transcytosis of the receptors was studied using this model. The effect of hormone, cAMP and agents acting on G proteins was examined.

Mechanisms of nuclear localization of the progesterone receptor.

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638-642 and was shown to be constitutively active. When amino acids 638-642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or anti-hormone), it was almost fully active. A second putative nuclear localization signal is located in the DNA binding domain activated either through ligand binding or through production of constitutive receptor. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand the receptor was transferred into the nucleus as a monomer. After administration of hormone (or anti-hormone) a "cytoplasmic" monomer was transferred into the nucleus through interaction with a "nuclear" monomer. These interactions occurred through the steroid binding domains of both monomers.

Expression of components of the insulin-like growth factor system and gonadotropin receptors in bovine cumulus-oocyte complexes during oocyte maturation.

IGF system expression has been largely explored in the bovine follicular wall whereas it remains poorly studied in the COC. Using semi-quantitative RT-PCR and Western blot analysis, we have investigated spatial and temporal expression of IGF-1, IGFR-1, IGFBP-2, IGFBP-4, as well as gonadotropin receptors in bovine COC during oocyte maturation. In addition, we have compared changes in the IGF system and FSHR expression during in vitro maturation in TCM199 alone or in the presence of 10 ng/ml of EGF. The transcripts for IGFR-1 and IGFBP-2 were detected in cumulus and germinal cells whereas IGF-1, IGFBP-4 and FSHR mRNA were restricted to cumulus cells. Topography of the IGF system and gonadotropin receptor expression within COC were unaffected by the maturation step. In contrast, levels of IGFBP-2 and FSHR expression decreased (P < 0.05) in matured COC. Under defined culture conditions, IGFBP-2 and FSHR mRNA expression remained at a high level in TCM199 alone and were significantly reduced (P < 0.05) in the presence of 10 ng/ml EGF after a 24 h period of in vitro maturation. In conclusion, our results demonstrate a cell-specific pattern of IGF system member gene expression within bovine COC suggesting interaction between the somatic and germinal compartments. In addition, synchronized changes in the pattern of COC IGFBP-2 and FSHR expression during oocyte maturation suggest possible synergistic actions between IGF-1 and FSH.

[LH and TSH receptors. A new family of G protein-coupled receptors]. / Récepteurs de la LH et de la TSH. Une nouvelle famille de récepteurs couplés à des protéines G.

Monoclonal antibodies have been raised against porcine LH receptor and allowed to clone the corresponding messenger RNA from testicular cells. Cross hybridisation with the LH receptor clone allowed to isolate a clone corresponding to the human TSH receptor from thyroids. The structure of both receptors have been determined. They show similarities but also differences to other G protein coupled receptors. In particular a large extracellular domain is specific of that new family of receptors. Variant forms of the LH receptor lacking transmembrane domains have been isolated. The obtention of monoclonal antibodies against both receptors allowed immunochemical and immunocytochemical studies to be performed. The human LH receptor gene have been localized to chromosome 2p21 and TSH receptor gene to chromosome 14q31. The complete organisation of the human TSH receptor gene has been determined.

[LH receptors. A new family of G-protein receptors]. / Récepteur de la LH. Une nouvelle famille de récepteurs couplés à des protéines G.

Monoclonal antibodies have been raised against porcine LH receptor and allowed to clone the corresponding messenger RNA from testicular cells. The structure of the LH receptor have been determined. It shows similarities but also differences to other G protein coupled receptors. In particular a large extracellular domain is specific for that family of receptors. Variants forms of the LH receptor generated by alternative splicing and lacking transmembrane domains have been isolated. Immunochemical and immunocytochemical studies have been performed. Three different forms of the LH receptor are physiologically expressed: a mature 85 kDa transmembrane species, a 68 kDa high mannose containing species corresponding to a precursor which accumulate inside the cells, and truncated 45-48 kDa molecular weight species corresponding to the variant messenger RNAs identified during the cloning of the receptor. A novel zonation of the ovary has been described by immunocytochemical studies. Cross hybridisation with the LH receptor clone allowed to isolate the related human TSH receptor from thyroïds. The human LH and FSH receptor genes have been localized to chromosome 2p21 and the TSH receptor gene to chromosome 14q31. The genes are very large and have introns only within their 5' part corresponding to the extracellular domain of the receptor.

[Pituitary glycoprotein hormone receptors]. / Les récepteurs des hormones glycoprotéiques hypophysaires.

Monoclonal antibodies have been raised against the porcine LH receptor and have allowed to clone the corresponding messenger RNA from testicular cells. The stricture of the LH receptor has been determined. It shows similarities but also differences with other G protein coupled receptors. Specially a large extracellular domain is specific of that new family of receptors. Variant forms of the LH receptor generated by alternative splicing and lacking transmembrane domain have been isolated. Immunochemical and immunocytochemical studies have been performed. Three different forms of the LH receptor are physiologically expressed: a mature 85kDa transmembrane species, a 68 kDa high mannose containing species corresponding to a precursor which accumulates inside the cells, and truncated 45-48kDa molecular weight species corresponding to the variant messenger RNAs identified during the cloning of the receptor. A novel zonation of the ovary has been described by immunocytochemical studies. Cross hybridization with the LH receptor clone allowed to isolate the related TSH receptor from human thyroid tissue. The human LH and FSH receptor genes have been localized to chromosome 2p21 and the TSH receptor gene to chromosome 14q31. The genes are very large (> 60 kbp) and have introns only within the 5' part encoding the extracellular domain of the receptor. Immunoelectron microscopic studies performed in Leydig cells and in stably transfected L cells have allowed to study intracellular traffic of the LH receptor. The same approach was used to study the transendothelial transfer of hCG in testicular microvasculature.

Gonadotropin receptors.

Gonadotropin receptors belong to a subgroup of G-protein coupled receptors characterized by a large extracellular domain responsible for the binding of the hormone. Soluble, hormone-binding, alternative splicing variants of the LH receptor, are present in high concentration. A mannose rich precursor form of LH and FSH receptor is accumulated inside target cells. FSH receptors are addressed to the basolateral domain of cells through specific signaling mechanisms. Gonadotropin receptors are also present in endothelial cells of target organ vessels and are involved in hormone transcytosis. Various genetic abnormalities of these receptors (and of the GnRH receptor) are discussed.

Phosphorylation of transfected wild type and mutated progesterone receptors.

An expression vector encoding wild type or mutated forms of the rabbit progesterone receptor was transfected into COS-7 cells and phosphorylation was studied by incubation with 32Pi followed by specific immunoprecipitation. The features of phosphorylation of the wild type receptor were identical to those previously observed in uterine cells: there was a basal level of phosphorylation which was increased approximately 7-fold by incubation with the hormone. The hyperphosphorylated receptor had decreased electrophoretic mobility ("upshift"). These experiments thus showed that the presence of the receptor specific kinase is not restricted to the target cells. Cleavage of the receptor by hydroxylamine and cyanogen bromide, and use of receptor mutants deleted in the N-terminal region, showed the absence of any detectable phosphorylation downstream from amino acid 520 (thus in the DNA and steroid binding domains). The majority of the phosphorylation sites were localized between amino acids 166 and 520. This localization was similar for basal and hormone-induced phosphorylation. DNA binding and hormone-induced hyperphosphorylation were not directly related, since deletion of the first zinc finger provided a hyperphosphorylated receptor. We showed that the constitutive receptor (totally deleted in the steroid binding region) exhibited only a low basal level of phosphorylation, and antagonist RU 486-receptor complexes were found to be hyperphosphorylated, leading us to conclude that the active form of the receptor was not the hyperphosphorylated one. Moreover receptor down regulation and hormone-induced receptor hyperphosphorylation were two independent phenomena. Basal phosphorylation was observed for both cytoplasmic and nuclear mutants, whereas nuclear localization was necessary but not sufficient for hyperphosphorylation. Finally, the second finger region and the hormone binding domain, which are necessary for receptor hyperphosphorylation, may be involved in the hormonally induced increased affinity of the receptor toward its kinase.

Mechanism of action of an antiprogesterone, RU486, in the rabbit endometrium. Effects of RU486 on the progesterone receptor and on the expression of the uteroglobin gene.

RU486 is a recently described antiprogesterone. In order to be able to understand its mechanism of action it is necessary to analyze its effect on a discrete gene product. We show here that the induction of uteroglobin mRNA by progesterone in the rabbit endometrium may be a suitable model for such studies since RU486 totally inhibits this effect without itself exerting any agonistic activity. Moreover, RU486, which does not bind to the estrogen receptor and is devoid of general antiestrogenic activity, partially inhibits the induction by estradiol of uteroglobin mRNA. Studies of the interaction between [3H]RU486 and the progesterone receptor have been undertaken with the aim of understanding the antagonistic effect of this compound. The binding to DNA-cellulose of heat-activated [3H]RU486-receptor complexes was slightly decreased (37%) when compared with that of the agonist [3H]R5020-receptor complexes (47%). Detailed analysis of this difference showed that it was due to both a decreased activation of complexes and to a diminished affinity of activated complexes towards DNA. The change in activation was shown by the fact that at high concentrations of DNA, where all activated complexes are bound, agonist-receptor complexes were bound to DNA in higher proportion than antagonist-receptor complexes. Moreover a difference was also observed when studying the binding of agonist-receptor and antagonist-receptor complexes to charged resins (phosphocellulose, DEAE-cellulose) which are known to discriminate between activated and non-activated complexes. Decreased affinity to DNA of antagonist-receptor complexes was shown by studying their binding at various concentrations of DNA, either in crude cytosol or after isolating a homogenous population of activated-receptor complexes by DNA-cellulose chromatography and by comparing the salt extraction from DNA-cellulose of agonist-receptor and antagonist-receptor complexes. Both effects (decreased activation and diminished affinity towards DNA) were relatively moderate and could account only for a small decrease in the agonistic activity of RU486. Thus, the fact that this compound is a complete antagonist without any agonistic activity can only be explained by a defect in some further step of hormone action as, for instance in the specific interaction with the regulatory regions of the uteroglobin gene. No immunological difference could be detected between [3H]R5020-receptor and [3H]RU486-receptor complexes, both interacted with the five monoclonal antibodies raised against purified R5020-receptor complexes.(ABSTRACT TRUNCATED AT 400 WORDS)

The chromosomal localization of the human follicle-stimulating hormone receptor gene (FSHR) on 2p21-p16 is similar to that of the luteinizing hormone receptor gene.

Two cDNA probes (5' and 3' region) corresponding to the human follicle-stimulating hormone receptor gene (FSHR) were used for chromosomal localization by in situ hybridization. The localization obtained on chromosome 2p21-p16 is similar to that of the luteinizing hormone/choriogonadotropin (LH/CG) receptor gene.

[Uteroglobin]. / L'utéroglobine.

Uteroglobin has been studied under two aspects: 1) as a model of specific interaction between a protein and a steroid hormone: crystals were obtained and analyzed by X-ray diffraction; 2) as a marker of progesterone action in the endometrium: messenger RNA was translated, purified and transcribed into complementary DNA.

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit.

A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell surface protein disulfide-isomerase is involved in the shedding of human thyrotropin receptor ectodomain.

In human thyroid glands the TSH receptor undergoes a cleavage reaction which yields to an extracellular alpha subunit and a membrane spanning beta subunit linked together by disulfide bridges. A similar reaction is observed in transfected L cells although some uncleaved monomers persist in these cells. We have recently shown that the alpha subunit of the TSH receptor undergoes partial shedding in human thyroid cells and heterologous cells permanently transfected with an expression vector encoding the receptor. This shedding is a two-step process. The first step consists in the cleavage of the proreceptor at the cell surface probably by a matrix metalloprotease and the second step in the reduction of the disulfide bridge(s) (Couet, J., Sar, S., Jolivet, A., Vu Hai, M. T., Milgrom, E., & Misrahi, M. 1996, J. Biol. Chem. 271, 4545-4552). We have used the transfected L cells to study the second step involved in sTSHR shedding. The membrane impermeant sulfhydryl reagent DTNB (5,5'-dithiobis(2-nitrobenzoic acid) allowed us to confirm that the reduction of the TSH receptor disulfide bonds occurred at the cell surface. The antibiotic bacitracin even at low concentrations also elicited a marked inhibition of TSH receptor shedding. This led us to implicate the enzyme protein disulfide isomerase (PDI, EC 5.3.4.1) in this process. We thus tested the inhibitory activity of specific monoclonal antibodies raised against PDI. All antibodies elicited a marked inhibition of sTSHR shedding. This confirmed that cell surface PDI is involved in the shedding of the TSH receptor ectodomain. The shed alpha subunit may be at the origin of circulating TSH receptor ectodomain detected in human blood.