Molecular characterization of two Russian sturgeon gonadotropin receptors: Cloning, expression analysis, and functional activity.

Sturgeons are being used in aquaculture because wild populations are now endangered due to overfishing for caviar. A challenge in working with sturgeon as an aquacultured species is its long and slow reproductive development. Reproduction is a hormonally regulated process that involves hierarchical signaling between the brain, pituitary gland, and gonads. In an effort to better understand the hormonal regulation of sturgeon reproduction, we have cloned the Russian sturgeon (st), Acipenser gueldenstaedtii, luteinizing hormone receptor (stLHR) and follicle stimulating hormone receptor (stFSHR) and measured their expression from previtellogenic to mature ovarian follicles. Sturgeon LHR and FSHR expression was elevated in early-vitellogenic and mature follicles compared with pre-vitellogenic and mid-vitellogenic follicles, and only LHR expression increased during late-vitellogenesis. Recombinant sturgeon FSH and LH both activated sturgeon LHR and FSHR in a cAMP reporter assay. Further molecular characterization of these receptors was accomplished by in silico modeling and cAMP reporter assays using heterologous recombinant gonadotropins from human and piscine species. There was no apparent trend in heterologous LH and/or FSH activation of the sturgeon LHR or FSHR. These data suggest that permissive activation of LHR and FSHR are a consequence of some yet undetermined biological characteristic(s) of different piscine species.

Exploring 3D structure of human gonadotropin hormone receptor at antagonist state using homology modeling, molecular dynamic simulation, and cross-docking studies.

Human gonadotropin hormone receptor, a G-protein coupled receptor, is the target of many medications used in fertility disorders. Obtaining more structural information about the receptor could be useful in many studies related to drug design. In this study, the structure of human gonadotropin receptor was subjected to homology modeling studies and molecular dynamic simulation within a DPPC lipid bilayer for 100 ns. Several frames were thereafter extracted from simulation trajectories representing the receptor at different states. In order to find a proper model of the receptor at the antagonist state, all frames were subjected to cross-docking studies of some antagonists with known experimental values (Ki). Frame 194 revealed a reasonable correlation between docking calculated energy scores and experimental activity values (|r| = 0.91). The obtained correlation was validated by means of SSLR and showed the presence of no chance correlation for the obtained model. Different structural features reported for the receptor, such as two disulfide bridges and ionic lock between GLU90 and LYS 121 were also investigated in the final model.

Constitutive activity in gonadotropin receptors.

Constitutively active mutants (CAMs) of gonadotropin receptors are, in general, rare conditions. Luteinizing hormone-choriogonadotropin receptor (LHCGR) CAMs provoke the dramatic phenotype of familial gonadotropin-independent isosexual male-limited precocious puberty, whereas in females, there is not yet any identified phenotype. Only one isolated follicle-stimulating hormone receptor (FSHR) CAM (Asp567Gly) has so far been detected in a single male patient, besides other FSHR weak CAMs linked to pregnancy-associated ovarian hyperstimulation syndrome or to impaired desensitization and internalization. Several animal models have been developed for studying enhanced gonadotropin action; in addition to unraveling valuable new information about the possible phenotypes of isolated FSHR and LHCGR CAMs in women, the information obtained from these mouse models has served multiple translational goals, including the development of new diagnostic and therapeutic targets as well as the prediction of phenotypes for mutations not yet identified in humans. Mutagenesis and computational studies have shed important information on the physiopathogenic mechanisms leading to constitutive activity of gonadotropin receptors; a common feature in these receptor CAMs is the release of stabilizing interhelical interactions between transmembrane domains (TMDs) 3 and 6 leading to an increase, with respect to the wild-type receptor, in the solvent accessibility at the cytosolic extension of TMDs 3, 5, and 6, which involves the highly conserved Glu/Asp-Arg-Tyr/Trp sequence. In this chapter, we summarize the structural features, functional consequences, and mechanisms that lead to constitutive activation of gonadotropin receptor CAMs and provide information on pharmacological approaches that might potentially modulate gonadotropin receptor CAM function.

In silico study on binding specificity of gonadotropins and their receptors: design of a novel and selective peptidomimetic for human follicle stimulating hormone receptor.

Gonadotropins bind to specific receptors in spite of sharing a high level of sequence and structural similarity. This specific binding is crucial for maintaining the reproductive health of an organism. In this study, residues that dictate the receptor binding specificity of the gonadotropins (FSH and LH) have been identified using combination of in silico methods. Docking studies (ZDOCK), based on the systematic replacement of these residues, confirmed its importance in receptor binding. An interesting observation is that the relative positioning of the residues conferring binding specificity varied for the gonadotropin-receptor complexes. This spatial difference of the key residues could be exploited for design of specific modulators. Based on the identified residues, we have rationally designed a peptidomimetic (FSHP) that displays good binding affinity and specificity for hFSHR. FSHP was developed by screening 3.9 million compounds using pharmacophore-shape similarity followed by fragment-based approach. It was observed that FSHP and hFSHâ can share the same receptor binding site thereby mimicking the native hFSHR-FSH interactions. FSHP also displayed higher binding affinity to hFSHR as compared to two reported hFSHR antagonists. MD simulation studies on hFSHR-FSHP complex revealed that FSHP is conformationally rigid and the intermolecular interactions are maintained during the course of simulation.

[Structural-functional organization of polypeptide hormones receptors containing LRR-repeats and their interaction with heterotrimeric G proteins].

The data on structural-functional organization of the serpentine type receptors containing LRR-repeats termed LGR receptors were summarized and analyzed. These receptors include the receptors of pituitary glycoprotein hormones (gonadotropins and thyrotropin) and the relaxin receptors RXFP1 and RXFP2. The mechanisms of the activation of the receptors by the hormones, and molecular basis of interaction between the receptors and heterotrimeric G proteins were considered. The role of membrane-proximal regions of cytoplasmic loops of the receptors and adjacent segments of transmembrane regions in the formation of G protein-binding surface of the receptor and in the activation of the G proteins are discussed. On the basis of literature data and our results, the molecular determinants responsible for selectivity and efficiency of interactions with the G proteins were detected in the LGR-receptors.

Thyrotropin and homologous glycoprotein hormone receptors: structural and functional aspects of extracellular signaling mechanisms.

The TSH receptor (TSHR) together with the homologous lutropin/choriogonadotropin receptor and the follitropin receptor are glycoprotein hormone receptors (GPHRs). They constitute a subfamily of the rhodopsin-like G protein-coupled receptors with seven transmembrane helices. GPHRs and their corresponding hormones are pivotal proteins with respect to a variety of physiological functions. The identification and characterization of intra- and intermolecular signaling determinants as well as signaling mechanisms are prerequisites to gaining molecular insights into functions and (pathogenic) dysfunctions of GPHRs. Knowledge about activation mechanisms is fragmentary, and the specific aspects have still not been understood in their entirety. Therefore, here we critically review the data available for these receptors and bring together structural and functional findings with a focus on the important large extracellular portion of the TSHR. One main focus is the particular function of structural determinants in the initial steps of the activation such as: 1) hormone binding at the extracellular site; 2) hormone interaction at a second binding site in the hinge region; 3) signal regulation via sequence motifs in the hinge region; and 4) synergistic signal amplification by cooperative effects of the extracellular loops toward the transmembrane region. Comparison and consolidation of data from the homologous glycoprotein hormone receptors TSHR, follitropin receptor, and lutropin/choriogonadotropin receptor provide an overview of extracellular mechanisms of signal initiation, conduction, and regulation at the TSHR and homologous receptors. Finally, we address the issue of structural implications and suggest a refined scenario for the initial signaling process on GPHRs.

Molecular characterization of gonadotropin subunits and gonadotropin receptors in black porgy, Acanthopagrus schlegeli: effects of estradiol-17beta on mRNA expression profiles.

The cDNAs of three gonadotropin (GTH) subunits (GTHalpha, FSHbeta, and LHbeta) and two GTH receptors (FSHR and LHR) from pituitary and gonads of black porgy were cloned. The nucleotide sequences of the GTHalpha, FSHbeta, and LHbeta cDNA were 354, 363, and 414 base pairs (bps) in length with open reading frames (ORF) encoding peptides of 117, 120, and 137 amino acids, respectively. The FSHR and LHR cDNA was 2118 and 2076 bps in length with ORFs encoding peptides of 705 and 691 amino acids, respectively. To study the mechanism of the estradiol-17beta (E(2)) action, we examined the expression pattern of GTH subunit mRNAs in pituitary and GTH-receptor mRNAs in gonads, and the changes of plasma E(2) level when E(2) treatment was applied to immature black porgy. E(2) treatment increased mRNA expression levels of the genes and plasma E(2) levels, indicating that E(2) stimulated the increases in GTH subunit and GTH-receptor mRNAs. These data indicate that E(2) plays an important regulatory role in the brain-pituitary-gonad axis of immature black porgy. We provide the molecular characterization and expression of the GTH subunits and GTH receptors during sex change in the protandrous black porgy.

Ligand-selective determinants in gonadotropin receptors.

In mammals, the interactions between gonadotropins and their cognate receptors are highly specific; unintended cross-reactivity under normal physiological conditions has not been observed. This paper summarizes the comparative structure-function studies that aim at elucidating the molecular basis of the ligand selectivity.

Identification of follicle-stimulating hormone-selective beta-strands in the N-terminal hormone-binding exodomain of human gonadotropin receptors.

Glycoprotein hormone receptors contain large N-terminal extracellular domains (ECDs) that distinguish these receptors from most other G protein-coupled receptors. Each glycoprotein hormone receptor ECD consists of a curved leucine-rich repeat domain flanked by N- and C-terminal cysteine-rich regions. Selectivity of the different glycoprotein hormone receptors for their cognate hormones is exclusively determined by their ECDs and, in particular, their leucine-rich repeat domain. To identify human (h)FSH-selective determinants we used a gain-of-function mutagenesis strategy in which beta-strands of the hLH receptor (hLH-R) were substituted with their hFSH receptor (hFSH-R) counterparts. Introduction of hFSH-R beta-strand 1 into hLH-R conferred responsiveness to hFSH, whereas hLH-R mutants harboring one of the other hFSH-R beta-strands displayed none or very limited sensitivity to hFSH. However, combined substitution of hFSH-R beta-strand 1 and some of the other hFSH-R beta-strands further increased the sensitivity of the mutant hLH-R to hFSH. The apparent contribution of multiple hFSH-R beta-strands in providing a selective hormone binding interface corresponds well with their position in relation to hFSH as recently determined in the crystal structure of hFSH in complex with part of the hFSH-R ECD.

Structural biology of glycoprotein hormones and their receptors.

Glycoprotein hormones regulate reproduction in vertebrates and exert their actions through specific G protein-coupled receptors on target cell surfaces. Structural information is now available for human chorionic gonadotropin (CG), follicle-stimulating hormone (FSH), and FSH bound to the extracellular binding domain of its receptor (FSHR(HB)). The recently determined structure of a human FSH-FSHR(HB) complex provides an explanation for the specificity of glycoprotein hormones binding to their receptors, and it suggests hypotheses concerning the mechanism of transmembrane signal transduction.

Identifying protein interactors in gonadotropin action.

Systems biology integrates a variety of diverse approaches to the study of the cellular pathways comprised of protein networks. Following an initial ligand-receptor binding event, transduction of the signal is modified in a variety of ways via downstream protein interactions. Protein interactions can occur between two proteins or, alternatively, an interaction between two proteins can be facilitated by an adapter protein. Protein interactions can affect the spatial and temporal distribution of ligand-receptor complexes in cells, attenuating or prolonging signaling. With regard to gonadotropin receptors, protein interactions have been primarily studied in terms of desensitization and termination of signal transduction, or for their role in trafficking. The purpose of this review is to describe protein interactions that mediate gonadotropin receptor functions and to highlight some emerging interactions, as well as some of the caveats inherent in the attempt to uncover these pathways.

A molecular dissection of the glycoprotein hormone receptors.

In glycoprotein hormone receptors, a subfamily of rhodopsin-like G protein-coupled receptors, the recognition and activation steps are carried out by separate domains of the proteins. Specificity of recognition of the hormones thyrotropin (TSH), lutropin (LH), human chorionic gonadotropin (hCG) and follitropin (FSH) involves leucine-rich repeats (LRRs) present in an N-terminal ectodomain, and can be associated with a limited number of residues at key positions of the LRRs. The mechanism by which binding of the hormones results in activation is proposed to involve switching of the ectodomain from a tethered inverse agonist to a full agonist of the serpentine, rhodopsin-like region of the receptor. Unexpectedly, the picture is complicated by the observation that promiscuous activation of one of the receptors (FSHr) by hCG or TSH can result from activating mutations affecting the serpentine region of the receptors.

Single chain variants of the glycoprotein hormones and their receptors as tools to study receptor activation and for analogue design.

One of the crucial steps in the biosynthesis of multisubunit proteins is their assembly. The glycoprotein hormone, thyroid-stimulating hormone, and the gonadotropins, luteinizing hormone, follicle-stimulating hormone and chorionic gonadotropin, are noncovalent heterodimers. Their assembly is critical for bioactivity because the heterodimers, but not the monomeric subunits, efficiently bind to and activate the cognate heptahelical receptor. Occasionally, mutated subunits cannot combine into a functional hormone, or the bioactivity of the assembled, yet modified, heterodimer is suboptimal.

Studies with chimeras of the gonadotropin receptors reveal the importance of third intracellular loop threonines on the formation of the receptor/nonvisual arrestin complex.

Using chimeras and more discrete exchange mutations of the rat (r) and human (h) gonadotropin receptors, we had previously identified multiple noncontiguous residues of the lutropin (LHR) and follitropin (FSHR) receptors that dictate their rates of internalization. Since the internalization of the LHR and the FSHR is driven by their abilities to associate with the nonvisual arrestins, we hypothesized that one or more of the residues previously identified by the internalization assays are involved in the formation of the receptor/nonvisual arrestin complex. In the studies reported herein, we tested this hypothesis by measuring the association of arrestin-3 with a large number of rLHR/hLHR and rFSHR/hFSHR exchange mutants that affect internalization. The results presented show that the same residues that dictate the rate of internalization of these two receptor pairs affect their ability to associate with arrestin-3. Although these residues are located in distinct topological domains, our analyses show that threonine residues in the third intracellular loop of both receptor pairs are particularly important for the formation of the receptor/arrestin-3 complexes and internalization. We conclude that the different rates of internalization of the gonadotropin receptors are dictated by their different abilities to associate with the nonvisual arrestins and that this association is, in turn, largely dictated by the presence of threonine residues in their third intracellular loops.

Common and differential mechanisms of gonadotropin receptors.

The gonadotropin receptors are G-protein-coupled receptors with unique structural and functional features, consisting of two halves. The N-terminal extracellular half (exodomain) binds the hormones, whereas the C-terminal membrane-associated half (endodomain) is responsible for receptor activation. In this review, the novel ternary interactions, contact points and mutual modulations among the exodomain, endodomain and hormone for hormone binding and signal generation are described based on the latest observations. This discussion is contrary to the yiew that the exodomain and endodomain are independent, at least functionally, and provides new insights into the receptor mechanisms for the gonadotropins and other G-protein-coupled receptors.

Piscine glycoprotein hormone (gonadotropin and thyrotropin) receptors: a review of recent developments.

Similar to the higher vertebrates, the pituitary in bony fishes express three glycoprotein hormones: thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In addition to the appropriate secretion of these hormones, the timely and quantitative expression of their specific receptors (TSHR, FSHR and LHR) in the target tissues is an essential requirement for their physiological action. In fishes that constitute a very diverse group of vertebrates, there are only a few published reports of primary structure of these receptors although other examples have been communicated briefly. This review will summarize these reports as well as to describe the insights gained from what is known about the mammalian receptors. The structural organization of the fish receptors (as deduced from the encoding cDNAs) is highly homologous to the higher vertebrate receptors in that there is a 7-pass transmembrane region and an N-terminal extracellular domain, which contributes to ligand specificity. In mammals, the FSHR and the TSHR genes are composed of 10 exons whereas the LHR gene is composed of 11 exons. The position of the 'extra intron' is conserved in the catfish LHR gene. In the mammals, the transmembrane domain of each of the three glycoprotein hormone receptors is encoded by a single exon, however, in the salmon genes and homologous invertebrate genes, this portion of the receptor is encoded by multiple exons. In general, the tissue-specific expression of these receptors is similar to that seen in mammals, however, the gonadal expression of TSHR in the striped bass and sunrise sculpin and the renal expression of LHR in the channel catfish are unique.

Fish gonadotropin and thyrotropin receptors: the evolution of glycoprotein hormone receptors in vertebrates.

We have cloned and characterized, for the first time in fish, two different gonadotropin receptors (GTHR) and a single thyrotropin receptor (TSHR) from amago salmon (Oncorhynchus rhodurus) and Nile tilapia (Oreochromis niloticus). Phylogenetic analyses and intron/exon structure suggest that the two GTHRs in fish are comparable to tetrapod follicle stimulating hormone and luteinizing hormone receptors. Temporal and spatial expression patterns, examined by Northern blot analysis and in situ hybridization, paralleled those seen in mammals and birds. Consequently, genetic and functional divergence of two GTHRs and TSHR probably occurred before the teleost and tetrapod split.

Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function.

The recent unraveling of structures of genes for the gonadotropin subunits and gonadotropin receptors has provided reproductive endocrinologists with new tools to study normal and pathological functions of the hypothalamic-pituitary-gonadal axis. Rare inactivating mutations that produce distinctive phenotypes of isolated LH or FSH deficiency have been discovered in gonadotropin subunit genes. In addition, there is a common polymorphism in the LHbeta subunit gene with possible clinical significance as a contributing factor to pathologies of LH-dependent gonadal functions. Both activating and inactivating mutations have been detected in the gonadotropin receptor genes, a larger number in the LH receptor gene, but so far only a few in the gene for the FSH receptor. These mutations corroborate and extend our knowledge of clinical consequences of gonadotropin resistance and inappropriate gonadotropin action. The information obtained from human mutations has been complemented by animal models with disrupted or inappropriately activated gonadotropin ligand or receptor genes. These clinical and experimental genetic disease models form a powerful tool for exploring the physiology and pathophysiology of gonadotropin function and provide an excellent example of the power of molecular biological approaches in the study of pathogenesis of diseases.

The rate of internalization of the gonadotropin receptors is greatly affected by the origin of the extracellular domain.

Previous results from this laboratory have shown that human kidney (293) cells transfected with the rat follitropin receptor (rFSHR) internalize agonist (i.e. human follitropin, hFSH) at a rate similar to that of other agonist-G protein-coupled receptor complexes while 293 cells transfected with the rat lutropin/choriogonadotropin receptor (rLHR) internalize agonist (human choriogonadotropin, hCG) at a rate that is about 1 order of magnitude slower. Taking advantage of this difference and the high degree of homology between the rLHR and rFSHR, we have now used chimeras of these two receptors to begin to delineate structural features that influence their internalization. Analysis of six chimeras that exchanged only the transmembrane domains (designated FLF and LFL), only the COOH-terminal domains (FFL or LLF) or both domains (FLL or LFF) show that the origin of the extracellular domain is at least as important, if not more, than the origin of the transmembrane and COOH-terminal domains in determining the rate of internalization of the gonadotropin receptors. Thus, the rates of internalization of agonist internalization mediated by FFL, FLF, and FLL more closely resemble rFSHR than rLHR, while the rates of agonist internalization mediated by LLF, LFL, and LFF more closely resemble rLHR than rFSHR. The importance of the extracellular domain was also evident even upon overexpression of arrestin-3, a protein that enhances the rate of internalization of the wild-type receptors and chimeras by binding to their intracellular regions.

Gonadotropin receptors and the control of gonadal steroidogenesis: physiology and pathology.

Over the past few years, knowledge of the structure of gonadotropin receptors and their mode of action has rapidly advanced. The cDNA corresponding to the luteinizeng hormone (LH) receptor (LHR) has been cloned, leading to the identification of a novel family of G-protein-coupled receptors. The follicle stimulating hormone (FSH) receptor (FSHR) was thereafter cloned by cross-hybridization with the LHR. Structure-function relationships have been studied by mutagenesis experiments in several laboratories. The cloning and chromosomal localization to chromosome 2p21 of the two human gonadotropin receptor genes has provided insights into their evolutionary relationships. The LHR and FSHR genes are very large and contain 10 and 11 exons respectively. The obtention of monoclonal antibodies against the receptors resulted in the characterization of the receptor proteins. These antibodies also allowed the study of receptor expression in target cells in physiological and pathological conditions. The internalization of the LHR has been studied by electron microscopy. A mechanism of receptor-mediated transcytosis through the endothelial cells of the testes has been described for the LHR. The polarized expression of receptors has been studied. The cloning of gonadotropin receptor genes has opened the field of genetic study of the receptors. Inactivating mutations of the LHR have been described in Leydig cell agenesis or hypoplasia. Different phenotypes, including complete pseudohermaphroditism, ambiguous genitalia and male phenotype, have been described. In the case of the FSHR, only one mutation has been reported in familial ovarian dysgenesis with primary amenorrhea. Related males have variable alterations of spermatogenesis and fertility. Constitutive mutations of the LHR have been reported in familial testotoxicosis. One similar mutation has also been described for the FSHR. Such mutations may lead to the development of a model of receptor activation.