Molecular characterization of two sea bass gonadotropin receptors: cDNA cloning, expression analysis, and functional activity

The follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) play central roles in vertebrate reproduction. They act through their cognate receptors to stimulate testicular and ovarian functions. The present study reports the cloning and characterization of two sea bass (Dicentrarchus labrax) cDNAs encoding a FSH receptor (sbsFSHR) and a LH receptor (sbsLHR). The mature proteins display typical features of the glycoprotein hormone receptor family members, but the sbsFSHR also contains some remarkable differences when compared with other fish or mammalian FSHRs. Among them, a distinct extracellular N-terminal cysteine domain as regards to its length and cysteine number, and the presence of an extra leucine-rich repeat. Expression analysis revealed that the sbsFSHR is exclusively expressed in gonadal tissues, specifically in the follicular wall of previtellogenic and early-vitellogenic follicles. On the contrary, sbsLHR mRNA was found to be widely distributed in sea bass somatic tissues. When stably expressed in mammalian cell lines, sbsFSHR was specifically stimulated by bovine FSH, while sbsLHR was activated by both bovine LH and FSH. Nevertheless, specific stimulation of the sbsLHR was observed when recombinant sea bass gonadotropins were used. The isolation of a FSHR and a LHR in sea bass opens new ways to study gonadotropin action in this species.     

Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50-55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the "classical" role of pituitary function regulation.     

Small-molecule agonists for the thyrotropin receptor stimulate thyroid function in human thyrocytes and mice

Seven-transmembrane-spanning receptors (7TMRs) are prominent drug targets. However, small-molecule ligands for 7-transmembrane-spanning receptors for which the natural ligands are large, heterodimeric glycoprotein hormones, like thyroid-stimulating hormone (TSH; thyrotropin), have only recently been reported, and none are approved for human use. We have used quantitative high-throughput screening to identify a small-molecule TSH receptor (TSHR) agonist that was modified to produce a second agonist with increased potency. We show that these agonists are highly selective for human TSHR versus other glycoprotein hormone receptors and interact with the receptor's serpentine domain. A binding pocket within the transmembrane domain was defined by docking into a TSHR homology model and was supported by site-directed mutagenesis. In primary cultures of human thyrocytes, both TSH and the agonists increase mRNA levels for thyroglobulin, thyroperoxidase, sodium iodide symporter, and deiodinase type 2, and deiodinase type 2 enzyme activity. Moreover, oral administration of the agonist stimulated thyroid function in mice, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Thus, we discovered a small molecule that activates human TSHR in vitro, is orally active in mice, and could be a lead for development of drugs to use in place of recombinant human TSH in patients with thyroid cancer.     

Molecular characterization of sea bass gonadotropin subunits (alpha,FSHbeta, and LHbeta) and their expression during the reproductive cycle

Reproduction is controlled by two pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This study reports the cloning, sequence analysis, and gene expression of gonadotropin (GTH) subunits from the European sea bass (Dicentrarchus labrax). The GTH subunits were cloned from a sea bass brain-pituitary cDNA library. The nucleotide sequences of the common alpha, the FSHbeta, and the LHbeta subunit cDNAs were 625, 521, and 591 base pair (bp) long, respectively, encoding for mature peptides of 94, 105, and 115 amino acids (aa), respectively. Sequence analysis showed that sea bass FSHbeta is more similar to higher vertebrate FSHbeta's (35-37%) than to LHbeta's (26-30%), whereas sea bass LHbeta is more similar to LHbeta's (40-53%) than to FSHbeta's (26-41%). Phylogenetic analysis of fish GTH sequences grouped the beta subunits into two groups, FSH and LH, distributed into four classes, corresponding to the accepted divisions of Elopomorphs, Ostariophysis, Salmonids, and Percomorphs. A dot-blot technique was developed to analyze GTH pituitary mRNA levels during the reproductive cycle of male sea bass. From October (initiation of gametogenesis) to February (spermiation), the expression of all three subunits in the pituitary increased in parallel, concomitantly with the gonadosomatic index (GSI) and the accumulation of LH protein in the pituitary, all values declining sharply at post-spermiation. This study demonstrates that the pituitary of sea bass contains two gonadotropin hormones and that both gonadotropins are probably involved in the control of gametogenesis, gamete maturation, and spermiation.     

Cloning, characterisation, and expression of three oestrogen receptors (ERalpha, ERbeta1 and ERbeta2) in the European sea bass, Dicentrarchus labrax

Three oestrogen receptor [ER] subtypes have been described in teleost fish, namely ERalpha, and two ERbeta subtypes, called ERbeta1 and ERbeta2 (or ERbeta and ERgamma in Atlantic croaker). Their expression during embryonic development and gonadal growth has evoked interest in their potential role in sexual differentiation and gonadal development in fish. We cloned three oestrogen receptors from adult liver (sb-ERalpha cDNA) and ovary (partial sb-ERbeta1 and sb-ERbeta2 cDNAs) of the European sea bass, and according to their phylogenetic relatedness to other ERs in teleosts, named them sea bass [sb-] ERalpha, ERbeta1 and ERbeta2. Deduced amino acid numbers for sb-ERalpha, sb-ERbeta1 and sb-ERbeta2 were 639, 517 and 608, respectively, representing in the case of sb-ERbeta1 and sb-ERbeta2 about 90% of the open reading frame. Highest amino acid identities were found for sb-ERalpha with eelpout ERalpha (88.7%), for sb-ERbeta1 with Atlantic croaker ERgamma (85.8%), and for sb-ERbeta2 with Atlantic croaker ERbeta (90.1%). Southern analysis confirmed that all three sea bass oestrogen receptors (sb-ERs) are the products of three distinct genes. In adult sea bass, ERalpha was predominantly expressed in liver and pituitary, while sb-ERbeta1 and sb-ERbeta2 were more ubiquitously expressed, with highest expression levels in pituitary. In a mixed-sex population of juvenile sea bass, sb-ERalpha expression was significantly elevated in gonads at 200 days posthatch (dph), while for sb-ERbeta1 and sb-ERbeta2 highest expression levels were observed in gonads at 250 dph. For sb-ERbeta2, expression was also significantly higher in the brain at 250 dph. The cloning of these three ER subtypes in the European sea bass together with the results obtained on expression levels in adult and juvenile animals has given us the foundation to investigate their possible role in sexual differentiation and development in this species in future studies.     

Thyrotropic activity of recombinant human glycoprotein hormone analogs and pituitary mammalian gonadotropins in goldfish (Carassius auratus): insights into the evolution of thyrotropin receptor specificity

Thyrotropin (TSH) is a pituitary glycoprotein hormone heterodimer that binds to its G-protein coupled receptor (TSH-R) at the thyroid to promote the synthesis and secretion of thyroid hormone. Very little is known about TSH-TSH-R interactions in teleost fish. Mammalian gonadotropins have been reported to have an intrinsic ability to activate teleost fish TSH-Rs, suggesting the TSH-R in teleost fish is more promiscuous than in other vertebrates. In this study we utilized the goldfish T(4)-release response and recombinant human TSH analogs as in vivo tools to evaluate the structural constraints on hormone-receptor interactions. We found that four positively charged lysines substituted for neutral or negatively charged amino acids within positions 11-20 of the glycoprotein hormone subunit α (GSUα) significantly increased biological activity of hTSH in fish, as it does in mammals. We further found that bovine follicle stimulating hormone but not luteinizing hormone, whose GSUα subunits also contain four lysine or arginine amino acid residues in the N-terminal portion of GSUα, was thyrotropic in goldfish, suggesting gonadotropin β subunit contributes to the heterothyrotropic activity. Though recombinant human FSH did not produce a dose-dependent increase in T(4), thyrotropic activity could be acquired with the addition of positively charged amino acids at the N-terminal portion of its GSUα, confirming the importance of the charge on those amino acids for activation of the goldfish TSH-R. These studies demonstrate that mammalian glycoprotein hormone analogs can be utilized to evaluate the conservation of receptor binding and activation mechanisms between fish and mammals.     

Conservation of the heterodimeric glycoprotein hormone subunit family proteins and the LGR signaling system from nematodes to humans

Glycoprotein hormones, follicle-stimulating hormones (FSHs), luteinizing hormones (LHs), thyroid-stimulating hormones (TSHs), and chorionic gonadotropin (CG) are key endocrine hormones secreted from the pituitary gonadotrophs and thyrotrophs and the placenta in primates. These hormones, consisting of a common alpha subunit and a specific beta subunit, act through the FSH receptor (FSHR), the LH receptor (LHR), and the TSH receptor (TSHR) that are highly specific for their cognate hormones. These glycoprotein hormones are structurally and functionally conserved in various vertebrates and have been identified in most lineages of actinopterygians (bony fish) and sarcopterygians (tetrapods). Of interest, recent genomic studies showed that vertebrate glycoprotein hormone receptors belong to an ancient subfamily of G protein-coupled receptors (GPCRs) named as leucine-rich repeat-containing GPCRs (LGRs). These findings have prompted the hypothesis that there could be additional glycoprotein hormones in vertebrate genomes. Indeed, searches of vertebrate genomes have led to the identification of two novel glycoprotein hormone subunits, glycoprotein alpha 2 (GPA2) and glycoprotein beta 5 (GPB5), as well as their homologs in invertebrates. Subsequently, it was demonstrated that GPA2 and GPB5 form a heterodimeric hormone, thyrostimulin/OGH, capable of activating TSHR in vivo and the thyroid axis in transgenic mice. However, the exact role of this novel glycoprotein hormone and its homolog in invertebrates is not clear. To gain a better understanding of the physiological role of the novel glycoprotein hormone subunits and their evolution, it is imperative to carry out systematic studies of these genes in representative model species. In the present report, we summarize our findings based on studies of genomes of model organisms from sea anemones to humans. We found that GPA2 and GPB5 represent the ancient forms of glycoprotein hormone alpha and beta subunits, respectively, and that vertebrate and invertebrate glycoprotein hormone subunit proteins shared common ancestors that evolved during early metazoan evolution. It is important to note that glycoprotein hormone alpha and beta subunit proteins from invertebrates formed a heterodimer with structural functional characteristics similar to that of vertebrate glycoprotein hormones. Taken together, both glycoprotein hormone alpha and beta subunits evolved before the evolution of nematodes, arthropods, and vertebrates. Parallel expansion of the alpha and beta subunits and their receptors through gene duplication and subsequent subfunctionalization and neofunctionalization of the duplicated genes allowed the development of multiple tissue-specific endocrine systems in vertebrates.     

Sea bass (Dicentrarchus labrax) sex hormone binding globulin: molecular and biochemical properties and phylogenetic comparison of its orthologues in multiple fish species

Sex hormone binding globulin (SHBG) binds and transports androgens and estrogens in the blood of vertebrate species including fish. We have used oligonucleotide primers corresponding to highly conserved regions of the SHBG coding sequences within the zebrafish and fugufish genomes to obtain a 1528 bp cDNA encoding SHBG from tissue RNA extracts from the European sea bass. Amino-terminal sequence analysis of recombinant sea bass SHBG indicated that its deduced precursor polypeptide includes a 35-residue secretion signal polypeptide, and the 361-residue mature sea bass SHBG sequence exhibits 45-67% sequence identity with SHBGs from other fish species that have been determined directly (for zebrafish) or deduced (for rainbow trout, medaka and fugufish) from sequences within public databases. The sea bass SHBG (39,894 Da) comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites, and exists as a 118,300 +/- 11,500 Da homodimer. Sea bass SHBG exhibits a high affinity (K(d) = 8.8 nM for 17beta-estradiol) and specificity for gonadal steroids and their precursors (e.g., 17beta-estradiol > testosterone > dehydroepiandrosterone > 5alpha-dihydrotestosterone > androstenedione >11-ketotesterone). Interestingly, the affinity of sea bass SHBG for the synthetic estrogen, 17alpha-ethynylestradiol was found to be essentially identical to that for 17beta-estradiol. The availability of SHBG sequences in sea bass and other fish set the stage for detailed studies of SHBG function in fish reproductive physiology, as well as its potential role as a target of endocrine disruptors.     

Long-term exposure to continuous light inhibits precocity in European male sea bass (Dicentrarchus labrax, L.): hormonal aspects

The effect of long-term exposure to continuous light on the hormonal aspects of the reproductive axis was investigated in juvenile male sea bass (Dicentrarchus labrax, L.) during the first annual cycle. Four-month-old fish were exposed to a simulated natural photoperiod (NP) and a continuous light (24 h) regime (LL) under natural conditions of temperature (13.3-25.8 degrees C). A dot-blot technique was used to analyse gonadotropin (the common glycoprotein alpha, GPalpha; the follicle stimulating hormone beta, FSHbeta; and the luteinizing hormone beta, LHbeta, subunits) mRNA levels in the pituitary during the experiment. A homologous ELISA was used to determine pituitary sea bream gonadotropin-releasing hormone (sbGnRH) and LH and plasma LH levels; gonadal and plasma sex steroids concentrations were determined by specific immunoassays. LL significantly inhibited the expression of all three gonadotropins subunits in the pituitary. However, no significant differences on plasma LH levels were observed between NP and LL groups throughout the period of the experience. Long-term exposure to LL regime was extremely effective in inhibiting gonadal growth and hence precocious maturation as well as the accumulation of Testosterone (T) and 11-ketotestosterone (11-KT) in the gonads compared to the control group. 11-KT plasma levels remained low and unchanged in the LL group during the study. This work describe important alterations of the endocrine system, particularly at the pituitary-gonad axis provoked by exposure to continuous illumination and discusses the mechanism by which precocity in male sea bass is generated.     

The extracellular domain suppresses constitutive activity of the transmembrane domain of the human TSH receptor: implications for hormone-receptor interaction and antagonist design

Among glycoprotein hormone receptors the TSH receptor (TSHR) is the most susceptible to constitutive activation by mutations in various regions of the molecule, including mutations in the extracellular domain (ECD) and extracellular loops of the transmembrane domain (TMD). To understand the role of the ECD in TSHR activation we have tested several TSHR constructs with major deletions of the ECD. Previous studies reported very low expression of such truncated glycoprotein hormone receptors, which prevented reliable assessment of their ligand-binding and basal constitutive activities. We have eliminated this problem using TSHR tagged at its N-terminus with a hemagglutinin tag (HA) recognized by the HA-specific monoclonal antibody. Based on such quantitation the TSHR deletion mutant missing 386 N-terminal amino acid residues, constituting 98% of the entire ECD, showed 4-7 fold higher normalized basal activity compared to activity of the corresponding wild-type (WT) TSHR construct. This increase in basal activity was significantly inhibited by linking the common alpha-subunit of glycoprotein hormones at the N-terminus of the truncated TSH receptor. The role of a hypothetical activating fragment (409-418) in TSHR activation was further studied using peptides and mutagenesis of charged residues. This study provides important evidence supporting the "two-state" model of TSHR activation and the potential role of proteolytic cleavage for receptor activation. Accordingly, the mechanism of hormone-induced receptor activation is dependent, at least in part, on the elimination of inhibitory interactions within the receptor. Such intra-molecular inhibition of TSHR may include electrostatic interactions between the ECD and extracellular loops of TMD. Moreover, the truncated, constitutively active receptors described herein provide new insights valuable in the design of TSHR antagonists.     

The cloned equine thyrotropin receptor is hypersensitive to human chorionic gonadotropin; identification of three residues in the extracellular domain involved in ligand specificity

The receptors for TSH, LH/chorionic gonadotropin (CG), and FSH belong to the same subfamily of G protein-coupled receptors. The specificity of recognition of their cognate hormone involves a limited number of residues in the leucine-rich repeats present in the N-terminal ectodomain of the receptor. It is admitted that receptors of this subfamily coevoluted with their respective ligands. The secretion of CG is restricted to gestation of primates and Equidae. We hypothesized that, facing the challenge of a new hormone, the glycoprotein hormone receptors would have evolved differently in Equidae and human so that distinct residues are involved in hormone specificity. In particular, it is known that equine CG has a dual (FSH and LH) activity when administered to other species. In the present work, we cloned and characterized functionally the equine TSH receptor (TSHR), which shares 89% homology with the human TSHR. The equine TSHR is not responsive to equine CG but is more sensitive to human CG than the human TSHR. Three residues, at positions 60, 229, and 235 of the ectodomain, are responsible for this difference in sensitivity as shown by modelization and targeted mutagenesis, followed by in vitro functional characterization. The phylogenetic approach is a suitable approach to identify determinants of specificity of receptors.     

The hinge region of the TSH receptor stabilizes ligand binding and determines different signaling profiles of human and bovine TSH

The hinge region (HinR) is a variable structure of glycoprotein hormone receptors. Its amino acid composition and length is different for glycoprotein hormone receptors and connects the ligand binding domain with the serpentine domain. The role of the HinR of the receptors for TSH, follitropin (FSH), and LH/choriogonadotropin (LHCG) in receptor and signaling specificity is unknown. To investigate the role of the HinR for ligand binding, signal generation, and for the transmission of the signal towards the serpentine domain, we replaced the HinR of the TSH receptor (TSHR) by those of LHCG receptor and FSH receptor and introduced constitutively activating mutations and one mutation deficient for bovine (b)TSH binding in these chimeras. Functional characterization of the TSHR variants was carried out after transient transfection of COS-7 cells by determination of the cell surface expression, ligand binding, and recombinant human (rh)TSH or bTSH activation of second messengers. We show that the HinR of the TSHR stabilizes hormone binding regarding ligand affinity and retention time of the bound ligand as determined by dissociation experiments. Introduction of a constitutively activating extracellular loop mutation in these constructs lead to partially restored binding patterns. These findings indicate that the HinR-extracellular loop interface is besides signaling also important for bTSH binding. Furthermore, data for G protein signaling reveal that the activity of bTSH, but not of rhTSH, depends on the TSHR HinR, which was indicated by a significant right shift in the dose-response curves for G(s) and G(q) activation for TSHR chimeras harboring the LHCG receptor and FSH receptor HinR, respectively. Moreover, we identified different G protein signaling profiles for bTSH and rhTSH, which cannot be explained by the characterized HinR. For future studies regarding structure and function of the TSHR, it will be necessary to characterize TSHR variants with both or more ligands.     

Thyrotropin receptor antibodies: new insights into their actions and clinical relevance

The thyrotropin receptor (TSHR) is a G-protein-coupled receptor with a large ectodomain. TSH, acting via TSHR, regulates thyroid growth and thyroid hormone production and secretion. The TSHR undergoes complex post-translational processing involving dimerization, intramolecular cleavage, and shedding of its ectodomain, and each of these processes may influence the antigenicity of the TSHR. The TSHR is also the major autoantigen in Graves' disease, as well as a leading candidate autoantigen in both Graves' ophthalmopathy and pretibial myxedema. The naturally conformed TSHR is most effectively presented as an autoantigen to the immune system, causing the production of stimulating TSHR-Abs. There are also autoantibodies which block the TSHR from TSH action, and neutral TSHR-Abs which have no influence on TSH action. TSHR-Abs can be detected by competition assays of TSHR-Abs for labeled TSH, or monoclonal TSHR-Ab binding to solubilized TSHRs, or by bioassays using thyroid cells or mammalian cells expressing recombinant TSHRs.     

Crystal structure of the TSH receptor in complex with a thyroid-stimulating autoantibody.

OBJECTIVE: To analyze interactions between the thyroid-stimulating hormone receptor (TSHR) and a thyroid-stimulating human monoclonal autoantibody (M22) at the molecular level. DESIGN: A complex of part of the TSHR extracellular domain (amino acids 1-260; TSHR260) bound to M22 Fab was prepared and purified. Crystals suitable for X-ray diffraction analysis were obtained and the structure solved at 2.55 A resolution. MAIN OUTCOME: TSHR260 comprises of a curved helical tube and M22 Fab clasps its concave surface at 90 degrees to the tube length axis. The interface buried in the complex is large (2,500 A(2)) and an extensive network of ionic, polar, and hydrophobic bonding is involved in the interaction. There is virtually no movement in the atoms of M22 residues on the binding interface compared to unbound M22 consistent with "lock and key" binding. Mutation of residues showing strong interactions in the structure influenced M22 activity, indicating that the binding detail observed in the complex reflects interactions of M22 with intact, functionally active TSHR. The receptor-binding arrangements of the autoantibody are very similar to those reported for follicle-stimulating hormone (FSH) binding to the FSH receptor (amino acids 1-268) and consequently to those of TSH itself. CONCLUSIONS: It is remarkable that the thyroid-stimulating autoantibody shows almost identical receptor-binding features to TSH although the structures and origins of these two ligands are very different. Furthermore, our structure of the TSHR and its complex with M22 provide foundations for developing new strategies to understand and control both glycoprotein hormone receptor activation and the autoimmune response to the TSHR.     

Human monoclonal thyroid stimulating autoantibody

A monoclonal autoantibody (MAb) with powerful thyroid stimulating activity has been produced from lymphocytes from a patient with Graves' disease. The autoantibody and its Fab fragment bind to the thyroid stimulating hormone (TSH) receptor (TSHR) with high affinity, inhibit labelled TSH binding to the receptor and stimulate cyclic AMP production in Chinese hamster ovary cells transfected with TSHR. TSHR autoantibodies with TSH agonist or antagonist activities from patients' serum samples are effective inhibitors of labelled monoclonal autoantibody binding to TSHR. Thus, the human monoclonal autoantibody has all the characteristics of serum TSHR autoantibodies. Its availability has important implications for new studies on the pathogenesis of Graves' disease.     

A hydrophobic cluster in the center of the third extracellular loop is important for thyrotropin receptor signaling

Previous reports on the follicle-stimulating hormone receptor and choriogonadotropic/LH receptor, which belong to the glycoprotein hormone receptor family, suggest that the extracellular loop (ECL) 3 could be a key domain for ligand binding and intramolecular receptor signaling. In contrast to ECLs 1 and 2 of glycoprotein hormone receptors, the ECL3 displays high sequence homology, particularly in the central portion of the loop. Therefore, we opted to identify amino acids with functional importance within ECL3 of the TSH receptor (TSHR). Single alanine substitutions of all residues in ECL3 were generated. Functional characterization revealed the importance of five amino acids in the central portion of ECL3 and K660 at the ECL3/transmembrane helix (TMH) 7 junction for TSHR signaling. Decrease of G(s) activation and loss of G(q) activation by substitutions of K660 demonstrates a role for this position for TSHR conformation and signal transduction. By molecular modeling, we predicted potential interaction partners of K660:E409 and D410 in the N terminus of TMH1 and D573 in the ECL2. Complementary double mutants did not reconstitute G(s)/G(q)-mediated signaling, suggesting that K660 is not directly involved in a structural unit between ECL3 and the N terminus of TMH1. These results support a TSHR model in which the side chain of K660 is orientated toward the backbone of ECL2. Moreover, our findings provide evidence that a hydrophobic cluster, comprising residues 652-656 of ECL3, strongly influences intramolecular signal transduction and G protein activation of the TSHR.