Tracking conformational transitions of the gonadotropin hormone receptors in a bilayer of (SDPC) poly-unsaturated lipids from all-atom molecular dynamics simulations.

Glycoprotein hormone receptors [thyrotropin (TSHR), luteinizing hormone/chorionic gonadotropin (LHCGR), and follicle stimulating hormone (FSHR) receptors] are rhodopsin-like G protein-coupled receptors. These receptors display common structural features including a prominent extracellular domain with leucine-rich repeats (LRR) stabilized by ß-sheets and a long and flexible loop known as the hinge region (HR), and a transmembrane (TM) domain with seven α-helices interconnected by intra- and extracellular loops. Binding of the ligand to the LRR resembles a hand coupling transversally to the α- and ß-subunits of the hormone, with the thumb being the HR. The structure of the FSH-FSHR complex suggests an activation mechanism in which Y335 at the HR binds into a pocket between the α- and ß-chains of the hormone, leading to an adjustment of the extracellular loops. In this study, we performed molecular dynamics (MD) simulations to identify the conformational changes of the FSHR and LHCGR. We set up a FSHR structure as predicted by AlphaFold (AF-P23945); for the LHCGR structure we took the cryo-electron microscopy structure for the active state (PDB:7FII) as initial coordinates. Specifically, the flexibility of the HR domain and the correlated motions of the LRR and TM domain were analyzed. From the conformational changes of the LRR, TM domain, and HR we explored the conformational landscape by means of MD trajectories in all-atom approximation, including a membrane of polyunsaturated phospholipids. The distances and procedures here defined may be useful to propose reaction coordinates to describe diverse processes, such as the active-to-inactive transition, and to identify intermediaries suited for allosteric regulation and biased binding to cellular transducers in a selective activation strategy.

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.

Extracellular loop 3 substitutions K589N and A590S in FSH receptor increase FSH-induced receptor internalization and along with S588T substitution exhibit impaired ERK1/2 phosphorylation.

The interaction of follicle stimulating hormone with its specific GPCR, the follicle stimulating hormone receptor (FSHR) is facilitated by the extracellular loops (ELs) which contact the transmembrane domain and relay the signal downstream. In order to determine the contribution of non conserved residues from the EL3 of FSHR in conferring specificity to FSH-FSHR interaction, they were swapped with respective residues from luteinizing hormone/choriogonadotropin receptor. The triple mutant EL3M exhibited increased internalization of FSH-FSHR complexes without affecting the cAMP signaling response. Here, substitution point mutants S588T, K589N and A590S of the EL3 of FSHR were generated and characterized. None of these substitutions affected FSHR expression, FSH binding ability and cAMP production as compared to wild type FSHR. However, the high internalization of EL3M was observed to be due to the K589N and A590S substitutions. Further, all the mutants showed an impaired FSH mediated ERK phosphorylation response and the extent of impairment was most striking in case of the A590S substitution. Interestingly, S588T mutant exhibited impaired ERK phosphorylation, without change in receptor internalization, indicating that these processes can be dissociated. Thus, the FSHR specific residues K589 and A590 in the EL3 of FSHR seem to be crucial for FSH-induced internalization and ERK phosphorylation.

In silico analysis, temporal expression and gonadotropic regulation of receptors for follicle-stimulating hormone and luteinizing hormone in testis of spotted snakehead Channa punctata.

This study in spotted snakehead Channa punctata was aimed to develop a comprehensive understanding of testicular gonadotropin receptors, from their sequence characterization, temporal expression to gonadotropic regulation, in seasonally breeding teleosts. A single form of follicle-stimulating hormone receptor (cpfshra) and luteinizing hormone/choriogonadotropin receptor (cplhcgr), was identified from testicular transcriptome data of C. punctata. Although deduced full-length protein sequence for cpFshra (694 amino acids) and cpLhcgr (691 amino acids) showed homology with their counterparts of other vertebrates, multiple insertion-deletion-substitution of residues suggest marked alterations in their structure and ligand specificity. The absolute quantification of testicular cpfshra and cplhcgr was estimated along the reproductive cycle following real-time PCR. The temporal expression profile showed highest testicular expression of both the gonadotropin receptors during resting phase. Their expression progressively decreased during preparatory and spawning phases concomitant with spermatogonial proliferation and differentiation and spermiogenesis. However, levels of cpfshra and cplhcgr sharply increased during post-spawning when seminiferous lobules were largely devoid of germ cells. To explore gonadotropic regulation of testicular cpfshra and cplhcgr, one group of fish of resting phase was administered with single dose of human chorionic gonadotropin (hCG; 5,000 IU/kg body mass) on day 0 and sacrificed on day 3 and day 5, while another group receiving two injections of hCG (day 0 and day 7) was sacrificed on day 14. The expression pattern of testicular gonadotropin receptors in hCG-treated fish sacrificed after 3, 5 and 14 days was similar to that of preparatory, spawning and postspawning phases, respectively. Likewise, testicular histology of hCG-treated fish sacrificed on day 3, day 5 and day 14 was comparable with that of preparatory, early spawning and late spawning phases, respectively. In light of the fact that gonadotropin receptors are largely expressed on somatic cells, an apparent decrease in testicular cpfshra and cplhcgr levels during preparatory and spawning phases or after 3 and 5 days from first hCG injection might not be due to downregulation of their expression. Rather, this could be due to dilution of somatic cell mRNA by large amount of germ cell mRNA. To verify this assumption, effect of hCG on plasma level of androgens was investigated employing enzyme-linked immunosorbent assay. A marked increase in plasma level of testosterone and 11-ketotestosterone was observed after hCG treatment in C. punctata. This would have been possible only when hCG upregulated the expression of testicular gonadotropin receptors.

First mutation in the FSHR cytoplasmic tail identified in a non-pregnant woman with spontaneous ovarian hyperstimulation syndrome.

BACKGROUND: Spontaneous ovarian hyperstimulation syndrome (sOHSS) is a rare event occurring mostly during natural pregnancy. Among described etiologies, some activating mutations of FSH receptor (FSHR) have been identified. CASE PRESENTATION: We report hereby the case of a non-pregnant women with three episodes of sOHSS. Hormonal evaluation was normal and no pituitary adenoma was detected. However, genetic analysis identified a novel heterozygous FSHR mutation (c.1901 G > A). This R634H mutation is the first described in the cytoplasmic tail of the receptor. Functional analysis failed to reveal constitutive activity of the mutant but a decreased cAMP production in response to FSH. The weak activity of this mutant is correlated with a markedly reduced cell surface expression. CONCLUSION: Pathophysiology of non gestationnal sOHSS is still ill established. The molecular characterization of this new mutant indicates that it might not be at play. Therefore, further investigations are needed to improve knowledge of the molecular mechanism of this syndrome.

The Follitropin Receptor: Matching Structure and Function.

Follitropin, or follicle-stimulating hormone (FSH) receptor (FSHR), is a G protein-coupled receptor belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although its primary location is the gonad, the FSHR has also been reported in extragonadal tissues including bone, placenta, endometrium, liver, and blood vessels from a number of malignant tumors. The recently resolved crystal structure of FSH bound to the entire FSHR ectodomain has been instrumental in more clearly defining the role of this domain in ligand binding and receptor activation. Biochemical, biophysical, and structural data also indicate that the FSHR exists as a higher order structure and that it may heterodimerize with its closely related receptor, the luteinizing hormone receptor; this association may have physiologic implications during ovarian follicle maturation given that both receptors may simultaneously coexist in the same cell. FSHR heterodimerization is unique to the ovary because in the testes, gonadotropin receptors are expressed in separate compartments. FSHR self-association appears to be required for receptor coupling to multiple effectors and adaptors, for the activation of multiple signaling pathways and the transduction of asymmetric signaling, and for negative and positive receptor cooperativity. It also provides a mechanism through which the glycosylation variants of FSH may exert distinct and differential effects at the target cell level. Given its importance in regulating activation of distinct signaling pathways, functional selectivity at the FSHR is briefly discussed, as well as the potential implications of this particular functional feature on the design of new pharmacological therapies in reproduction.

A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing.

STUDY QUESTION: Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility? SUMMARY ANSWER: A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking. WHAT IS KNOWN ALREADY: WES may diagnose up to 25-35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure. STUDY DESIGN, SIZE, DURATION: A WES study was followed by flow cytometry studies of mutant protein function. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G>T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro validation. All novel purported genetic variants can be clinically reported only as 'variants of uncertain significance' until more patients with a similar phenotype are discovered with the same variant. WIDER IMPLICATIONS OF THE FINDINGS: We report the first WES-discovered FSHR mutation, validated by quantitative flow cytometry. WES is a valuable tool for diagnosis of rare genetic diseases, and flow cytometry allows for quantitative characterization of purported variants. WES-assisted diagnosis allows for treatments aimed at the underlying molecular etiology of disease. Future studies should focus on pharmacological and assisted reproductive treatments aimed at the disrupted FSHR, so that patients with FSH resistance can be treated by personalized medicine. STUDY FUNDING/COMPETING INTERESTS: E.V. is partially funded by the DSD Translational Research Network (NICHD 1R01HD068138). M.S.B. is funded by the Neuroendocrinology, Sex Differences and Reproduction training grant (NICHD 5T32HD007228). The authors have no competing interests to disclose.

Association of a promoter polymorphism in FSHR with ovarian reserve and response to ovarian stimulation in women undergoing assisted reproductive treatment.

Previous studies have suggested an association between a variant in the promoter region of the FSHR gene and diminished response to controlled ovarian hyperstimulation (COH) in women undergoing assisted reproduction. FSHR -29G>A was genotyped in 559 women undergoing their first cycle of COH for IVF/intracytoplasmic sperm injection (ICSI) using TaqMan allelic discrimination assay. Correlation and regression analysis was performed to assess the relationship between FSHR promoter genotypes and markers of ovarian reserve and measures of response to COH, including the number of oocytes retrieved, gonadotrophin dose used and the live-birth rate. There were no statistically significant differences between the genotype frequencies and the markers of ovarian reserve or the early measures of response to COH. However, the live-birth rate was higher for women carrying the variant A allele (odds ratio [OR] 1.37; 95% confidence interval [CI] 1.02-1.84 per allele). This relationship did not reach statistical significance after adjustment for the number of embryos transferred (OR 1.33; 95% CI 0.98-1.83 per allele). Results from this study do not provide evidence that the FSHR -29G>A variant can be used in the individualization of the treatment protocol for women undergoing IVF/ICSI.

Evidence for Follicle-stimulating Hormone Receptor as a Functional Trimer.

Follicle-stimulating hormone receptor (FSHR), a G-protein coupled receptor, is an important drug target in the development of novel therapeutics for reproductive indications. The FSHR extracellular domains were observed in the crystal structure as a trimer, which enabled us to propose a novel model for the receptor activation mechanism. The model predicts that FSHR binds Asnα(52)-deglycosylated FSH at a 3-fold higher capacity than fully glycosylated FSH. It also predicts that, upon dissociation of the FSHR trimer into monomers, the binding of glycosylated FSH, but not deglycosylated FSH, would increase 3-fold, and that the dissociated monomers would in turn enhance FSHR binding and signaling activities by 3-fold. This study presents evidence confirming these predictions and provides crystallographic and mutagenesis data supporting the proposed model. The model also provides a mechanistic explanation to the agonist and antagonist activities of thyroid-stimulating hormone receptor autoantibodies. We conclude that FSHR exists as a functional trimer.

Single-molecule analyses of fully functional fluorescent protein-tagged follitropin receptor reveal homodimerization and specific heterodimerization with lutropin receptor.

We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.

Structure of follicle-stimulating hormone in complex with the entire ectodomain of its receptor.

FSH, a glycoprotein hormone, and the FSH receptor (FSHR), a G protein-coupled receptor, play central roles in human reproduction. We report the crystal structure of FSH in complex with the entire extracellular domain of FSHR (FSHR(ED)), including the enigmatic hinge region that is responsible for signal specificity. Surprisingly, the hinge region does not form a separate structural unit as widely anticipated but is part of the integral structure of FSHR(ED). In addition to the known hormone-binding site, FSHR(ED) provides interaction sites with the hormone: a sulfotyrosine (sTyr) site in the hinge region consistent with previous studies and a potential exosite resulting from putative receptor trimerization. Our structure, in comparison to others, suggests FSHR interacts with its ligand in two steps: ligand recruitment followed by sTyr recognition. FSH first binds to the high-affinity hormone-binding subdomain of FSHR and reshapes the ligand conformation to form a sTyr-binding pocket. FSHR then inserts its sTyr (i.e., sulfated Tyr335) into the FSH nascent pocket, eventually leading to receptor activation.

LH (Trp8Arg/Ile15Thr), LHR (insLQ) and FSHR (Asn680Ser) polymorphisms genotypic prevalence in women with endometriosis and infertility.

PURPOSE: To verify if polymorphisms of LH (Trp8Arg/Ile15Thr), LH receptor (insLQ), and FSH receptor (Asn680Ser) are associated with endometriosis and infertility. METHODS: This is a prospective case-control study. Sixty-seven patients with endometriosis and infertility (study group) and 65 healthy fertile patients (control group) were enrolled in the study between July 2010 and July 2013. All patients had their endometriosis diagnosis made or excluded by laparoscopic surgery; study group was submitted to the surgery for infertility investigation and control group for tubal ligation. Day-3 serum hormones were collected from all patients. Analysis of nucleotide mutations for LH polymorphisms (Trp8Arg and Ile15Thr), LHR polymorphism (insLQ), and FSHR polymorphism (Asn680Ser) were performed by PCR. RESULTS: Day-3 FSH, estradiol and LH serum levels were not different between the groups, while CA-125 was higher in patients with endometriosis and infertility. All polymorphisms studied were in Hardy-Weinberg equilibrium. The prevalence of insLQ was significantly higher in patients with endometriosis and infertility (P = 0.005). Allele occurrence in control group was 0.10 versus 0.25 in infertile endometriosis group (P = 0.001). There was no difference regarding Trp8Arg/Ile15Thr (P > 0.05) and Asn680Ser (P > 0.05) prevalence between groups. CONCLUSION: This is the first time that prevalence of insLQ was shown to be higher in patients with endometriosis and infertility than in healthy fertile patients. There was no difference in LH and FSHR polymorphisms' prevalence between groups.

A single-domain intrabody targeting the follicle-stimulating hormone receptor impacts FSH-induced G protein-dependent signalling.

Intracellular variable fragments of heavy-chain antibody from camelids (intra-VHH) have been successfully used as chaperones to solve the 3D structure of active G protein-coupled receptors bound to their transducers. However, their effect on signalling has been poorly explored, although they may provide a better understanding of the relationships between receptor conformation and activity. Here, we isolated and characterized iPRC1, the first intra-VHH recognizing a member of the large glycoprotein hormone receptor family, the follicle-stimulating hormone receptor (FSHR). This intra-VHH recognizes the FSHR third intracellular loop and decreases cAMP production in response to FSH, without altering Gαs recruitment. Hence, iPRC1 behaves as an allosteric modulator and provides a new tool to complete structure/activity studies performed thus far on this receptor.

Mutations and polymorphisms in FSH receptor: functional implications in human reproduction.

FSH brings about its physiological actions by activating a specific receptor located on target cells. Normal functioning of the FSH receptor (FSHR) is crucial for follicular development and estradiol production in females and for the regulation of Sertoli cell function and spermatogenesis in males. In the last two decades, the number of inactivating and activating mutations, single nucleotide polymorphisms, and spliced variants of FSHR gene has been identified in selected infertile cases. Information on genotype-phenotype correlation and in vitro functional characterization of the mutants has helped in understanding the possible genetic cause for female infertility in affected individuals. The information is also being used to dissect various extracellular and intracellular events involved in hormone-receptor interaction by studying the differences in the properties of the mutant receptor when compared with WT receptor. Studies on polymorphisms in the FSHR gene have shown variability in clinical outcome among women treated with FSH. These observations are being explored to develop molecular markers to predict the optimum dose of FSH required for controlled ovarian hyperstimulation. Pharmacogenetics is an emerging field in this area that aims at designing individual treatment protocols for reproductive abnormalities based on FSHR gene polymorphisms. The present review discusses the current knowledge of various genetic alterations in FSHR and their impact on receptor function in the female reproductive system.

Identification of two novel, alternatively spliced mRNA transcripts of the human follicle-stimulating hormone receptor.

Glycoprotein hormone receptors contain large extracellular domains encoded by multiple exons that can be alternatively spliced. Using human ovarian surface epithelium, we cloned two new splice variants of the human follicle-stimulating hormone receptor (FSH-R) gene, hFSH-R2 and hFSH-R3. The hFSH-R2 splice variant differed from the full-length FSH-R mRNA by the deletion of exon 10 and inclusion of two small exons after exon 9 whereas the hFSH-R3 splice variant retained only exons 1-6 of the full-length transcript. Both variants were expressed at low levels, but were detected in cells from follicular fluid derived from 30 different subjects. Transfection of these two variants individually into KGN cells, an ovarian cancer cell line that expresses wild-type FSH-R, reduced FSH-mediated phosphorylation of ERK(1/2), Akt, and p38/MAPK. Furthermore, in vitro co-expression of either hFSH-R2 or hFSH-R3 and full-length FSH-R in HEK293T cells reduced signal transduction through full-length FSH-R. Further studies are needed to fully elucidate the functions of these receptor isoforms.

Follicular expression of follicle stimulating hormone receptor variants in the ewe.

BACKGROUND: Several alternatively-spliced mRNA transcripts of the follicle stimulating hormone receptor (FSHR) have been identified in sheep, including FSHR-1 (G protein-coupled form), FSHR-2 (dominant negative form), and FSHR-3 (growth factor type-1 form). Our objective was to determine which of these variants is predominantly expressed in follicles collected from ewes at various times after estrus. METHODS: Suffolk-cross ewes (n = 8) were allowed to come into estrus naturally and were euthanized 24 (n = 3), 36 (n = 3), or 48 (n = 2) hours after the onset of estrus. All visible follicles were measured, aspirated and pooled according to follicular diameter: small (<= 2.0 mm), medium (2.1-4.0 mm), large (4.1-6.0 mm), and preovulatory (> = 6.1 mm). Aspirated cells were separated from follicular fluid by centrifugation. Total RNA was extracted from cell pellets and reverse transcribed. The resulting cDNA was subjected to qPCR, using primer sets designed to amplify each variant specifically. Gene expression was normalized to that of beta-actin within samples, and compared by analysis of variance with the level of significant differences set at p < .05. RESULTS: Relative expression of FSHR-3 exceeded that of both FSHR-1 and FSHR-2 in medium follicles, and tended to be higher in small follicles (p = .09) regardless of time after onset of estrus, and thus results from different time points were pooled. Expression of FSHR-3 was greater than that of FSHR-2 and luteinizing hormone receptor (LHR) in small and medium follicles. Expression of LHR was greatest in preovulatory follicles. CONCLUSIONS: These experiments show that in addition to the well characterized G protein-coupled form of the FSHR, alternatively spliced variants of the FSHR may participate in follicular dynamics during follicular waves of the sheep estrous cycle. Furthermore, these results indicate that an "alternatively" spliced form of the FSHR (FSHR-3) is the predominant form of the FSHR in the sheep.

FSHR activation through small molecule modulators: Mechanistic insights from MD simulations.

Follicle-stimulating hormone receptor (FSHR) is a glycoprotein hormone receptor that plays a vital role in reproduction, cancer progression and osteoporosis. Owing to its therapeutic importance, several small molecule modulators have been identified by researchers through high throughput studies that usually include virtual screening of chemical libraries followed by in vitro validation through radio-ligand binding assays, cAMP accumulation and luciferase-based luminescence assays. The binding site of these modulators and structural changes that accompany modulator binding remains elusive. Here, we address these aspects through molecular docking and MD simulations on well-studied FSHR modulators and comparing the domain motions between agonist/FSH bound and antagonist bound FSHR structures. It was observed that agonist and antagonist modulators bind to the same site, but interact with distinct residues in transmembrane domain(TMD). FSHR(TMD) residues Ile522, Ala595, Ile602 and Val604 were found to interact only with agonist. Notably, these residues are conserved in the close homolog luteinizing hormone/choriogonadotropin receptor (LHCGR) and participate in interaction with its agonist Org43553. We observed distinctly prominent domain motions and conformational changes in TM helices 3, 4 and 6 for agonist bound FSHR structure. These structural changes have also been reported for LHCGR, and few GPCR members suggesting an important and well conserved mechanism of GPHR activation that could be exploited for design of novel modulators.

[The molecular design and drug development of recombinant long-acting follicle stimulating hormone].

Follicle-stimulating hormone (FSH) is a glycoprotein which regulates the development, growth, pubertal maturation and reproductive processes of the body. Exogenous FSH has been used to promote ovarian follicular growth and maturation in female and spermatogenesis in male. The relative short elimination half life and rapid metabolic clearance of current versions of FSH require a daily or twice-daily scheduled subcutaneous injection to maintain stable FSH level being not below the threshold during ovarian stimulation. The development of recombinant long-acting FSH with enhanced biological activities may be helpful for less injection therefore to improve patient compliance, while reducing patient stress and error rates. A number of technological strategies have been explored to develop recombinant longer-acting FSH. For examples, attachment of the C-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit or a sequence containing potential glycosylation sites to either subunit of FSH, creation of a single chain containing the alpha and beta subunits of FSH combined with CTP or N-linked glycosylation signal sequence as a linker, or fusion of the Fc domain of IgGi to FSH. Based on the modifiable molecular structure and pharmacokinetic and pharmacodynamic properties of recombinant FSH, it is hopeful that more FSH drugs with prolonged half-life and increased bioactivity will be developed to meet the modern clinical demands.

Overlapping synthetic peptides as a tool to map protein-protein interactions ̶ FSH as a model system of nonadditive interactions.

In earlier work, we used partially overlapped synthetic peptides as a tool to find regions of interaction between the human FSH hormone and its receptor, aiming to find possible antagonists or agonists. Years later, the FSH and FSH receptor 3D structures were reported by other laboratories. The 3D results were in close agreement with the interacting regions predicted by using synthetic peptides. These earlier studies are reviewed here, and the predicted regions of interaction compared to the FSH and FSH receptor 3D structures to illustrate the usefulness of the synthetic peptide strategy to find binding regions. Different contact regions contribute multiplicatively to the high affinity of the entire ligand; thus, peptides covering a fraction of the anchor sites and with low free energy density cannot reach the affinity of the entire molecule. The earlier use of multiple linear regression to find the relevant predictors for effective binding, and a new way to estimate ΔG° and nonadditive interactions for the synthetic peptides in solution, by using the buried surface area (BSA), will be discussed.

Human FSH Glycoform α-Subunit Asparagine52 Glycans: Major Glycan Structural Consistency, Minor Glycan Variation in Abundance.

Follicle-stimulating hormone (FSH), an α/ß heterodimeric glycoprotein hormone, consists of functionally significant variants resulting from the presence or absence of either one of two FSHß subunit N-glycans. The two most abundant variants are fully-glycosylated FSH24 (based on 24 kDa FSHß band in Western blots) and hypo-glycosylated FSH21 (21 kDa band, lacks ßAsn24 glycans). Due to its ability to bind more rapidly to the FSH receptor and occupy more FSH binding sites than FSH24, hypo-glycosylated FSH21 exhibits greater biological activity. Endoglycosidase F1-deglycosylated FSH bound to the complete extracellular domain of the FSH receptor crystallized as a trimeric complex. It was noted that a single biantennary glycan attached to FSHα Asn52 might preemptively fill the central pocket in this complex and prevent the other two FSH ligands from binding the remaining ligand-binding sites. As the most active FSH21 preparations possessed more rapidly migrating α-subunit bands in Western blots, we hypothesized that Asn52 glycans in these preparations were small enough to enable greater FSH21 receptor occupancy in the putative FSHR trimer model. Highly purified hFSH oligosaccharides derived from each FSH subunit, were characterized by electrospray ionization-ion mobility-collision-induced dissociation (ESI-IM-CID) mass spectrometry. FSHß glycans typically possessed core-linked fucose and were roughly one third bi-antennary, one third tri-antennary and one third tetra-antennary. FSHα oligosaccharides largely lacked core fucose and were bi- or tri-antennary. Those αAsn52 glycans exhibiting tetra-antennary glycan m/z values were found to be tri-antennary, with lactosamine repeats accounting for the additional mass. Selective αAsn52 deglycosylation of representative pituitary hFSH glycoform Superdex 75 gel filtration fractions followed by ESI-IM-CID mass spectrometry revealed tri-antennary glycans predominated even in the lowest molecular weight FSH glycoforms. Accordingly, the differences in binding capacity of the same receptor preparation to different FSH glycoforms are likely the organization of the FSH receptor in cell membranes, rather than the αAsn52 oligosaccharide.