A Novel Mutation in the FSH Receptor (I423T) Affecting Receptor Activation and Leading to Primary Ovarian Failure.

CONTEXT: Follicle-stimulating hormone (FSH) plays an essential role in gonadal function. Loss-of-function mutations in the follicle-stimulating hormone receptor (FSHR) are an infrequent cause of primary ovarian failure. OBJECTIVE: To analyze the molecular physiopathogenesis of a novel mutation in the FSHR identified in a woman with primary ovarian failure, employing in vitro and in silico approaches, and to compare the features of this dysfunctional receptor with those shown by the trafficking-defective D408Y FSHR mutant. METHODS: Sanger sequencing of the FSHR cDNA was applied to identify the novel mutation. FSH-stimulated cyclic adenosine monophosphate (cAMP) production, ERK1/2 phosphorylation, and desensitization were tested in HEK293 cells. Receptor expression was analyzed by immunoblotting, receptor-binding assays, and flow cytometry. Molecular dynamics simulations were performed to determine the in silico behavior of the mutant FSHRs. RESULTS: A novel missense mutation (I423T) in the second transmembrane domain of the FSHR was identified in a woman with normal pubertal development but primary amenorrhea. The I423T mutation slightly impaired plasma membrane expression of the mature form of the receptor and severely impacted on cAMP/protein kinase A signaling but much less on ß-arrestin-dependent ERK1/2 phosphorylation. Meanwhile, the D408Y mutation severely affected membrane expression, with most of the FSH receptor located intracellularly, and both signal readouts tested. Molecular dynamics simulations revealed important functional disruptions in both mutant FSHRs, mainly the loss of interhelical connectivity in the D408Y FSHR. CONCLUSIONS: Concurrently, these data indicate that conformational differences during the inactive and active states account for the distinct expression levels, differential signaling, and phenotypic expression of the I423T and D408Y mutant FSHRs.

In Vitro Impact of FSH Glycosylation Variants on FSH Receptor-stimulated Signal Transduction and Functional Selectivity.

FSH exists as different glycoforms that differ in glycosylation of the hormone-specific ß-subunit. Tetra-glycosylated FSH (FSH24) and hypo-glycosylated FSH (FSH18/21) are the most abundant glycoforms found in humans. Employing distinct readouts in HEK293 cells expressing the FSH receptor, we compared signaling triggered by human pituitary FSH preparations (FSH18/21 and FSH24) as well as by equine FSH (eFSH), and human recombinant FSH (recFSH), each exhibiting distinct glycosylation patterns. The potency in eliciting cAMP production was greater for eFSH than for FSH18/21, FSH24, and recFSH, whereas in the ERK1/2 activation readout, potency was highest for FSH18/21 followed by eFSH, recFSH, and FSH24. In ß-arrestin1/2 CRISPR/Cas9 HEK293-KO cells, FSH18/21 exhibited a preference toward ß-arrestin-mediated ERK1/2 activation as revealed by a drastic decrease in pERK during the first 15-minute exposure to this glycoform. Exposure of ß-arrestin1/2 KO cells to H89 additionally decreased pERK1/2, albeit to a significantly lower extent in response to FSH18/21. Concurrent silencing of ß-arrestin and PKA signaling, incompletely suppressed pERK response to FSH glycoforms, suggesting that pathways other than those dependent on Gs-protein and ß-arrestins also contribute to FSH-stimulated pERK1/2. All FSH glycoforms stimulated intracellular Ca2+ (iCa2+) accumulation through both influx from Ca2+ channels and release from intracellular stores; however, iCa2+ in response to FSH18/21 depended more on the latter, suggesting differences in mechanisms through which glycoforms promote iCa2+ accumulation. These data indicate that FSH glycosylation plays an important role in defining not only the intensity but also the functional selectivity for the mechanisms leading to activation of distinct signaling cascades.

Structure-Function Relationships of the Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) plays a crucial role in reproduction. This structurally complex receptor is a member of the G-protein coupled receptor (GPCR) superfamily of membrane receptors. As with the other structurally similar glycoprotein hormone receptors (the thyroid-stimulating hormone and luteinizing hormone-chorionic gonadotropin hormone receptors), the FSHR is characterized by an extensive extracellular domain, where binding to FSH occurs, linked to the signal specificity subdomain or hinge region. This region is involved in ligand-stimulated receptor activation whereas the seven transmembrane domain is associated with receptor activation and transmission of the activation process to the intracellular loops comprised of amino acid sequences, which predicate coupling to effectors, interaction with adapter proteins, and triggering of downstream intracellular signaling. In this review, we describe the most important structural features of the FSHR intimately involved in regulation of FSHR function, including trafficking, dimerization, and oligomerization, ligand binding, agonist-stimulated activation, and signal transduction.

The follicle-stimulating hormone receptor Asn680Ser polymorphism is associated with preterm birth in Hispanic women.

OBJECTIVE: Recently, a study based on the analysis of accelerated evolution of related genes at birth identified the follicle-stimulating hormone receptor (FSHR) as a possible candidate for the development of preterm delivery. Additionally, FSHR expression has been described in extragonadal tissue including the placenta. Therefore, the aim of the present study was to determine the association between the N680S polymorphism of the follicle-stimulating hormone receptor and preterm birth in a population of Hispanic women. METHODS: Placenta samples were obtained from 64 women who had preterm births and 54 control cases. DNA was extracted and genotyped for the N680S FSHR gene polymorphism by polymerase chain reaction-restriction fragment length polymorphism. The χ2 test and t-test were used to calculate statistical significance. RESULTS: Statistically significant differences in genotype frequencies for the N680S polymorphism were observed between preterm and term groups (p = .04). Based on the Akaike information criterion values, the dominant model showed that the NN genotype had a significantly increased risk of preterm birth compared with the SS + NS genotype (OR 2.52, 95% CI 1.20-5.33, p = .02). CONCLUSIONS: The results herein suggest that the FSHR polymorphism N680S is significantly associated with preterm birth in the Hispanic population.

Role of Cysteine Residues in the Carboxyl-Terminus of the Follicle-Stimulating Hormone Receptor in Intracellular Traffic and Postendocytic Processing.

Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the absence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and ß-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these cysteine residues are S-palmitoylated, the data presented emphasize on this posttranslational modification as an important factor for both upward and downward trafficking of this receptor.

Effects of polymorphisms of LHR and FSHR genes on sexual precocity in a Bos taurus x Bos indicus beef composite population

The purpose of the present research was to investigate the effects of polymorphisms of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) genes, evaluated by polymerase chain reaction-restriction fragment length polymorphism in European-Zebu composite beef heifers from six different breed compositions. The polymorphism site analysis from digestion with HhaI and AluI restriction endonucleases allowed the genotype identification for LHR (TT, CT and CC) and FSHR (GG, CG and CC) genes. A high frequency of heterozygous animals was recorded in all breed compositions for both genes, except in two compositions for LHR. The probability of pregnancy (PP) at first breeding was used to evaluate the polymorphism effect on sexual precocity. The PP was analyzed as a binary trait, with a value of 1 (success) assigned to heifers that were diagnosed pregnant by rectal palpation and a value of 0 (failure) assigned to those that were not pregnant at that time. Heterozygous heifers showed a higher pregnancy rate (67 and 66% for LHR and FSHR genes, respectively), but no significant effects were observed for the genes studied (P = 0.9188 and 0.8831 for LHR and FSHR, respectively) on the PP. These results do not justify the inclusion of LHR and FSHR restriction fragment length polymorphism markers in selection programs for sexual precocity in beef heifers. Nevertheless, these markers make possible the genotype characterization and may be used in additional studies to evaluate the genetic structure in other bovine populations.