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

Molecular dynamics simulation of the follicle-stimulating hormone receptor. Understanding the conformational dynamics of receptor variants at positions N680 and D408 from in silico analysis.

Follicle-stimulating hormone receptor (FSHR) is a G-protein coupled receptor (GPCR) and a prototype of the glycoprotein hormone receptors subfamily of GPCRs. Structural data of the FSHR ectodomain in complex with follicle-stimulating hormone suggests a "pull and lift" activation mechanism that triggers a conformational change on the seven α-helix transmembrane domain (TMD). To analyze the conformational changes of the FSHR TMD resulting from sequence variants associated with reproductive impairment in humans, we set up a computational approach combining helix modeling and molecular simulation methods to generate conformational ensembles of the receptor at room (300 K) and physiological (310 K) temperatures. We examined the receptor dynamics in an explicit membrane environment of polyunsaturated phospholipids and solvent water molecules. The analysis of the conformational dynamics of the functional (N680 and S680) and dysfunctional (mutations at D408) variants of the FSHR allowed us to validate the FSHR-TMD model. Functional variants display a concerted motion of flexible intracellular regions at TMD helices 5 and 6. Disruption of side chain interactions and conformational dynamics were detected upon mutation at D408 when replaced with alanine, arginine, or tyrosine. Dynamical network analysis confirmed that TMD helices 2 and 5 may share communication pathways in the functional FSHR variants, whereas no connectivity was detected in the dysfunctional mutants, indicating that the global dynamics of the FSHR was sensitive to mutations at amino acid residue 408, a key position apparently linked to misfolding and variable cell surface plasma membrane expression of FSHRs with distinct mutations at this position

Normal testicular function without detectable follicle-stimulating hormone. A novel mutation in the follicle-stimulating hormone receptor gene leading to apparent constitutive activity and impaired agonist-induced desensitization and internalization

Activating mutations in the follicle-stimulating hormone (FSH) receptor (FSHR) gene are rarely detected due to the absence of a clearly defined phenotype, particularly in men. We here report the biochemical features of a novel mutation in the first extracellular loop of the FSHR. The mutation (N431I) was detected in an asymptomatic man exhibiting normal spermatogenesis, suppressed serum FSH, and normal or elevated levels of biochemical markers of FSH action. Employing different experimental strategies on HEK-293 cells transiently expressing the N431I FSHR mutant, we found that the mutation led to decreased cell surface plasma membrane expression of the receptor protein, but conferred a low level of constitutive activity associated with markedly altered agonist-stimulated desensitization and internalization. These latter features may contribute and/or amplify the persistent activation of the receptor in both absence and presence of agonist and provide new insights into opportunities for adjuvant therapies based on disruption of these processes

Frequency of the T307A, N680S, and -29G>A single-nucleotide polymorphisms in the follicle-stimulating hormone receptor in Mexican subjects of Hispanic ancestry

Abstract Background FSHR SNPs may influence the ovarian sensitivity to endogenous and exogenous FSH stimulation. Given the paucity of data on the FSHR c.919A > G, c.2039A > G and − 29G > A SNPs in Hispanic population, we here analyzed their frequency distribution in Mexican mestizo women. Methods Samples from 224 Mexican mestizo women enrolled in an IVF program as well as a genotype database from 8182 Mexican mestizo subjects, were analyzed for FSHR SNPs at positions c.919, c.2039 and − 29G > A. Association between the genetic variants and reproductive outcomes was assessed. Results The c.919 and c.2039 SNPs were in strong linkage disequilibrium and their corresponding genotype frequencies in the IVF group were: AA 46.8%, AG 44.2%, and GG 8.9%, and AA 41.9%, AG 48.2% and GG 9.8%, respectively. For the -29G > A SNP, genotype frequencies were 27% (GG), 50% (GA) and 23% (AA). In normal oocyte donors with the c.2039 GG genotype, the number of oocytes recovered after ovarian stimulation (COS) were significantly (p  A SNP. Analysis of the large scale database revealed that both allelic and genotype frequencies for the three SNPs were very similar to those detected in the IVF cohort (p ≥ 0.38) and that female carriers of the c.2039 G allele tended to present lower number of pregnancies than women bearing the AA genotype; this trend was stronger when women with more Native American ancestry was separately analyzed (OR = 2.0, C.I. 95% 1.03–3.90, p = 0.04). There were no differences or trends in the number of pregnancies among the different genotypes of the -29G > A SNP. Conclusions The frequency of the GG/GG combination genotype for the c.919 and c.2039 SNPs in Mexican hispanics is among the lowest reported. The GG genotype is associated with decreased number of oocytes recovered in response to COS as well as to lower pregnancy rates in Hispanic women from the general population. The absence of any effect of the -29AA genotype on the response to COS, indicates that there is no need to perform this particular genotype testing in Hispanic women with the purpose of providing an individually-tailored COS protocol

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 β-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

A novel mutation in the FSH receptor (I423T) affecting receptor activation and leading to primary ovarian failure

International audienceContext :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 β-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

Multiple Molecular Mechanisms Are Involved in the Activation of the Kidney Sodium-Chloride Cotransporter by Hypokalemia

Low potassium intake activates the kidney sodium-chloride cotransporter (NCC) whose phosphorylation and activity depend on the With-No-Lysine kinase 4 (WNK4) that is inhibited by chloride binding to its kinase domain. Low extracellular potassium activates NCC by decreasing intracellular chloride thereby promoting chloride dissociation from WNK4 where residue L319 of WNK4 participates in chloride coordination. Since the WNK4-L319F mutant is constitutively active and chloride-insensitive in vitro, we generated mice harboring this mutation that displayed slightly increased phosphorylated NCC and mild hyperkalemia when on a 129/sv genetic background. On a low potassium diet, upregulation of phosphorylated NCC was observed, suggesting that in addition to chloride sensing by WNK4, other mechanisms participate which may include modulation of WNK4 activity and degradation by phosphorylation of the RRxS motif in regulatory domains present in WNK4 and KLHL3, respectively. Increased levels of WNK4 and kidney-specific WNK1 and phospho-WNK4-RRxS were observed in wild-type and WNK4 mice on a low potassium diet. Decreased extracellular potassium promoted WNK4-RRxS phosphorylation in vitro and ex vivo as well. These effects might be secondary to intracellular chloride depletion, as reduction of intracellular chloride in HEK293 cells increased phospho-WNK4-RRxS. Phospho-WNK4-RRxS levels were increased in mice lacking the Kir5.1 potassium channel, which presumably have decreased distal convoluted tubule intracellular chloride. Similarly, phospho-KLHL3 was modulated by changes in intracellular chloride in HEK293 cells. Thus, our data suggest that multiple chloride-regulated mechanisms are responsible for NCC upregulation by low extracellular potassium