Follicle-stimulating Hormone Receptor Function Research Articles

Abstract 1791 Investigation on the impact of sphingolipid disrupting agents on follicle-stimulating hormone receptor function

Abstract 1791 Investigation on the impact of sphingolipid disrupting agents on follicle-stimulating hormone receptor function

Investigating the Role of CRAC in FSH Receptor Function and Structure

Human infertility is a complex disorder that can often be attributed to a dysfunction of the endocrine system. Follicle‐stimulating hormone (FSH) is one of many hormones that participate in a complex process in both women and men to regulate normal reproduction. The dysfunction of this hormone and its receptor are some of the many causes of infertility. FSH is secreted by the anterior pituitary and, in women, initiates a cascade of biological events that enable ovulation. FSH carries out its function by binding and activating specific receptors. The FSH receptor (FSHR) is a G protein‐coupled receptor (GPCR) that is located in the cell membrane of target cells in the ovaries and testes. GPCRs often interact with a variety of molecules beyond their ligands including, in some cases, cholesterol in the plasma membrane. The Cholesterol Recognition/Interaction Amino Acid Consensus Sequence (CRAC) is one such binding domain and is represented by the sequence LV‐XXXX‐Y‐XXXXX‐K/R. The human FSHR contains a sequence consistent with the CRAC motif in the first intracellular loop L‐TTLQ‐Y‐KLTVP‐R. We hypothesized that mutating this domain will negatively affect the signaling of the receptor upon ligand activation. To test this hypothesis tyrosine 375 was mutated to phenylalanine (Y375F) which should render the CRAC inactive. After establishing a cell line stably expressing the mutant receptor, activation of the p44/42 (ERK1/2) pathway was measured. Preliminary data suggest that signaling of the hFSHR‐Y375F mutant is decreased relative to the wild‐type receptor. As we learn more about this receptor, we continue to gain knowledge about conditions such as infertility. This information can facilitate the development of innovative treatments which can help many families who struggle to conceive.

The Roles of Luteinizing Hormone, Follicle-Stimulating Hormone and Testosterone in Spermatogenesis and Folliculogenesis Revisited.

Spermatogenesis and folliculogenesis involve cell–cell interactions and gene expression orchestrated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). FSH regulates the proliferation and maturation of germ cells independently and in combination with LH. In humans, the requirement for high intratesticular testosterone (T) concentration in spermatogenesis remains both a dogma and an enigma, as it greatly exceeds the requirement for androgen receptor (AR) activation. Several data have challenged this dogma. Here we report our findings on a man with mutant LH beta subunit (LHβ) that markedly reduced T production to 1–2% of normal., but despite this minimal LH stimulation, T production by scarce mature Leydig cells was sufficient to initiate and maintain complete spermatogenesis. Also, in the LH receptor (LHR) knockout (LuRKO) mice, low-dose T supplementation was able to maintain spermatogenesis. In addition, in antiandrogen-treated LuRKO mice, devoid of T action, the transgenic expression of a constitutively activating follicle stimulating hormone receptor (FSHR) mutant was able to rescue spermatogenesis and fertility. Based on rodent models, it is believed that gonadotropin-dependent follicular growth begins at the antral stage, but models of FSHR inactivation in women contradict this claim. The complete loss of FSHR function results in the complete early blockage of folliculogenesis at the primary stage, with a high density of follicles of the prepubertal type. These results should prompt the reassessment of the role of gonadotropins in spermatogenesis, folliculogenesis and therapeutic applications in human hypogonadism and infertility.

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.

Production and validation of a polyclonal serum against bovine FSH receptor

In ovarian granulosa cells, follicle-stimulating hormone (FSH) regulates the proliferation and differentiation events required for follicular growth and oocyte maturation. FSH actions are mediated exclusively through the FSH receptor (FSHR). In cattle, the FSHR gene expression pattern during folliculogenesis and the implications of this receptor in reproductive disorders have been extensively studied. However, the limited availability of specific antibodies against bovine FSHR has restricted FSHR protein analysis. In the present study, we developed an anti-FSHR polyclonal serum by using a 14-kDa peptide conjugated to maltose binding protein. The antiserum obtained was characterized by western blot of protein extracts from bovine follicles, BGC-1 cells and primary cultures of granulosa cells stimulated with testosterone. Also, the blocking effect of serum on estradiol secretion and cell viability after gonadotropin stimulus was characterized in a functional in vitro assay. A 76-kDa protein, consistent with the predicted molecular size of full-length FSHR, was detected in ovarian tissue. Besides, two immunoreactive bands of 60-kDa and 30-kDa (only in cultured cells) were detected. These bands would be related to some of the isoforms of the receptor. Therefore, immunohistochemical assays allowed detecting FSHR in the cytoplasm of granulosa cells and an increase in its expression as follicles progressed from primordial to large preantral follicles. These results suggest that the anti-FSHR serum here developed has good reactivity and specificity against the native FSHR. Therefore, this antiserum may serve as a valuable tool for future studies of the biological function of FSHR in physiological conditions as well as of the molecular mechanism and functional involvement of FSHR in reproductive disorders.

G protein-dependent signaling triggers a β-arrestin-scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation.

Many interaction partners of β-arrestins intervene in the control of mRNA translation. However, how β-arrestins regulate this cellular process has been poorly explored. In this study, we show that β-arrestins constitutively assemble a p70S6K/ribosomal protein S6 (rpS6) complex in HEK293 cells and in primary Sertoli cells of the testis. We demonstrate that this interaction is direct, and experimentally validate the interaction interface between β-arrestin 1 and p70S6K predicted by our docking algorithm. Like most GPCRs, the biological function of follicle-stimulating hormone receptor (FSHR) is transduced by G proteins and β-arrestins. Upon follicle-stimulating hormone (FSH) stimulation, activation of G protein-dependent signaling enhances p70S6K activity within the β-arrestin/p70S6K/rpS6 preassembled complex, which is not recruited to the FSHR. In agreement, FSH-induced rpS6 phosphorylation within the β-arrestin scaffold was decreased in cells depleted of Gαs. Integration of the cooperative action of β-arrestin and G proteins led to the translation of 5' oligopyrimidine track mRNA with high efficacy within minutes of FSH input. Hence, this work highlights new relationships between G proteins and β-arrestins when acting cooperatively on a common signaling pathway, contrasting with their previously shown parallel action on the ERK MAP kinase pathway. In addition, this study provides insights into how GPCR can exert trophic effects in the cell.-Tréfier, A., Musnier, A., Landomiel, F., Bourquard, T., Boulo, T., Ayoub, M. A., León, K., Bruneau, G., Chevalier, M., Durand, G., Blache, M.-C., Inoue, A., Fontaine, J., Gauthier, C., Tesseraud, S., Reiter, E., Poupon, A., Crépieux, P. G protein-dependent signaling triggers a β-arrestin-scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation.

Role of the Extracellular and Intracellular Loops of Follicle-Stimulating Hormone Receptor in Its Function.

Follicle-stimulating hormone receptor (FSHR) is a leucine-rich repeat containing class A G-protein coupled receptor belonging to the subfamily of glycoprotein hormone receptors (GPHRs), which includes luteinizing hormone/choriogonadotropin receptor (LH/CGR) and thyroid-stimulating hormone receptor. Its cognate ligand, follicle-stimulating hormone binds to, and activates FSHR expressed on the surface of granulosa cells of the ovary, in females, and Sertoli cells of the testis, in males, to bring about folliculogenesis and spermatogenesis, respectively. FSHR contains a large extracellular domain (ECD) consisting of leucine-rich repeats at the N-terminal end and a hinge region at the C-terminus that connects the ECD to the membrane spanning transmembrane domain (TMD). The TMD consists of seven α-helices that are connected to each other by means of three extracellular loops (ELs) and three intracellular loops (ILs) and ends in a short-cytoplasmic tail. It is well established that the ECD is the primary hormone binding domain, whereas the TMD is the signal transducing domain. However, several studies on the ELs and ILs employing site directed mutagenesis, generation of chimeric receptors and in vitro characterization of naturally occurring mutations have proven their indispensable role in FSHR function. Their role in every phase of the life cycle of the receptor like post translational modifications, cell surface trafficking, hormone binding, activation of downstream signaling, receptor phosphorylation, hormone–receptor internalization, and recycling of hormone–receptor complex have been documented. Mutations in the loops causing dysregulation of these processes lead to pathophysiological conditions. In other GPHRs as well, the loops have been convincingly shown to contribute to various aspects of receptor function. This review article attempts to summarize the extensive contributions of FSHR loops and C-terminal tail to its function.

Investigation of FSH Responsiveness in a Granulosa Cell Line

The follicle stimulating hormone receptor (FSHR) is a g protein‐coupled receptor found in granulosa cells and Sertoli cells in females and males, respectively. FSH binds to its receptor on the surface of granulosa cells leading to proliferation and maturation of follicles towards ovulation. Understanding this pathway is significant because it can lead to developments in regulating fertility. Previously, our lab studied FSHR using an HEK293 cell line stably transfected with FSHR cDNA. More recently we have investigated an immortalized human granulosa cell line (hGrC1) to study FSHR signaling in a more physiologically relevant context. Due to the novelty of the hGrC1 cell line, not much is known about the cells, specifically what stage of follicular development they are most closely related to. Based on previous datafrom our lab regarding FSHR responsiveness, the question arose of whether hGrC1 cells undergo developmental changes in culture. It was hypothesized that this developmental change is analogous to follicular maturation in vivo. The change is significant because FSHR is not present in granulosa cells until later stages of follicular development. hGrC1 cells were grown in culture for a variable number of days before treatment with FSH, after which the presence of phosphorylated CREB and phosphorylated p44 were determined by western blot analysis as measures of FSH dependent signaling. An FSH dependent increase in signaling from both pathways was seen after cells were treated 4 days post‐confluency suggesting that there is a developmental shift leading to more hFSHR protein the longer the cells were grown in culture. This is significant because it suggests that hGrC1 cells mimic granulosa cell differentiation in vitro and may be a viable model for testing both FSHR function as well as understanding the factors driving folliculogenesis.

Decreased Degradation of Internalized Follicle-Stimulating Hormone Caused by Mutation of Aspartic Acid 6.30550 in a Protein Kinase-CK2 Consensus Sequence in the Third Intracellular Loop of Human Follicle-Stimulating Hormone Receptor1

A naturally occurring mutation in follicle-stimulating hormone receptor (FSHR) gene has been reported: an amino acid change to glycine occurs at a conserved aspartic acid 550 (D550, D567, D6.30(567)). This residue is contained in a protein kinase-CK2 consensus site present in human FSHR (hFSHR) intracellular loop 3 (iL3). Because CK2 has been reported to play a role in trafficking of some receptors, the potential roles for CK2 and D550 in FSHR function were evaluated by generating a D550A mutation in the hFSHR. The hFSHR-D550A binds hormone similarly to WT-hFSHR when expressed in HEK293T cells. Western blot analyses showed lower levels of mature hFSHR-D550A. Maximal cAMP production of both hFSHR-D550A as well as the naturally occurring mutation hFSHR-D550G was diminished, but constitutive activity was not observed. Unexpectedly, when (125)I-hFSH bound to hFSHR-D550A or hFSHR-D550G, intracellular accumulation of radiolabeled FSH was observed. Both sucrose and dominant-negative dynamin blocked internalization of radiolabeled FSH and its commensurate intracellular accumulation. Accumulation of radiolabeled FSH in cells transfected with hFSHR-D550A is due to a defect in degradation of hFSH as measured in pulse chase studies, and confocal microscopy imaging revealed that FSH accumulated in large intracellular structures. CK2 kinase activity is not required for proper degradation of internalized FSH because inhibition of CK2 kinase activity in cells expressing hFSHR did not uncouple degradation of internalized radiolabeled FSH. Additionally, the CK2 consensus site in FSHR iL3 is not required for binding because CK2alpha coimmunoprecipitated with hFSHR-D550A. Thus, mutation of D550 uncouples the link between internalization and degradation of hFSH.

Mutations of FSH Receptor Intracellular Loop 2 Which Abrogate Interaction with 14-3-3τ Reveal a Novel Signaling Defect When Placed in Context of Full Length Receptor.

Follicle stimulating hormone receptor (FSHR) is a member of the superfamily of G-protein-coupled receptors. The intracellular domains of these receptors are believed to interact with a number of cytoplasmic proteins, which could regulate receptor trafficking and signaling in a spatial and temporal manner. We previously reported the interaction of FSHR intracellular loop 2 (iL2) with 14-3-3τ. 14-3-3 proteins have been associated with many diverse functions including critical roles in signal transduction pathways, exocytosis and cell cycle regulation. In order to determine the significance of 14-3-3τ in FSHR function, the present aim was to identify the specific residues of FSHR-iL2 that are required for interaction with 14-3-3τ. Triple alanine scanning mutagenesis encompassing the entire iL2 region of FSHR (residues 441-471) was carried out. The interaction of each triple mutant with 14-3-3τ was studied and compared with the wild type FSHR-iL2 by using a yeast genetic screen. Interaction was completely abolished when the mutants 447TLE449/AAA and 450RWH452/AAA were screened. This site includes a canonical G-protein binding motif. Site directed mutagenesis was used to introduce single alanine substitutions into FSHR-iL2 447-452 in order to delineate the role of each of these six amino acids in full-length FSHR. These single mutants were expressed in HEK-293 cells. The mutants E449A, R450A and W451A expressed little or no mature receptor and therefore cells transfected with these mutants did not bind FSH. One of the important observations was that the mutant H452A trafficked to the cell surface normally and bound FSH, but was unable to transduce the FSH binding event into activation of adenylate cyclase. Further studies revealed that this mutant receptor did not enter the cell following hormone binding. These novel findings suggest that there is a link between receptor internalization and signaling upstream of the protein kinase A pathway. Taken together, these data suggest that 14-3-3τ may facilitate such processes.

Role of the intracellular domains of the human FSH receptor in GαS protein coupling and receptor expression

The human (h) follicle-stimulating hormone receptor (FSHR) belongs to the superfamily of G protein-coupled receptors (GPCRs). This receptor consists of 695 amino acid residues and is preferentially coupled to the Gs protein. This receptor is highly conserved among species (overall homology, 85%), with a 25–69% homology drop when compared to the human LH and TSH receptors. Although studies in prototypical rhodopsin/β-adrenergic receptors suggest that multiple domains in the intracellular loops (iL) and the carboxyl-terminus (Ctail) of these receptors contribute to G protein coupling and receptor expression, there is a paucity of structure/function data on the role of these domains in FSHR function. Employing point mutations we have found that several residues present in the iL2 of the hFSHR are important for both coupling the receptor to the Gs protein and maintaining the receptor molecule in an inactive conformation. In fact, HEK-293 cells expressing several hFSHR mutants with substitutions at R450 (central to the highly conserved ERW triplet motif) and T453 (a potential target for phosphorylation) failed to mediate ligand-provoked Gs protein activation but not agonist binding, whereas substitutions at the hydrophobic L460 (a conserved residue present in all glycoprotein hormone receptors) conferred elevated basal cAMP to the transfected cells. Thus, this particular loop apparently acts as a conformational switch for allowing the receptor to adopt an active conformation upon agonist stimulation. Residues in both ends of the iL3 are important for signal transduction in a number of GPCRs, including the FSHR. We have recently explored the importance of the reversed BBXXB motif (BXXBB; where B represents a basic residue and X a non-basic residue) present in the juxtamembrane region of the hFSHR iL3. A hFSHR mutant with all basic amino acids present in the iL3 BXXBB motif replaced by alanine failed to bind agonist and activate effector, and was expressed as an immature ≤62kDa form of the receptor. Individual substitutions of basic residues resulted in mutants that bound agonist normally but failed to activate effector when replaced at R552 or R556. Triple mutations in the same motif located in the NH2-end of the Ctail resulted in a complete inability of the receptor to bind agonist and activate effector, whereas individual substitutions resulted in decreased or virtually abolished agonist binding and cAMP accumulation, with both functions correlating with the detected levels of mature (80kDa) forms of the receptor. Thus, the BXXBB motif at the iL3 of the FSHR is essential for coupling the activated receptor to the Gs protein, whereas the same motif in the Ctail is apparently more important for membrane expression. The role of cysteine residues present in the Ctail of the FSHR is an enigma since there are no conserved cysteines amongst LHR, FSHR and TSHR. C629 and C655 are conserved in the gonadotropin receptors but not in the TSHR. Alanine replacement of C627 had no effect on hFSHR expression and function, whereas the same mutation at C629 altered membrane expression and signal transduction. Serine or threonine substitutions of C655 did not modify any of the parameters analyzed. In the hFSHR, C629 may be a target for palmitoylation, and apparently it is the only cysteine residue in the Ctail domain that might play an important role in receptor function.

A common SNP in exon 10 of the follicle stimulating hormone receptor is a major determinant of length and hormonal dynamics of womens menstrual cycles

Data from ovarian hyperstimulation cycles suggest that follicle stimulating hormone (FSH) action is modulated by a frequent polymorphism of the FSH receptor gene exchanging Asn to Ser at codon 680. Women homozygous for the Ser680 variant, display an attenuated FSH receptor function. We hypothesized, that the FSH receptor genotype influences also the normal, ovulatory menstrual cycle, with different ovarian and hormonal dynamics.

The effect of follicle-stimulating hormone (FSH) on the expression of FSH receptor in cultured rat granulosa cells

The acquisition of FSH receptors during folliculogenesis is believed to be a key event in the subsequent development of the follicle. The regulation by FSH of FSH receptor expression and function were further studied using cultured granulosa cells of diethylstilbestrol (DES)-primed immature rats. Incubation of rat granulosa cells with FSH led to a reduction in FSH receptor levels for a short time (6 h), followed by an increase in FSH receptor levels that reached maximum of around 150% of the initial level within 3 days after the addition of FSH. FSH stimulation caused a reduced cAMP response to subsequent FSH treatment and a time course experiment demonstrated that this response was detectable within 30 min of exposure to FSH and reached a plateau after 4 h to 24 h. The recovery of FSH responsiveness in cAMP production of granulosa cells was seen after 48 h of FSH-free interval. Treatment with forskolin (FSK) enhanced the effect of subsequent FSH on the production of intracellular cAMP. Treatment with PMA did not affect the response to subsequent FSH treatment. These data showed that the FSH is essential for the suppression of the FSH receptor function in the adenylyl cyclase pathway. Desensitization of cellular response to continuous agonist stimulation may occur because of changes in the numbers of FSH receptor, as well as changes in the functional properties of the effector system.

Structure-function relationship of follicle-stimulating hormone and its receptor.

Follicle stimulating hormone (FSH) is one of the two pituitary gonadotrophins involved in the regulation of gonadal function. Structurally, this gonadotrophin is a heterodimer composed of two non-covalently associated subunits containing several heterogenous oligosaccharide residues which play an important role in both the in-vivo and in-vitro bioactivity of the hormone. Its cognate receptor, which belongs to the superfamily of the G protein-linked cell surface receptors, also displays a high degree of functional and molecular complexity. Studies employing monoclonal antibodies, synthetic peptides and/or site directed mutagenesis, have unveiled some of the multiple structural determinants involved in FSH and FSH receptor function and interaction. Despite their structural complexity, both molecules exhibit a high degree of plasticity and diversity that allows formation of distinct ligand-receptor complexes capable of selectively activating or deactivating a variety of signalling pathways. Knowledge and mapping of the structural determinants and functional epitopes for intra- and extracellular hormone action are of paramount importance not only for a better and more detailed understanding of the molecular basis of FSH action and FSH receptor function but also for the rational design of analogues with predicted properties and effects.

Identification of the sites of N-linked glycosylation on the follicle- stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function

Identification of the sites of N-linked glycosylation on the follicle- stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function

Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function.

The FSH receptor (FSHR) contains a large extracellular domain in which exist three potential sites for N-linked glycosylation. A truncated form of the FSHR representing only the extracellular domain was created and expressed in mammalian cells. We show that this truncated receptor is glycosylated, through the carbohydrates are not as fully processed as those of the full-length receptor. This truncated receptor, which remains intracellular, binds FSH with an affinity comparable to that of the full-length FSHR. Therefore, although other regions of the FSHR may contribute to hormone binding, the extracellular domain alone can confer high affinity binding. The above results suggest that N-linked FSHR carbohydrates may, in some way, be required for FSH binding. Therefore, further experiments, done in the context of the full-length receptor, were performed to determine the actual sites of glycosylation in the FSHR as well as to elucidate their role in the functions of the FSHR. Site-directed mutagenesis was done to individually or collectively disrupt the potential sites for N-linked glycosylation. Western blot analyses of the wild type vs. mutant receptors demonstrate that, of the three potential sites for N-linked glycosylation, Asn's 174 and 276 are actually glycosylated. Binding assays demonstrate that these two N-linked FSHR carbohydrates are redundant in function since carbohydrate at either Asn174 or Asn276 allows the receptor to be expressed on the cell surface and to bind FSH with normal affinity. However, FSH binding activity is not observed with nonglycosylated mutant receptors where both sites have been collectively disrupted. Similarly, when cells expressing the wild type FSHR were treated with tunicamycin to prevent N-linked glycosylation, the resulting nonglycosylated FSHR was not able to bind FSH. In contrast, normal high affinity binding of FSH was maintained when N-linked carbohydrates were enzymatically removed from wild type receptors. Our results demonstrate that while N-linked carbohydrates on the FSH receptor are not required directly for the binding of hormone, a carbohydrate at either Asn174 or Asn276 is required for the efficient folding of the nascent receptor protein into a conformation that allows high affinity binding of hormone.