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Glycoprotein hormone receptors (provisional nomenclature [47]) are activated by a non-covalent heterodimeric glycoprotein made up of a common α chain (glycoprotein hormone common alpha subunit CGA, P01215), with a unique β chain that confers the biological specificity to FSH, LH, hCG or TSH. There is binding cross-reactivity across the endogenous agonists for each of the glycoprotein hormone receptors. The deglycosylated hormones appear to exhibit reduced efficacy at these receptors [122, 31].

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Testotoxicosis is a rare cause of peripheral precocious puberty in boys caused by constitutively activating mutations of the LHCG receptor. Affected males usually have normal gonadotropin profiles and fertility in their adult life. Here, we described the long-term follow-up of a 24-year-old young man with severe testotoxicosis due to a de novo activating mutation in the third transmembrane helix of the LHCGR (p.Leu457Arg). This patient was treated with different medications, including medroxyprogesterone acetate, ketoconazole, cyproterone acetate and aromatase inhibitor from age 2.5 to 9.5 years. His basal and GnRH-stimulated gonadotropin levels were continually suppressed during and after medical treatment. At adulthood, extremely high serum testosterone levels (>35 nmol/L), undetectable gonadotropin levels (LH < 0.15 IU/L and FSH < 0.6 IU/L) and oligozoospermia were evidenced. Despite his suppressed FSH levels and an unfavorable spermogram, the patient fathered a healthy girl and biological paternity was confirmed through analysis of microsatellites. Spontaneous fertility in a young man with severe testotoxicosis and chronic suppression of FSH levels reinforces the key role of high intratesticular testosterone levels in human spermatogenesis.

Glycoprotein hormone receptors (provisional nomenclature [45]) are activated by a non-covalent heterodimeric glycoprotein made up of a common α chain (glycoprotein hormone common alpha subunit CGA, P01215), with a unique β chain that confers the biological specificity to FSH, LH, hCG or TSH. There is binding cross-reactivity across the endogenous agonists for each of the glycoprotein hormone receptors. The deglycosylated hormones appear to exhibit reduced efficacy at these receptors [120].

We demonstrate 2 novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to here as SP) that results in an alteration of the coding sequence of the N terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant human LHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated.

By mediating estrogen synthesis and follicular growth in response to FSH, the ovarian FSH receptor (FSHR) is essential for female fertility. Indeed, ovarian stimulation via administration of FSH to women with infertility is part of the primary therapeutic intervention used in assisted reproductive technology. In physiological and therapeutic contexts, current dogma dictates that once ovulation has occurred, FSH/FSHR signaling is no longer required for successful pregnancy outcomes. However, a continued role for FSH during pregnancy is suggested by recent studies demonstrating extraovarian FSHR in the female reproductive tract. Furthermore, functional roles for FSHR in placenta and in uterine myometrium have now been demonstrated. In placenta, vascular endothelial FSHR of fetal vessels within the chorionic villi (human) or labyrinth (mouse) mediate angiogenesis, and it has further been shown that deletion of placental Fshr in mice has deleterious effects on pregnancy. In uterine myometrium, changes in the densities of FSHR in muscle fiber and stroma in the nonpregnant state, early pregnancy, and term pregnancy differentially regulate contractile activity, suggesting that signaling through myometrial FSHR may contribute to the quieting of contractile activity required for successful implantation and that the temporal upregulation of the FSHR at term pregnancy may be required for the appropriate timing of parturition. In addition, extraovarian expression of mRNAs encoding the glycoprotein hormone α subunit and the FSH β subunit has been demonstrated, suggesting that these novel aspects of extraovarian FSH/FSHR signaling during pregnancy may be mediated by locally synthesized FSH.

It has been shown in both human and mouse placentas that follicle stimulating hormone receptor (FSHR) is expressed in fetal vascular endothelium. There are conflicting reports, however, on the role of FSH to stimulate angiogenesis in vitro in cultured endothelial cells from umbilical veins. Therefore, in this study we undertook an in vivo approach utilizing Fshr null mice to definitively address this question. In the context where all pregnant dams have identical Fshr genotypes, we generated fetuses and associated fetal portions of placenta that were Fshr wt or Fshr null and analyzed angiogenesis within the placental labyrinths. Quantitative morphometric analyses of placentas obtained at mid-gestation revealed that the percentage of the placenta composed of labyrinth is significantly decreased in Fshr null placentas relative to wt placentas. Furthermore, data presented demonstrate that within the Fshr null labyrinths, fetal vessel angiogenesis was significantly reduced relative to wt labyrinths. The results obtained with this combination of in vivo and genetic approaches conclusively demonstrate that signaling through endothelial FSHR does indeed stimulate angiogenesis and that placental Fshr is essential for normal angiogenesis of the fetal placental vasculature.

Context: The FSH receptor (FSHR) is traditionally thought to play a role in female reproductive physiology solely within the context of ovarian FSHR. However, FSHR is also expressed in endothelial cells of the placental vasculature and human umbilical cord vessels, suggesting additional facets of female reproduction regulated by extragonadal FSHR. Objective: We sought to determine the functional role of FSHR on human umbilical cord endothelial cells (HUVECs), hypothesizing that activation of the FSHR would stimulate angiogenesis. Design: The ability of FSH to stimulate several angiogenic processes in HUVECs was determined. Setting: This was a laboratory-based study using commercially prepared HUVECs. Results: Tube formation, wound healing, cell migration, cell proliferation, nitric oxide production, and cell survival were stimulated in response to FSH. Quantitative comparisons between HUVECs incubated with maximally stimulatory concentrations of FSH vs vascular endothelial growth factor (VEGF), a well-characterized angiogenic factor, revealed that FSH is as efficacious as VEGF in promoting angiogenic processes. FSH did not provoke increased secretion of VEGF by HUVECs, suggesting the direct stimulation of angiogenic processes by FSH in endothelial cells. In contrast to gonadal cells, the FSHR on HUVECs did not mediate an FSH-stimulated increase in cAMP. However, increased phosphorylation of AKT in response to FSH was observed, suggesting that FSH stimulation of HUVEC FSHR stimulates the PI3K/AKT signaling pathway. Conclusions: Our studies reveal a novel role for FSHR in female reproductive physiology. Its ability to promote angiogenesis in placental endothelial cells suggests that the FSHR may have an influential role in pregnancy.

Previous studies from our laboratory revealed that the follicle-stimulating hormone receptor (FSHR) is expressed at low levels in nonpregnant human myometrium and that it is up-regulated in pregnant term nonlaboring myometrium; however, the physiological relevance of these findings was unknown. Herein, we examined signaling pathways stimulated by FSH in immortalized uterine myocytes expressing recombinant FSHR at different densities and showed that cAMP accumulation is stimulated in all cases but that inositol phosphate accumulation is stimulated only at high FSHR densities. Because an increase in cAMP quiets myometrial contractile activity but an increase in 1,4,5-triphosphoinositol stimulates contractile activity, we hypothesized that FSHR density dictates whether FSH quiets or stimulates myometrial contractility. Indeed, in human and mouse nonpregnant myometrium, which express low levels of FSHR, application of FSH resulted in a quieting of contractile activity. In contrast, in pregnant term nonlaboring myometrium, which expresses higher levels of FSHR, application of FSH resulted in increased contractile activity. Examination of pregnant mouse myometrium from different stages of gestation revealed that FSHR levels remained low throughout most of pregnancy. Accordingly, through mid-gestation, the application of FSH resulted in a quieting of contractile activity. At Pregnancy Day (PD) 16.5, FSHR was up-regulated, although not yet sufficiently to mediate stimulation of contractility in response to FSH. This outcome was not observed until PD 19.5, when FSHR was further up-regulated. Our studies describe a novel FSHR signaling pathway that regulates myometrial contractility, and suggest that myometrial FSHR levels dictate the quieting vs. stimulation of uterine contractility in response to FSH.

The glycoprotein hormone receptors are G protein-coupled receptors containing a large extracellular domain fused to a prototypical serpentine domain. cis-activation occurs when binding of hormone to the extracellular domain stabilizes the serpentine domain in an active conformation. Studies by others suggested that these receptors can also signal by trans-activation, where hormone binding to one receptor protomer activates the serpentine domain of an associated protomer, as documented by the partial rescue of hormone-dependent signaling when a binding defective mutant is coexpressed with a signaling defective mutant. However, our characterizations of several LH receptor (LHR) mutants used in previous studies differ markedly from those originally reported. Also, when examining a pair of LHR mutants previously shown to functionally rescue in vitro as well as in vivo, in addition to finding that the properties of the individual mutants differ significantly from those originally described, we determined that when this pair of mutants was coexpressed in vitro, quantitative analyses did not indicate functional rescue. Additional data are presented that provide a plausible alternate explanation for the apparent in vivo trans-activation that was reported. Finally, using LHR mutants that we have documented to be expressed at the cell surface but to lack human chorionic gonadotropin binding activity or to be severely impaired in their ability to activate Gs, we did not observe functional rescue of human chorionic gonadotropin-stimulated cAMP when the mutants were coexpressed, even though bioluminescence resonance energy transfer analyses confirmed that the coexpressed mutants formed dimers. Taken altogether, our data substantively question the concept of functional rescue between LHR mutants.

Strong gain-of-function mutations have not been identified in humans in the FSH receptor (FSHR), whereas such mutations are common among many other G protein-coupled receptors. In order to predict consequences of such mutations on humans, we first identified constitutively activated mutants of the mouse (m) Fshr and then expressed them under the human anti-Müllerian hormone promoter in transgenic mice or created knock-in mutation into the mouse genome. We show here that mutations of Asp580 in the mFSHR significantly increase the basal receptor activity. D580H and D580Y mutations of mFSHR bind FSH, but the activity of the former is neither ligand-dependent nor promiscuous towards LH/human choriogonadotropin stimulation. Transgenic expression of mFshrD580H in granulosa cells leads to abnormal ovarian structure and function in the form of hemorrhagic cysts, accelerated loss of small follicles, augmented granulosa cell proliferation, increased estradiol biosynthesis, and occasional luteinized unruptured follicles or teratomas. The most affected mFshrD580H females are infertile with disturbed estrous cycle and decreased gonadotropin and increased prolactin levels. Increased estradiol and prolactin apparently underlie the enhanced development of the mammary glands, adenomatous pituitary growth, and lipofuscin accumulation in the adrenal gland. The influence of the mFSHRD580Y mutation is milder, mainly causing hemorrhagic cysts in transgenic mFSHRD580Y and mFSHRD580Y -knock-in mice. The results demonstrate that gain-of-function mutations of the FSHR in mice bring about distinct and clear changes in ovarian function, informative in the search of similar mutations in humans.

The human follitropin receptor (hFSHR) is a G protein-coupled receptor (GPCR) central to reproductive physiology that is composed of an extracellular domain (ECD) fused to a serpentine region. Using bioluminescence resonance energy transfer (BRET) in living cells, we show that hFSHR dimers form constitutively during their biosynthesis. Mutations in TM1 and TM4 had no effect on hFSHR dimerization, alone or when combined with mutation of Tyr(110) in the ECD, a residue predicted to mediate dimerization of the soluble hormone-binding portion of the ECD complexed with FSH (Q. Fan and W. Hendrickson, Nature 433:269-277, 2005). Expressed individually, the serpentine region and a membrane-anchored form of the hFSHR ECD each exhibited homodimerization, suggesting that both domains contribute to dimerization of the full-length receptor. However, even in the context of only the membrane-anchored ECD, mutation of Tyr(110) to alanine did not inhibit dimerization. The full-length hFSHR and the membrane-anchored ECD were then each engineered to introduce a consensus site for N-linked glycosylation at residue 110. Despite experimental validation of the presence of carbohydrate on residue 110, we failed to observe disruption of dimerization of either the full-length hFSHR or membrane-anchored ECD containing the inserted glycan wedge. Taken altogether, our data suggest that both the serpentine region and the ECD contribute to hFSHR dimerization and that the dimerization interface of the unoccupied hFSHR does not involve Tyr(110) of the ECD.

Rare activating mutations of the human (h)FSHR have been reported in some women with spontaneous ovarian hyperstimulation in pregnancy, where follicular growth is inappropriately stimulated by elevated concentrations of human chorionic gonadotropin acting through the hFSHR. It is not known whether ovarian hyperstimulation in peripubertal girls with untreated primary hypothyroidism is caused by hFSHR mutations and/or influenced by hFSHR allelic variants, rendering the hFSHR more sensitive to circulating TSH.The aim of the study was to determine whether mutations of the hFSHR and/or hFSHR allelic variants are associated with greater sensitivity of the hFSHR to TSH.The hFSHR gene was sequenced from eight pediatric patients displaying gonadal hyperstimulation due to primary hypothyroidism. HEK293 cells expressing different hFSHR allelic combinations were studied for their responsiveness to recombinant (r)hTSH.The study was conducted at university research centers.Eight unrelated patients (seven girls and one boy) who exhibited primary hypothyroidism and gonadal hyperstimulation were included in the study.There were no interventions.DNA sequencing of the hFSHR gene was the main outcome measure. Basal, rhFSHR- and rhTSH receptor-stimulated cAMP levels were assayed in HEK293 cells transfected with the hTSH receptor or different hFSHR allelic combinations. Cell surface receptor numbers were also determined.No hFSHR mutations were identified in the patient population, but we did identify two known polymorphisms. In vitro experiments demonstrated a dose-dependent and specific rhTSH-dependent increase in cAMP production in HEK293 cells expressing the wild-type hFSHR, regardless of hFSHR isoform.Pediatric gonadal hyperstimulation associated with severe primary hypothyroidism is likely due to the actions of the elevated concentrations of TSH on the wild-type hFSHR, and this response is not dependent upon the hFSHR isoform.

The L457(3.43)R mutation of the hLHR was initially identified in a Brazilian boy with gonadotropin-independent precocious puberty. As would be expected, L457(3.43)R, when expressed in 293 cells, caused a marked elevation in basal cAMP levels. Interestingly, in spite of the fact that the elevated basal levels of cAMP elicited by L457R were not as great as those elicited by the wild-type hLHR when stimulated with hCG, L457(3.43)R cells were unresponsive to further hormonal stimulation. We have since determined that the L457(3.43)R mutant, as well as other constitutively active mutants of the hLHR, causes an increase in phosphodiesterase activity that attenuates the target cell to hormonal stimulation of the wild-type hLHR or other Gs-coupled GPCRs. We have also shown that the constitutive activity and lack of hormonal responsiveness of L457(3.43)R are due to the formation of a salt bridge between the introduced arginine in the mid portion of helix 3 with D578(6.44) in the mid portion of helix 6. The formation of this salt bridge results in the disruption of interactions between the cytoplasmic ends of helices 3 and 6 that are associated in general with activation of the hLHR. As such, this mutant has provided novel insights into the properties of target cells expressing activating hLHR mutants and into the structural basis for hLHR activation.

The human lutropin receptor (hLHR) plays a pivotal role in reproductive endocrinology. A number of naturally occurring mutations of the hLHR have been identified that cause the receptor to become constitutively active. To gain further insights into the structural basis for the activation of the hLHR by activating mutations, we chose to examine a particularly strong constitutively activating mutation of this receptor, L457R, in which a leucine that is highly conserved among rhodopsin-like G protein-coupled receptors in helix 3 has been substituted with arginine. Using both disruptive as well as reciprocal mutagenesis strategies, our studies demonstrate that the ability of L457R to stabilize an active form of the hLHR is because of the formation of a salt bridge between the replacing amino acid and Asp-578 in helix 6. Such a lock between the transmembrane portions of helices 3 and 6 is concurrent with weakening the connections between the cytosolic ends of the same helices, including the interaction found in the wild-type receptor between Arg-464, of the (E/D)R(Y/W) motif, and Asp-564. This structural effect is properly marked by the increase in the solvent accessibility of selected amino acids at the cytosolic interfaces between helices 3 and 6. The integrity of the conserved amino acids Asn-615 and Asn-619 in helix 7 is required for the transfer of the structural change from the activating mutation site to the cytosolic interface between helices 3 and 6. The results of in vitro and computational experiments further suggest that the structural trigger of the constitutive activity of the L457R mutant may also be responsible for its lack of hormone responsiveness.

Although melanocortin-4 receptor mutations are the cause of the most common monogenic form of obesity, the involvement of the melanocortin-3 receptor (MC3R) in the pathogenesis of obesity is unknown. Earlier studies failed to identify any mutations in obese patients except for the identification of two variants (K6T and I81V) that likely represent polymorphisms. However, a potential mutation (I183N) was recently reported from patients having high-fat contents. We report here the functional characterization of these variants. We show that K6T and I81V have ligand binding and signaling properties similar to wild-type (wt) MC3R, indicating that they are indeed polymorphisms. However, the other variant, I183N, completely lacks signaling in response to agonist stimulation, although it binds ligand with normal affinity and with only slightly decreased capacity. Coexpression of the wt and I183N MC3Rs showed that I183N does not exert dominant-negative activity on wt MC3R. These results provide supporting evidence for the hypothesis proposed in the original case report that MC3R mutation might be a genetic factor that confers susceptibility to obesity, likely due to haploinsufficiency. Further mutations at I183 revealed a discrete requirement for I183 in agonist-induced MC3R activation. The corresponding residue is also important for agonist-induced human melanocortin-4 receptor and lutropin receptor activation. In summary, we identify a residue that is critical for activation of G protein-coupled receptors. (J Clin Endocrinol Metab 89: 3936 –3942, 2004)

The human LH receptor (hLHR) consists of a heptahelical segment prototypical of G protein-coupled receptors and a large ectodomain. The binding of hormone to the ectodomain or the presence of activating mutations stabilize the hLHR in an active conformation. Although it has been inferred that activation of the hLHR involves conformational alterations, direct evidence supporting this has not been reported. We have addressed this issue by comparing the protease sensitivity of the wild-type hLHR as compared to three activating mutants of the hLHR. Our data demonstrate that the ectodomains of the activating hLHR mutants are more susceptible to proteolysis as compared to the wild-type hLHR. As such, they provide the first experimental data demonstrating that the conformations of the active and inactive states of the hLHR are distinct and suggest that activation of the hLHR (at least as induced by mutations) involves a tighter interaction of the ectodomain with the heptahelical segment of the receptor as opposed to a release of the ectodomain from the heptahelical segment.

Expression and function of the follicle-stimulating hormone receptor (FSHR) in females were long thought to be limited to the ovary. Here, however, we identify extragonadal FSHR in both the human female reproductive tract and the placenta, and test its physiological relevance in mice. We show that in nonpregnant women FSHR is present on: endothelial cells of blood vessels in the endometrium, myometrium, and cervix; endometrial glands of the proliferative and secretory endometrium; cervical glands and the cervical stroma; and (at low levels) stromal cells and muscle fibers of the myometrium. In pregnant women, placental FSHR was detected as early as 8-10 wk of gestation and continued through term. It was expressed on: endothelial cells in fetal portions of the placenta and the umbilical cord; epithelial cells of the amnion; decidualized cells surrounding the maternal arteries in the maternal decidua; and the stromal cells and muscle fibers of the myometrium, with particularly strong expression at term. These findings suggest that FSHR expression is upregulated during decidualization and upregulated in myometrium as a function of pregnancy. The presence of FSHR in the placental vasculature suggests a role in placental angiogenesis. Analysis of genetically modified mice in which Fshr is lacking in fetal portions of the placenta revealed adverse effects on fetoplacental development. Our data further demonstrate FSHB and CGA mRNAs in placenta and uterus, consistent with potential local sources of FSH. Collectively, our data suggest heretofore unappreciated roles of extragonadal FSHR in female reproductive physiology.

One of the hallmarks of the differentiation of granulosa cells is the estradiol and FSH/cAMP-dependent induction of the LH receptor (LHR). Previous studies using granulosa cells isolated from diethylstilbestrol-pretreated immature rats identified a novel cAMP-responsive element termed the SAS site (Sp1c adjacent site) in the promoter region of the rat (r) LHR gene. The studies presented herein show that there is an additional distal site located at nucleotide (nt) -933/-924 that appears to interact with the same transcription factor that binds to the promoter SAS site. Similar to the SAS site, the complex formed between granulosa cell nuclear extracts and this distal site is enhanced by cAMP treatment of the granulosa cells. The core sequence required for the formation of the DNA/protein complex at this distal rLHR site was determined to be AGTGG(A)GGGG. With the exception of adenine at -928, substitution of any residue within this sequence prevented formation of this complex. The core sequence of this distal site differs from that of the proximal SAS site, which is GGGGG, and hence the distal site has been termed a SAS-like site. Reporter gene assays using constructs containing the -2,109/-1 region of the rLHR demonstrate that mutation of the distal SAS-like site abolishes the cAMP-induced transcription of the rLHR gene in rat granulosa cells, underscoring the functional significance of this site. Given the lack of sequences in the 5'-flanking region of the rLHR gene consistent with known cAMP-responsive elements, the identification of the novel SAS and SAS-like sites in the rLHR gene provides important clues toward understanding the mechanisms by which the rLHR gene is induced by FSH/cAMP.

The presence of the LH receptor (LHR) in nongonadal tissues of the reproductive tract has been reported, but localization studies have not been performed. Our objectives were to demonstrate the presence of LHR in the reproductive tract and to localize receptor expression. Reproductive age rats and mice were obtained and 125 I-hCG binding assays were performed on membrane preparations from the uterus, ovary, liver, and testis. In situ hybridizations were performed using 35 S-labeled antisense and sense RNA probes prepared from nucleotides 1‐591 of the mouse LHR cDNA. Specific hCG binding was detected in membrane preparations from the ovary, uterus, and testis but not in the liver in both the rat and mouse. In the ovary, LHR mRNA was localized in theca cells, large follicles, and corpora lutea as expected. In the uterus, LHR mRNA was expressed in stromal cells of the endometrium and in the uterine serosa. Uterine smooth muscle cells had low levels of expression, and the endometrial epithelium was negative. In the oviduct, high levels of LHR expression were noted on the serosa and in subepithelial cells. Oviductal smooth muscle had low expression, and the epithelium was negative. We conclude that functional, nongonadal LHR are expressed in the mouse reproductive tract. The presence and localization of LHR expression in the mouse reproductive tract lay the foundation for transgenic models to address the physiologic role of these receptors. fallopian tubes, female reproductive tract, hormone action, LH

Naturally occurring activating mutations in the human LH receptor (hLHR) gene are the cause of sporadic or familial male gonadotropin-independent precocious puberty. We have previously reported three different activating mutations of the hLHR gene in four unrelated Brazilian boys with male-limited precocious puberty. In the current study, we examined three other Brazilian boys, two brothers and one unrelated boy, with gonadotropin-independent precocious puberty. Direct sequencing of the entire exon 11 of the hLHR gene in the two brothers revealed a heterozygous substitution of T for C at nucleotide 1103, resulting in the substitution of leucine at position 368 by proline in the first transmembrane helix. Their mother carried the same mutation, establishing the familial nature of this mutation. Human embryonic 293 cells expressing hLHR(L368P) bound hCG with the same high affinity as cells expressing the wild-type hLHR. Cells expressing the novel L368P mutation displayed up to a 12-fold increase in basal cA...

The induction of the lutropin receptor (LHR) in granulosa cells by FSH is mediated, at least in part, by cAMP. However, the classic cAMP-responsive element (CRE) is not present in the 5′-flanking region of the rat LHR gene. Previous studies from our laboratory had shown that three Sp1 sites within the promoter region of the rat LHR (rLHR) bind Sp1 and Sp3 and are involved in the basal and cAMP-mediated transcription of the rLHR gene. In the present studies we show that the rLHR promoter region forms a complex (designated complex A) with nuclear extracts from rat granulosa cells, and the abundance of complex A is markedly increased when using cells that had been pretreated with 8-bromo (Br)-cAMP. We have localized the binding of the protein(s) in complex A to a DNA sequence immediately upstream and partially overlapping with the Sp1c binding site. The core site (designated SAS for Sp1c adjacent sequence) is localized to nucleotide (nt) −146 to −142 and contains the sequence GGGGG. The consensus sequence for the core portion of this element appears to be (G/T)GGGG. Mutations of the SAS site, but not SP1c site, abolish complex A formation. Experiments utilizing rat granulosa cells transfected with luciferase reporter genes driven by the 5′-flanking region of the rLHR gene demonstrate a functional role for the SAS site in the cAMP responsiveness of the rLHR gene.

The luteinizing hormone (LH) receptor (LHR) is a heptahelical receptor present primarily in the ovaries of females and the testes of males. This same receptor can bind with high affinity either pituitary LH or the nearly identical placental hormone human chorionic gonadotropin (hCG). In both males and females, the levels of LH remain quite low during childhood years, until puberty, at which time the hypothalamic-pituitary-gonadal axis matures. After puberty, the functions of LH are critical to normal reproductive function. In postpubertal males, LH stimulates testosterone synthesis in the Leydig cells of the testes, which, in turn, is necessary for both formation of male secondary sexual characteristics and spermatogenesis. In nonpregnant postpubertal females, LH plays several roles. During the follicular phase of the ovarian cycle, LH stimulates theca cells to synthesize androgens, which are then aromatized into estradiol in granulosa cells under the influence of follicle-stimulating hormone (FSH). The midcycle surge of LH induces follicular maturation and ovulation. Subsequently, during the luteal phase, LH induces the formation of the corpus luteum and stimulates progesterone synthesis. In the pregnant female, placental hCG binds to the LHR on ovarian luteal cells and causes the corpus luteum, which otherwise undergoes atresia, to be maintained and to continue steroid synthesis, which is necessary for the continuation of pregnancy. During pregnancy, if the fetus is male, placental hCG also stimulates fetal testicular Leydig cells to produce testosterone, which, in turn, mediates the differentiation of the external genitalia and induces the descent of the testes (see Roberts et al. 1999 [in this issue]). Clearly, the LHR plays a critical role in reproductive physiology in both males and females. The importance of the LHR signal-transduction pathway in normal reproductive functioning has been further underscored by the discovery, in recent years, of naturally occurring mutations of the human LHR gene, hLHR.

The expression of the lutropin receptor (LHR) in granulosa cells is a complex phenomenon under the hormonal control of FSH and estradiol. Using primary cultures of granulosa cells from immature female rats pretreated with diethylstilbestrol (a compound with estrogen-like activity), the role of FSH in LHR induction was studied. Previous studies from our laboratory have shown that FSH or 8-bromo-cAMP addition to these cells causes a marked increase in the rate of transcription of the rat LHR (rLHR) gene. The present studies were undertaken to compare the properties of the rLHR gene in undifferentiated vs. differentiated rat granulosa cells as a means of determining those elements that confer basal activity and cAMP responsiveness. Our studies show that the proximal 155 bp (relative to the translational initiation codon) of the 5′-flanking region of rLHR gene represent a minimal promoter that accounts for the basal expression of this receptor in rat granulosa cells. A major domain located between nucleotides...

The lutropin/choriogonadotropin receptor (LHR) and follitropin receptor (FSHR) are members of the superfamily of G protein-coupled receptors. The carboxyl half of each receptor is composed of the classical seven membrane spanning regions connected by intracellular and extracellular loops. In addition, each receptor contains a large extracellular domain. Despite the complexity of the structure of G protein-coupled receptors, little is known about how these receptors assume their correct conformations during biosynthesis. Although the role of chaperone proteins in the folding of other proteins has been well documented, their role in the folding of G protein-coupled receptors has been an enigma. To better understand the folding of the LH and FSH receptors, we examined their association with the general chaperone proteins calnexin, binding protein (BiP), and the 94-kDa glucose-regulated protein (GRP94). Clonal 293 cell lines expressing comparably high levels of each receptor were solubilized, and the extracts...

In this report, the genomic DNA was examined from two siblings with gonadal LH resistance. A 46,XY pseudohermaphrodite presented with female external genitalia and his 46,XX sister exhibited menstrual irregularities (oligoamenorrhea) and infertility. Exons 1–11 of the LH receptor (LHR) gene were amplified by the PCR using different sets of intronic primers and were directly sequenced. Sequencing revealed that both individuals carried a deletion of nucleotides 1822–1827, resulting in the deletion of Leu-608 and Val-609 within the seventh transmembrane helix. This mutation was introduced into a recombinant human (h) LHR cDNA. Transfections of 293 cells with hLHR(wt) vs. hLHR(ΔL608,V609) revealed that very little of the mutant receptor was expressed at the cell surface. This was due to both a decrease in the total amount of receptor expressed as well as to an increased intracellular retention of the mutant receptor. In spite of the decreased cell surface expression of the mutant, sufficient amounts were present to allow for assessment of its functions. Equilibrium binding assays showed that the cell surface hLHR(ΔL608,V609) binds hCG with an affinity comparable to that of the wild-type receptor. However, the cells expressing the hLHR(ΔL608,V609) exhibit only a 1.5- to 2.4-fold stimulation of cAMP production in response to hCG. In contrast, cells expressing comparably low levels of hLHR(wt) responded to hCG with 11- to 30-fold increases of cAMP levels. Therefore, the testicular and ovarian unresponsiveness to LH in these patients appears to be due to a mutation of the hLHR gene in which Leu-608 and Val-609 are deleted. As a consequence, the majority of the mutant receptor is retained intracellularly. The small percentage of mutant receptor that is expressed at the cell surface binds hormone normally but is unable to activate Gs.

The lutropin/choriogonadotropin receptor (LHR) is a G protein-coupled receptor central to reproductive physiology. In addition to the complex structure formed by seven membrane-spanning helices, this receptor also contains a large amino-terminal domain whose tertiary structure is unknown. Many mutations of this receptor result in partial or complete intracellular retention of the mutant, regardless of whether the mutations are located in the extracellular domain, interhelical loops, transmembrane helices, or cytoplasmic tail. Nonetheless, as long as a mutation has not disturbed a hormone binding site, the intracellularly trapped mutant retains high affinity binding for human CG (hCG), suggesting that it is not extremely misfolded. Because temperature is known to affect protein folding, we examined the effects of reduced temperature on cell surface expression of intracellularly retained mutants of the LHR. Our studies examined three different 293 cells lines, each stably expressing a different LHR mutant t...

The LH receptor (LHR) and FSH receptor (FSHR) are each G protein-coupled receptors that play critical roles in reproductive endocrinology. Each of these receptors has previously been shown to self-associate into homodimers and oligomers shortly after their biosynthesis. As shown herein using bioluminescence resonance energy transfer to detect protein-protein interactions, our data show that the LHR and FSHR, when coexpressed in the same cells, specifically heterodimerize with each other. Further experiments confirm that at least a portion of the cellular LHR/FSHR heterodimers are present on the cell surface and are functional. We then sought to ascertain what effects, if any, heterodimerization between the LHR and FSHR might have on signaling. It was observed that when the LHR was expressed under conditions promoting the heterodimerization with FSHR, LH or human chorionic gonadotropin (hCG) stimulation of Gs was attenuated. Conversely, when the FSHR was expressed under conditions promoting heterodimerization with the LHR, FSH-stimulated Gs activation was attenuated. These results demonstrate that the coexpression of the LHR and FSHR enables heterodimerizaton between the 2 gonadotropin receptors and results in an attenuation of signaling through each receptor.

The thyroid-stimulating hormone receptor (TSHR), luteinizing hormone receptor (LHR), and follicle-stimulating hormone receptor (FSHR) are collectively referred to as the glycoprotein hormone receptors because they bind the structurally similar glycoprotein hormones. The glycoprotein hormones consist of the pituitary hormones thyroid-stimulating hormone (thyrotropin, TSH), luteinizing hormone (lutropin, LH), and follicle-stimulating hormone (follitropin, FSH) as well as the placental hormone, chorionic gonadotropin (choriogonadotropin, CG). The glycoprotein hormones are each composed of two dissimilar subunits ( α and β ) that are noncovalently associated. Within a given species, the α -subunit is identical and the β -subunits are distinct but homologous. Due to the nearly identical nature of LH β and CG β , the LHR binds either LH or CG. The binding specificity of LH/CG to the LHR, FSH to the FSHR, and TSH to the TSHR is fairly strict. However, in some cases, extremely high levels of hormone can cause activation of the inappropriate receptor. Because the LHR and FSHR are localized primarily to the gonads, these two receptors are also referred to as the gonadotropin receptors. The glycoprotein hormone receptors belong to the family A (rhodopsin-like) group of G-protein-coupled receptors (GPCRs). However, they share the relatively unique feature of having large extracellular domains that mediate the high affinity binding of hormone. In spite of their different physiological roles, the glycoprotein hormone receptors share a similar structural organization and mechanism of action.

The rat lutropin/choriogonadotropin receptor (rLHR) is a G protein-coupled receptor which binds either human choriogonadotropin (hCG) or lutropin (luteinizing hormone, LH) and, therefore, plays a central role in reproductive physiology. In addition to the seven transmembrane helices, three extracellular loops, three intracellular loops, and a cytoplasmic tail characteristic of all G protein-coupled receptors, the rLHR also contains a relatively large N-terminal extracellular domain. Since high affinity hormone binding occurs to this N-terminal extracellular domain and since G proteins are activated by intracellular regions of the receptor, it has been hypothesized that upon hormone binding a portion of the hormone or the receptor's extracellular domain might interact with the receptor's extracellular loops and/or transmembrane helices, thus evoking an intracellular conformational change. To explore this possibility, we prepared and characterized several mutants of the rLHR in which portions of the extracellular loops were deleted. Ultimately, it was not possible to examine the signal transduction properties of the mutants because all but one mutant were retained intracellularly. Although the intracellularly retained mutants must be somewhat misfolded, all were found to bind hCG with high affinity if the cells were first solubilized in detergent. However, the binding of oLH to the detergent solubilized mutants was altered. Thus, whereas the wild-type rLHR bound oLH with two apparent affinities, the solubilized deletion mutants bound oLH with only one apparent affinity. Although these data could be interpreted to suggest that an ovine LH (oLH) binding site on the extracellular loops of the rLHR was deleted, data shown argue against this hypothesis. Rather, the results presented suggest that the two apparent affinities of the wild-type rLHR for oLH represent the binding affinities of two populations of rLHR where the mature, cell surface form binds oLH with a higher affinity than the immature, intracellular form. Furthermore, we show that mutations of the rLHR which cause intracellular retention of the receptor result in a decrease from two to one apparent binding sites for oLH due to the absence of the high affinity oLH binding component contributed by the mature cell surface receptor. Therefore, whereas hCG cannot discriminate between the mature cell surface wild-type receptor and an intracellularly retained rLHR mutant, oLH can make this discrimination, thus suggesting a conformational difference between the two forms of the receptor.

The LH receptor (LHR) and FSH receptor (FSHR), collectively termed the gonadotropin receptors, are members of the Family A of GPCRs. The gonadotropin receptors each contain N-linked carbohydrates that are not directly involved in hormone binding, but contribute to the proper folding, and therefore, cell surface expression of the receptor. Loss-of-function mutations of an LHR or FSHR results in decreased target cell responsiveness. Most inactivating mutations cause receptor misfolding, resulting in the retention of the mutant in its immature form in the endoplasmic reticulum. A membrane-permeable allosteric agonist of the LHR has been shown to serve as a pharmacological chaperone for misfolded and intracellularly retained LHRs by promoting their cell surface expression. Wild-type LHR and FSHR each form homodimers and heterodimers while in the ER. Therefore, when wild-type receptor is co-expressed with a misfolded mutant, the misfolded receptor dimerizes with immature wild-type receptor in the ER, causing a dominant-negative effect on cell surface expression of the mature wild-type receptor. Notably, the propensity for homodimerization is not affected by the activation status of the receptor. However, within a receptor dimer, the activity of one protomer may allosterically regulate the other protomer. Therefore, the dimerization of the gonadotropin receptors appears to be an obligate process that is part of the normal itinerary for trafficking to the cell surface and, once there, the dimerized receptors allow for additional modulations of cell signaling.

The LH receptor (LHR) and FSH receptor (FSHR), collectively termed the gonadotropin receptors, are members of the Family A of GPCRs. The gonadotropin receptors each contain N-linked carbohydrates that are not directly involved in hormone binding, but contribute to the proper folding, and therefore, cell surface expression of the receptor. Loss-of-function mutations of an LHR or FSHR results in decreased target cell responsiveness. Most inactivating mutations cause receptor misfolding, resulting in the retention of the mutant in its immature form in the endoplasmic reticulum. A membrane-permeable allosteric agonist of the LHR has been shown to serve as a pharmacological chaperone for misfolded and intracellularly retained LHRs by promoting their cell surface expression. Wild-type LHR and FSHR each form homodimers and heterodimers while in the ER. Therefore, when wild-type receptor is co-expressed with a misfolded mutant, the misfolded receptor dimerizes with immature wild-type receptor in the ER, causing a dominant-negative effect on cell surface expression of the mature wild-type receptor. Notably, the propensity for homodimerization is not affected by the activation status of the receptor. However, within a receptor dimer, the activity of one protomer may allosterically regulate the other protomer. Therefore, the dimerization of the gonadotropin receptors appears to be an obligate process that is part of the normal itinerary for trafficking to the cell surface and, once there, the dimerized receptors allow for additional modulations of cell signaling.

The present studies were undertaken to characterize the properties of a recombinant truncated rat LH/CG (rLH/CG) receptor representing the extracellular domain (rLHR-t338) expressed in mammalian cells. Using detergent-soluble extracts of stably transfected cells, we show that the binding of ovine (o) LH to the extracellular domain is different from the binding of oLH to the full-length receptor. Thus, whereas the full-length receptor from either rat corpora lutea or stably transfected 293 cells binds oLH with two apparent affinities, rLHR-t338 binds oLH with only one affinity, which is intermediate between the high and low affinities of the full-length receptors. However, rLHR-t338 as well as the full-length receptor binds hCG and hLH with similar high affinities and by a model assuming the presence of only one binding site. These data are consistent with the possibility that the carboxyl-terminal half of the LH/CG receptor may contribute (either directly or indirectly) to a second low affinity hormone-binding site. The truncated receptor retains normal binding specificity, as it does not bind hFSH, demonstrating that the extracellular domain alone contains all of the necessary elements for conferring specificity among the gonadotropin hormones. Our studies also show that rLHR-t338 is glycosylated and migrates as two proteins of 48 and 44 kilodaltons (kDa) on sodium dodecyl sulfate gels (where the 44-kDa species is probably a partially degraded form of the 48-kDa protein). However, the N-linked oligosaccharides of the truncated receptor do not appear to be fully processed, as they remain in a high mannose form. rLHR-t338 is not secreted from cells, but remains trapped intracellularly. It can be detected, though, in detergent-soluble extracts of cells or in homogenates. Upon centrifugation of homogenates, it was found that about 90% of the recoverable truncated receptor can be obtained in the supernatant fraction. Thus, mammalian cells could provide a source of a water-soluble extracellular domain of the LH/CG receptor.

Naturally occurring mutations of G protein-coupled receptors (GPCRs) causing misfolding and failure to traffic to the cell surface can result in disease states. Some small-molecule orthosteric ligands can rescue such misfolded receptors, presumably by facilitating their correct folding and shuttling to the plasma membrane. Here we show that a cell-permeant, allosterically binding small-molecule agonist (Org 42599) rescues the folding and cell surface expression, and therefore target cell signaling, of mutant human luteinizing hormone (LH) receptors (A593P and S616Y) that cause Leydig cell hypoplasia in man. Both mutant receptors were retained in the cytoplasm whereas WT receptor localized at the cell membrane, and binding of LH to cells expressing the mutant receptors was markedly lower than to those expressing the WT receptor. Incubation with Org 42599 increased mutant receptor expression, cell surface localization, and the proportion of mutant receptor in the mature glycosylated form. Importantly, although LH stimulated little (S616Y) or no (A593P) activation of cells expressing mutant receptors, incubation of cells with Org 42599 facilitated rescue of expression and stimulation by the native ligand, LH. Although Org 42599 could activate these receptors, it could not displace 125 I-labeled human LH binding to the WT receptor, indicating that it acts in an allosteric manner. Here we demonstrate a small-molecule GPCR allosteric agonist that functionally rescues intracellularly retained mutant LH receptors by facilitating their cell surface expression. This approach may have application for treatment of infertile patients bearing such mutations and, more broadly, for other misfolded GPCR mutants resulting in human pathologic processes.

I. Introduction IN 1989 we published a review in this journal in which we summarized, criticized, and attempted to reconcile the conflicting data that existed on the structure of the mammalian LH/CG receptor (1). Based on our analysis of the existing data we concluded that the LH/CG receptor was composed of a single polypeptide chain. Since that review was published, complementary DNAs (cDNAs) for the LH/CG receptor from several species have been cloned, sequenced, and expressed, conclusively establishing that this receptor is indeed a single polypeptide chain (2–5). The cloning of the cDNAs for the LH/CG receptor has also generated new knowledge and experimental tools that are rapidly being used to probe novel aspects of the functions and regulation of this important receptor. Since the studies leading to the cloning of the first cDNA for the LH/CG receptor have been recently summarized (6), this review will only briefly touch on this aspect and focus mainly on what has been learned since the initial clo...

The rat lutropin/choriogonadotropin receptor (rLHR) is a G protein-coupled receptor, the large extracellular domain of which binds human choriogonadotropin (hCG) with high affinity. Within the extracellular domain are six potential sites for N-linked glycosylation. Although several studies have attempted to determine if N-linked carbohydrates are necessary for hormone binding, the results have been in apparent disagreement. In this study we have used site-directed mutagenesis to singly and collectively alter the consensus sequences for N-linked glycosylation in the rLHR. In particular, we examined the binding activity in both intact cells as well as detergent-solubilized extracts so that the effects on trafficking to the plasma membrane could be determined. In addition, we independently examined the effects of substituting a particular Asn versus Thr or Ser residue within a given glycosylation consensus sequence. Our data suggest that substitution of Asn-173 with Gln results in both a decreased ability of the receptor to be expressed on the plasma membrane as well as a vastly decreased binding affinity of the receptor for hCG. However, if the consensus sequence for N-linked glycosylation at Asn-173 is altered by substitution of Thr-175 with Ala (instead of Asn-173 to Gln), the resulting receptor binds hCG with high affinity although it is still impaired in its ability to be expressed at the plasma membrane. Furthermore, if all consensus sequences for N-linked glycosylation are mutated collectively while maintaining Asn-173 (by substituting Thr-175 with Ala instead of Asn-173 to Gln), the resulting deglycosylated receptor, although not expressed on the plasma membrane, binds hCG with high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

The lutropin receptor (LHR) is a G protein-coupled receptor (GPCR) that mediates the actions of pituitary LH in males and females and that of placental hCG in pregnant women and, therefore, plays an essential role in reproductive physiology. Mutations of the lhcgr gene that result in constitutive activation of the LHR have been shown to be causative of gonadotropin-independent precocious puberty in young boys. Studies on constitutively active mutants (CAMs) of the LHR have been extremely informative in elucidating the roles of the LHR in reproductive physiology as well as in understanding the molecular basis underlying activation of this GPCR. The constitutive activities of hLHR CAMs can be attenuated by introducing mutations into the CAMs that stabilize the resting state of the hLHR or by coexpressing the hLHR CAMs with an hLHR mutant that is stabilized in the resting state, allowing the two forms of the hLHR to heterodimerize. This chapter describes the experimental methods and strategies underlying studies of hLHR CAMs.

The follicle stimulating hormone receptor (FSHR) plays a critical role in reproductive function. In the males, FSH supports spermatogenesis, whereas in females, FSH is absolutely required for ovarian follicle growth. In females, inactivating mutations in the FSHR result in ovarian dysgenesis with amenorrhea and infertility. The few males reported with severe inactivating mutations exhibited varying spermatogenic defects, but not azoospermia. While these findings may potentially suggest that FSH action is not absolutely required for spermatogenesis, it cannot be ruled out that these individuals have some residual FSHR activity. Gain-of-function mutations in the FSHR cause spontaneous ovarian hyperstimulation syndrome in females due to the inappropriate stimulation of the mutant FSHR by human choriogonadotropin.

The human lutropin receptor (LHCGR) plays an integral role in male and female reproductive physiology. In response to either placental hCG or pituitary LH, gonadal LHCGR mediates its effects primarily through Gs activation. Heterozygous mutations leading to constitutive activation of the LHCGR cause gonadotropin-independent precocious puberty in males, but have no detectable effects on prepubertal or postpubertal females. Homozygous or compound heterozygous inactivating mutations of the LHCGR cause gonadal resistance to hCG and LH, where the clinical phenotypes associated with these mutations are closely correlated with the severity of the mutation. Inactivating mutations in 46,XY individuals cause Leydig cell hypoplasia and impairments in the differentiation of male external genitalia, the development of secondary sexual characteristics and sperm production. 46,XX siblings with inactivating LHCGR mutations exhibit infertility and varying degrees of menstrual irregularities.

The lutropin receptor (LHR) is a G protein-coupled receptor (GPCR) that mediates the actions of pituitary LH in males and females and that of placental hCG in pregnant women and, therefore, plays an essential role in reproductive physiology. Mutations of the lhcgr gene that result in constitutive activation of the LHR have been shown to be causative of gonadotropin-independent precocious puberty in young boys. Studies on constitutively active mutants (CAMs) of the LHR have been extremely informative in elucidating the roles of the LHR in reproductive physiology as well as in understanding the molecular basis underlying activation of this GPCR. The constitutive activities of hLHR CAMs can be attenuated by introducing mutations into the CAMs that stabilize the resting state of the hLHR or by coexpressing the hLHR CAMs with an hLHR mutant that is stabilized in the resting state, allowing the two forms of the hLHR to heterodimerize. This chapter describes the experimental methods and strategies underlying studies of hLHR CAMs.

An agonist-induced change in the functional properties of a constant number of receptors seems to be a ubiquitous phenomenon involved in the regulation of cell surface receptors. Although the mechanisms responsible for this phenomenon (called uncoupling or desensitization) have been studied in detail using beta 2-adrenergic receptors it is unclear if the models derived from these studies are applicable to other members of the family of G protein-coupled receptors. Since it has been shown previously that truncation of the C-terminal cytoplasmic tail of the beta 2-adrenergic receptor results in a delay in the onset of agonist-induced uncoupling (Bouvier, M., Hausdorff, W.P., De Blasi, A., O'Dowd, B.F., Kobilka, B.K., Caron , M.G., and Lefkowitz, R.J. (1988) Nature 333, 370-373), we now present experiments designed to test the effects of a similar truncation of the lutropin/choriogonadotropin (LH/CG) receptor on its functional properties. The results presented herein show that (i) clonal lines of human embryonic kidney cells stably transfected with cDNAs encoding for the wild-type (rLHR-wt) or a mutant receptor truncated at amino acid residue 631 (rLHR-t631) express functional LH/CG receptors as judged by their ability to bind hCG and to respond to it with increased cAMP accumulation; (ii) a preincubation of the cells expressing rLHR-wt with hCG leads to a reduction in the ability of hCG to activate adenylylcyclase; and (iii) this reduction is severely blunted in cells expressing rLHR-t631. These results demonstrate that the C-terminal cytoplasmic tail of the LH/CG receptor is necessary for agonist-induced uncoupling.

The LH/CG receptor is a G protein-coupled receptor present on gonadal cells whose levels are modulated by a number of hormones, growth factors, and second messenger analogs. With the recently cloned cDNA for the LH/CG receptor, it has been shown that changes in the levels of the cognate mRNA are involved, at least in part, in the observed changes in receptor density. In order to study the transcriptional regulation of the LH/CG receptor we have isolated a 2-kilobase region of the 5'-flanking region of the rat LH/CG receptor gene and subcloned nucleotide -1 (relative to the translational initiation codon) to -1370 into a luciferase reporter plasmid. We show here that this region of the LH/CG receptor gene is able to enhance luciferase activity in MA-10 cells, a line of Leydig tumor cells that normally express LH/CG receptors, as opposed to human kidney 293 cells, which do not. Furthermore, the addition of 8-bromo-cAMP to MA-10 cells, under conditions known to decrease LH/CG receptor numbers and receptor mRNA levels, decreases the relative luciferase activity to about 26% of control. This decrease in reporter gene activity is severely blunted in a subclone of MA-10 cells with a cAMP-resistant phenotype. Our studies show, for the first time, that sequence(s) present with 1370 base pairs of the translational start site of the rat LH/CG receptor gene are sufficient for conferring expression of this gene in Leydig cells and for the negative modulation of LH/CG receptor gene transcription by high concentrations of cAMP.

The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG-stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.

The D405N and Y546F mutations of the human lutropin receptor (hLHR) have previously been shown to partially attenuate hCG-stimulated cAMP synthesis despite normal cell surface expression and hCG binding affinity (Min, L. and Ascoli, M. Mol. Endocrinol. 14:1797–1810, 2000). We now show that these mutations each stabilize a resting state of the hLHR. A combined mutant D405N,Y546F is similarly expressed at the cell surface and exhibits normal ligand-binding, but is profoundly signaling impaired. Introduction of hLHR(wt) into cells stably expressing the signaling inactive D405N,Y546F resulted in the attenuation of hCG-stimulated cAMP production by hLHR(wt) even if excess Gs is co-expressed. Similarly, co-expression of D405N,Y546F with hLHR constitutively active mutants (CAMs) attenuated their constitutive activity. Quantitative bioluminescence resonance energy transfer (BRET) analyses demonstrated that D405N,Y546F formed heterodimers with both wt and CAM hLHR. In contrast hLHR(D405N,Y546F) did not heterodimerize with the melanocortin 3 receptor (MC3R) and agonist-stimulated cAMP production through the MC3R was not attenuated when these two receptors were co-expressed. Taken altogether, our data demonstrate that a signaling inactive hLHR mutant (that is trafficked normally to the plasma membrane) attenuates the signaling of the cell surface localized wt or the constitutively active hLHR due to receptor heterodimerization. Our studies, therefore, suggest a novel ramification of GPCR signaling resulting from receptor dimerization.

The follicle stimulating hormone receptor (FSHR) plays a critical role in reproductive function. In the males, FSH supports spermatogenesis, whereas in females, FSH is absolutely required for ovarian follicle growth. In females, inactivating mutations in the FSHR result in ovarian dysgenesis with amenorrhea and infertility. The few males reported with severe inactivating mutations exhibited varying spermatogenic defects, but not azoospermia. While these findings may potentially suggest that FSH action is not absolutely required for spermatogenesis, it cannot be ruled out that these individuals have some residual FSHR activity. Gain-of-function mutations in the FSHR cause spontaneous ovarian hyperstimulation syndrome in females due to the inappropriate stimulation of the mutant FSHR by human choriogonadotropin.

The human lutropin receptor (LHCGR) plays an integral role in male and female reproductive physiology. In response to either placental hCG or pituitary LH, gonadal LHCGR mediates its effects primarily through Gs activation. Heterozygous mutations leading to constitutive activation of the LHCGR cause gonadotropin-independent precocious puberty in males, but have no detectable effects on prepubertal or postpubertal females. Homozygous or compound heterozygous inactivating mutations of the LHCGR cause gonadal resistance to hCG and LH, where the clinical phenotypes associated with these mutations are closely correlated with the severity of the mutation. Inactivating mutations in 46,XY individuals cause Leydig cell hypoplasia and impairments in the differentiation of male external genitalia, the development of secondary sexual characteristics and sperm production. 46,XX siblings with inactivating LHCGR mutations exhibit infertility and varying degrees of menstrual irregularities.

To determine whether hormone-dependent changes in the levels of LH/CG receptor in the rat ovary are associated with changes in expression of LH/CG receptor mRNAs, total RNA from rat follicles and corpora lutea at various stages of development was prepared and analyzed by Northern blots and/or solution hybridization. Whereas small antral follicles contained lo amounts of LH/CG receptor mRNAs, the growth of preovulatory (PO) follicles was associated with an increase in all LH/CG receptor mRNA transcripts. Induction of LH/CG receptor mRNAs in granulosa cells of hypophysectomized rats was dependent on the synergistic effects of estradiol and FSH. An LH/CG surge in vivo or LH treatment of PO follicles in vitro caused a rapid decline of all LH/CG receptor mRNAs in PO follicles, which was prevented by cycloheximide. Newly formed corpora lutea (days 1-4 postovulation) contained low amounts of LH/CG receptor mRNAs unless the rats were pregnant or treated exogenously with PRL. During pregnancy, corpora lutea isolated on days 4-19 of gestation contained high levels of LH/CG receptor mRNAs, which decreased markedly on days 21 and 24, the time of functional luteolysis and decreasing LH/CG receptor levels. These studies demonstrate that hormonal regulation of LH/CG receptor mRNA in rat ovarian cells parallels changes in LH/CG receptor levels and involves diverse molecular mechanisms, including 1) low concentrations of cAMP (elicited by FSH) in developing follicles, 2) inhibition by high concentrations of cAMP (elicited by LH/CG) in PO follicles, and 3) induction and maintenance by PRL in corpora lutea of gestation.

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.

Polyclonal antibodies were raised against peptides corresponding to specific regions of the rat luteal lutropin/choriogonadotropin (LH/CG) receptor. One of these (anti-LHR02) was directed against a 13 amino acid sequence that occurs within the large N-terminal hydrophilic domain, and another (anti-LHR06) was directed against the 14 most C-terminal amino acids of the receptor (as deduced from the open reading frame of the cDNA). These antisera, which we show to be highly specific for the LH/CG receptor, were used in indirect immunofluorescence experiments with primary cultures of rat luteal cells to determine the orientation of this receptor within the plasma membrane. Anti-LHR02 was shown to specifically bind to intact cells. In contrast, anti-LHR06 did not bind to intact cells. When the cells were first permeabilized with detergent and then incubated with this antibody, however, an intense and specific immunofluorescence was observed. These studies clearly show that the orientation of the LH/CG receptor in the plasma membrane is such that the N-terminal domain is located extracellularly, while the C terminus is located intracellularly. Thus, we have experimentally confirmed that the orientation of this receptor as predicted from the amino acid sequence is indeed correct.

Cloned cDNA encoding the rat Sertoli cell receptor for FSH was isolated from a cognate library and functionally expressed in cultured mammalian cells. The FSH receptor (FSH-R), as predicted from the cDNA, is a single 75K polypeptide with a 348 residue extracellular domain which contains three N-linked glycosylation sites. This domain is connected to a structure containing seven putative transmembrane segments which displays sequence similarity to G protein-coupled receptors. Thus, the FSH-R is identical in its structural design to the LH/CG receptor (LH/CG-R). Furthermore, both receptors display 50% sequence similarity in their large extracellular domains and 80% identity across the seven transmembrane segments. Expression of the cloned cDNA in mammalian cells conferred FSH-dependent cAMP accumulation. The selectivity for FSH is attested by the fact that the related human glycoprotein hormones human CG and human TSH do not stimulate adenylyl cyclase in FSH-R expressing cells even when these hormones are present at high concentrations.