Follicle Stimulating Hormone Receptor (FSHR)
Although the importance of follicle-stimulating hormone (FSH) in the reproduction has been well recognized for many years, FSH receptor (FSHR) had not been identified until 1970. High-affinity, low-capacity binding sites for FSH were demonstrated in 1972 using rat testicular tubules incubated with radiolabeled FSH (Simoni et al. 1997). In the following years, FSHR was identified, isolated, and purified in several species and cellular localization, ligand-receptor interaction, and intracellular signaling were studied extensively. FSHR gene was cloned in 1990 by screening rat Sertoli cell cDNA library. Since then, FSHR/Fshr has been cloned in several species ranging from human to reptile (Simoni et al. 1997). These findings led to identification of FSHR mutations and polymorphisms that either enhance (activating or gain-of-function mutation) or impair (inactivating or loss-of-function mutation) the function of the receptor (Lussiana et al. 2008; Desai et al. 2013; Simoni and Casarini 2014). Advent in the molecular biology has made it possible to produce site-directed mutagenesis and gene-transfer/knockout mouse, contributing to the clarification of functional structure and physiological roles of FSH and FSHR (Vassart et al. 2004; Siegel et al. 2013). Revelations of crystal structures of FSH in 2001, followed by FSH-FSHR hormone-binding domain complex in 2005, and FSH-FSHR extracellular domain in 2012 have farther advanced our understanding in the FSHR structure and mechanisms in ligand binding and activation (Jiang et al. 2014). Utilization of bioluminescence/fluorescence resonance energy transfer (BRET/FRET) made it possible to investigate the activation process of FSHR in real time in living cells (Ayoub et al. 2015). The stereochemical structure and activating mechanism of FSHR have not been fully clarified yet. Crystallization of the entire FSHR and investigation of molecular mechanisms of FSHR-mediated signal transduction by using powerful investigation tools such as single fluorescent-molecule video imaging will shed light on the activation mechanism of FSHR and other members of glycoprotein hormone receptors (GPHR) family (Jiang et al. 2014).
Structures and Function of FSH and FSHR
FSH and FSHR belong to two distinct and structurally unrelated protein families (Figs. 2a and 2b). FSH is a member of glycoprotein hormone (GPH) family, which includes luteinizing hormone (LH), human chorionic gonadotropin (hCG), and thyroid stimulating hormone (TSH). FSHR is a member of the GPH receptor family, a subfamily of large G protein-coupled receptor (GPCR) family, which includes LH/CG receptor (LHR) and TSH receptor (TSHR) (Banerjee and Mahale 2015). The pairwise relationships between ligands and their cognate receptors indicate coevolution of these distinct protein families (Jiang et al. 2014).
FSH is synthesized and released by gonadotropic cells in the anterior pituitary in response to gonadotropin-releasing hormone (GnRH) from the hypothalamus. It consists of two subunits: α-subunit, which is common to other members of GPHs, LH, hCG, and TSH, and a hormone specific β-subunit. They are cystine-knot proteins with cysteine-linked folded protein backbones (Davis et al. 2014) (Fig. 2b). Both subunits appear to participate to the binding and activation of FSHR. They are glycosylated in various degrees and the difference in glycosylation is thought to be one of the causes that elicit biased signaling in FSH action as mentioned below (Arey and Lopez 2011; Davis et al. 2014; Landomiel et al. 2014).
The TMD constitutes of 264 aa and contains seven membrane-spanning hydrophobic α helices connected by intra- (ILs) and extracellular loops (ELs), the structure characteristic to the members of GPCR family (Figs. 1b and 2a). The TMD transduces extracellular signal across the membrane into the cell. The ELs appear to play important roles in FSH binding, receptor activation, second messenger production, signal transduction, cell surface receptor trafficking, and receptor internalization, whereas the ILs involve in FSH binding, second messenger production, signal transduction, phosphorylation, and intracellular protein interactions (Banerjee and Mahale 2015). The TMD has been shown to be a target of several small molecule allosteric agonists and antagonists (Nataraja et al. 2015).
The ICD constitutes of 65 aa. The ICD plays crucial roles in signal transduction and cell surface receptor trafficking. Together with the ILs, this domain interacts with G proteins and other signaling molecules involved in signal transduction and receptor desensitization/internalization/recycling (Ulloa-Aguirre et al. 2007; Banerjee and Mahale 2015).
FSHR is subjected to extensive posttranslational modifications such as glycosylation, disulfide formation, palmitoylation, and phosphorylation (Figs. 1a and 2a). These modifications are essential for protein folding, trafficking, cell surface expression, activation, sequestration, and signal transduction of the receptor (Ulloa-Aguirre et al. 2007).
Accumulating evidences indicate that the FSHR occurs as di/oligomeric units on the cellular membrane (Ulloa-Aguirre and Zarinan 2016). The FSHR has been also shown to form heterodimer with closely related LHR in ovarian granulosa cells where both receptors are coexpressed during the final phase of the follicular development (Feng et al. 2013). The transmembrane α helices and the extracellular domain are thought to be involved in di/oligomerization of the receptor (Ulloa-Aguirre and Zarinan 2016). Although physiological significance of the receptor di/oligomerization is not well understood, it may enable positive cooperativity between receptor protomers to augment FSH action, or in case of FSHR/LHR heterodimer, negative cooperativity between protomers to suppress an excessive LH action, which may cause premature luteinization (Ulloa-Aguirre and Zarinan 2016). Oligomerization of the FSHR is thought to modulate the mode of ligand binding to the receptor. Based on the crystal structure, it is predicted that the trimeric FSHR is able to hold only one fully glycosylated FSH molecule to one of the receptor protomer, leading to the activation of one set of signal transduction pathway due to the bulky glycan on the FSH α-subunit (N52α) blocks the binding of more FSH to other receptors. The trimeric FSHR is capable to bind three deglycosylated FSH molecules; however, it cannot induce receptor activation since the fully deglycosylated FSH is biological inactive (Jiang et al. 2014).
FSH binding to the ECD of the FSHR causes conformational changes of the receptor and triggers the FSHR activation and subsequent signal transduction. Although the exact mechanism that activates FSHR has not been fully elucidated, a two-step receptor activation mechanism is currently proposed. At first, FSH binds to the inner concave surface of HBSD causing a conformational change in FSH, which enhances FSH-FSHR interaction and formation of a sulfated tyrosine-binding pocket at the interface of two FSH subunits (Fig. 3b). To this pocket, the sulfated tyrosine residue on the hairpin loop of SSSD is docked, lifting the hairpin loop from the ELs of the TMD. This releases TMD from the inhibitory effect of the hairpin loop, causing a conformational change of the TMD, which results in the activation of recruited protein partners and downstream signaling pathways (Jiang et al. 2014) (Fig. 3c).
FSHR Activation and Biased Signaling in FSH Action
Upon activation, FSHR activates various signal transduction pathways mediated by protein kinases, adapter proteins, second messengers, and downstream effectors. For many years, it was thought that FSH signaling is essentially mediated by Gαs coupling cAMP dependent protein kinase (Gαs/cAMP/PKA) signaling pathway. In this pathway, binding of FSH to FSHR induces a conformational change of the receptor, which activates heterotrimeric G proteins coupling to the receptor. The activated G proteins dissociate into Gαs subunit and Gβγ heterodimeric complex, and the former activates membrane-bound enzyme adenylyl cyclase, leading to synthesis of a second messenger cAMP. Cyclic AMP in turn activates PKA, which phosphorylates transcription factors such as cAMP responsive element binding proteins (CREBs), initiating transcription of FSH target genes (Gloaguen et al. 2011; Ulloa-Aguirre et al. 2011; Landomiel et al. 2014). It is now considered that the FSHR-mediated signal transduction is far more complicated with multiple signaling pathways interacting each other at various signaling steps. Firstly, Gαs led cAMP signaling activates not only PKA but also activates exchange protein directly activated by cAMP (EPAC) and they directly or indirectly activate protein kinase B (PKB) and various mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase 1/2 (ERK1/2), MAPK kinase 1/2 (MEK1/2), and p38MAPK (Gloaguen et al. 2011; Ulloa-Aguirre et al. 2011; Landomiel et al. 2014). The level of cAMP is also regulated by FSHR-mediated activation of phosphodiesterase (PDE), which catalyzes cAMP to AMP, thereby downregulating Gαs stimulated cellular responses (Gloaguen et al. 2011).
FSHR is also able to couple to other Gα subunits such as Gαi, Gαq, and Gαh. Gαi involves in counteracting Gαs-stimulated cAMP accumulation as well as activating ERK1/2, while Gαq and Gαh involve in phospholipase C (PLC)-mediated inositol-1,4,5-trisphosphate (IP3) accumulation (Ulloa-Aguirre et al. 2007; Gloaguen et al. 2011).
FSHR has been shown to activate a number of Gs-independent signaling pathways through coupling to adapter proteins such as β-arrestin and the adapter protein containing a Pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif 1 (APPL1) (Gloaguen et al. 2011; Ulloa-Aguirre et al. 2011). Upon FSHR activation, Gβγ complex dissociate from Gα and recruits GPCR-kinases (GRKs) to phosphorylate the ICD and ILs of the receptor. This leads to the recruitment of β-arrestins to the phosphorylation sites, triggering desensitization/internalization/recycling of FSHR (Shenoy and Lefkowitz 2011; Kang et al. 2014). In addition, β-arrestins have been shown to involve in activation of ERK and ribosomal protein S6 (rpS6) (Gloaguen et al. 2011; Landomiel et al. 2014). APPL1 has been implicated to mediate FSH-dependent phosphatidylinositol 3-kinase (PI3K) and PKB signaling (Gloaguen et al. 2011; Ulloa-Aguirre et al. 2011).
In addition, FSHR appears to regulate activation of the mammalian target of the rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway, which is known to involved in translation initiation (Gloaguen et al. 2011; Ulloa-Aguirre et al. 2011).
Together, these signaling pathways activate a range of cytosolic effectors and nuclear transcription factors, leading to complex and multifaceted cellular responses.
It is now understood that a ligand can activate certain signaling pathway(s) rather than indiscriminately activates diverse and often contradictory signaling pathways as mentioned above. This selective mechanism is termed as biased signaling or ligand bias (Ulloa-Aguirre et al. 2011; Landomiel et al. 2014). In the classical two-state model, FSHR exist either active (ligand bound) or inactive (ligand unbound) state and the active conformation is only able to trigger the signaling. In the biased signaling, structural variants of the ligands or mutations/polymorphisms of the receptor induce multiple active conformations of FSHR, triggering various signaling pathways. For example, FSH occurs as a heterogenic population of glycosylated variants in vivo and this might be a natural source of signaling bias (Davis et al. 2014; Landomiel et al. 2014). The concept of signaling bias has accelerated the development of agonists and antagonists that selectively regulate FSHR and other GPCRs. To date, many small molecules acting as agonists or antagonists have been developed. Interestingly, many of them are allosteric regulators that act at the TMD of the receptor rather than acting at the HBSD of the ECD. The development of these FSHR modulators that can be administered orally and are capable to regulate specific FSH signaling without side effects provide great opportunity to improve infertility treatments and contraception as well as for nonreproductive use such as controlling cancer development (Nataraja et al. 2015; Papadimitriou et al. 2016).
Temporal and Special Expression of FSHR and Its Functions
Temporal and special expression of FSHR has been examined in several species including rodents, ruminants, nonhuman primates, and human by using various techniques such as ligand-binding assay, in situ hybridization, immunohistochemistry, northern hybridization, RNase protection assay, and quantitative RT-PCR (Simoni et al. 1997; George et al. 2011). Irrespective of species examined, FSHR is localized on the cellular membrane of the ovarian granulosa cells and the testicular Sertoli cells. The expression of FSHR is developmentally regulated in both cell types. In female mice and rats, FSHR mRNA appears to be expressed in the fetal ovary and undifferentiated granulosa cells of primordial follicle. The presence of the functional FSHR has been confirmed in small preantral follicles of various species and its expression increases as follicular development progresses into antral stage (Simoni et al. 1997; Araujo et al. 2014; McGee and Raj 2015). During preantral stage, FSH is not required for follicular growth although it has been shown to enhance granulosa cell proliferation and steroidogenesis (Kumar 2005; Araujo et al. 2014). On the other hand, FSH is absolutely necessary for antral formation and subsequent follicular development and maturation (Kumar 2005; Ginther 2016). Roles of FSH in antral follicle development and maturation have been extensively studied in mono-ovulatory species such as cattle where continuous monitoring of follicle development and circulating FSH levels are possible (Ginther 2016). During the final stage of follicular maturation, granulosa cells acquired LHR under the influence of FSH and other local factors such as estradiol and become being able to survive in the face of FSH decline caused by the negative feedback of estradiol and inhibin (Ginther 2016). During this crucial period, tone of FSH action is mainly regulated by the level of FSH reached to granulosa cells rather than the level of FSHR expressed on the cells (Ginther 2016). The expression of FSHR sharply declines in preovulatory follicles after preovulatory LH-surge and in atretic follicles (Simoni et al. 1997). Corpus luteum (CL) does not express FSHR and gonadotropin dependency is totally sifted to LH in the CL (Simoni et al. 1997).
Unlike in the female, FSH-R is not absolutely necessary for spermatogenesis in the male. FSHR knockout caused smaller testis and accessory glands, decrease in testosterone production, and reduction in qualitative and quantitative aspects of spermatogenesis, but they were nevertheless fertile (Kumar 2005).
In male rats, FSHR mRNA is expressed in the fetal testis but it is not clear whether it plays any functional role (Simoni et al. 1997). In immature rats, number of FSHR increases until day 7 and stays constant for 2 weeks (Simoni et al. 1997). The increase in FSHR number coincides with the proliferation of Sertoli cells, indicating the functional role for FSH in this process. The expression of FSHR decreases dramatically around day 40 when major initiation of spermatogenesis occurs (Simoni et al. 1997).
Spermatogenesis can be classified into several stages, defined by combination of germ cells at different stages of development in the seminiferous tubule. In the rat, where the spermatogenesis is organized into 14 stages, FSHR is expressed stage dependently, being highest in stages XIII-II and lowest in VI and IX (Simoni et al. 1997; George et al. 2011). FSH responsiveness of the cells, in terms of cAMP production, follows similar pattern but a few stages behind the expression pattern, being highest in stages IV and V (Simoni et al. 1997). Although FSH is not crucial in the spermatogenesis, it facilitates the production and/or survival of spermatogonia and their transition into spermatocytes through FSHR expressed on Sertoli cells (Ramaswamy and Weinbauer 2014).
Ectopic expression of FSHR has been reported in some tissues such as myometrium, placenta, decidua, fetal membrane, umbilical cord, prostate, bone, and ovarian surface epithelium (Simoni et al. 1997; Bose 2008; Kumar 2014). Physiological significance of FSHR in these tissues has been investigated and implied to be involved in the feto-placental development (Kumar 2014), carcinogenesis in various tissues (Gartrell et al. 2013; Papadimitriou et al. 2016), and accelerated bone loss in postmenopausal women (Wang et al. 2015).
Regulation of FSHR Expression
The expression of FSHR is regulated by various endocrine, paracrine, and autocrine factors. FSH appears to be a prime hormone that regulates FSHR expression. Effect of FSH on the receptor expression differs among species and between sexes. In male rodents, FSH downregulates expression of Fshr in Sertoli cells both in vivo and in vitro (Simoni et al. 1997). In the female, FSH effect appears to be biphasic; treatment of immature or hypophysectomized rats with eCG or FSH increased FSHR mRNA and FSH binding, whereas subsequent treatment to induce ovulation with hCG or high dose of FSH downregulated FSHR mRNA and FSH binding (Simoni et al. 1997). In cultured rat granulosa cells, FSH prevented FSHR downregulation that otherwise occurs in the absence of FSH (Simoni et al. 1997). FSH was also shown to induce a dose-dependent decrease in FSHR mRNA expression in cultured rat granulose cells (Minegishi 2004). These findings suggest that the expression of FSHR is autoregulated by FSH to maintain optimum levels of FSH stimulation.
Number of locally produced factors, either alone or together with FSH, modulate FSHR expression in granulosa cells (Simoni et al. 1997; Minegishi 2004). Transforming growth factor (TGF)β increases while TGFα decreases basal expression of FSHR mRNA and protein in cultured rat granulosa cells. TGFα also dose dependently suppresses the effect of TGFβ. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) suppress FSH-stimulated FSHR mRNA expression. Retinoic acid similarly suppresses FSH action (Minegishi 2004). GnRH likewise antagonizes FSH action on the expression of FSHR. Insulin-like growth factor I (IGF-I), on the other hand, synergistically increases FSH stimulated FSHR mRNA expression (Minegishi 2004). Activin together with FSH increases FSHR mRNA expression at the transcription and posttranscription levels and this effect is counteracted by follistatin, the activin-binding protein (Minegishi 2004; George et al. 2011).
Mutations and Polymorphisms of FSHR
Mutagenesis may alter expression and function of FSHR; mutagenesis in the promoter region may compromise promoter activity, while that in the coding region may result in substitutions of amino acids that enhance (activating mutations) or impair (inactivating mutations) receptor functions (Hermann and Heckert 2007; Desai et al. 2013; Siegel et al. 2013). Amino acid substitutions in functional domains of FSHR may alter structural and/or physicochemical nature of the receptor and may affect its functions. Reported activating mutations of human occur mostly at the TMD. Nevertheless, the mutations increase sensitivity to FSH or other tropic hormones such as hCG and TSH, allowing promiscuous activation of the receptor. Patients with these mutations suffer from spontaneous ovarian hyperstimulation syndrome. The other type of activating mutation increases constitutive activity of the receptor unbound to FSH (Desai et al. 2013; Siegel et al. 2013). Inactivating mutations occur at any of the three functional domains. These mutations may reduce the receptor response to FSH through altering FSHR expression on the cell surface, reducing signal transduction efficiency, or impairing FSH binding (Desai et al. 2013; Siegel et al. 2013). Patients carrying these mutations suffer from symptoms such as primary/secondary amenorrhea and precocious ovarian failure (Desai et al. 2013; Siegel et al. 2013).
Single nucleotide polymorphism (SNP) in FSHR has been also implied to affect ovarian response in women undergoing the IVF program. Of more than 1,300 SNPs identified in the coding and noncoding region of the FSHR, three SNPs located in the 5′ UTR (g. G>A -29), SSSD (p. Thr307Ala) and ICD (p.Asp680Ser) have been associated with altered FSHR function or FSH responsiveness to date (Desai et al. 2013; Siegel et al. 2013).
FSHR is a transmembrane glycoprotein that directs FSH signal to the intracellular signaling pathways. FSHR consists of three functional domains, extracellular domain (ECD), transmembrane domain (TMD), and intracellular domain (ICD). The ECD is further divided into hormone-binding subdomain (HBSD) and signal specificity subdomain (SSSD), which are responsible for high-affinity specific interaction with FSH and the receptor activation. The TMD and ICD transduces activating signal to intracellular signaling pathways by changing their conformations. Upon activation, FSHR activates a number of signal transduction pathways mediated by PKA, PKB, and MAPKs through activating various FSHR associated proteins such as G proteins, β-arrestins, and APPL1, which results in cellular proliferation and differentiation as well as desensitization/internalization/recycling of FSHR. FSH adopts various active conformations depending on types of ligand it binds (e.g., glycosylation variants and agonists) or mutations/polymorphisms of the receptor itself and this in turn selectively activates various signaling pathways. This selective mechanism is termed as biased signaling or ligand bias. Ovarian granulosa cells and testicular Sertoli cells are primary cell types that express FSHR where it plays pivotal roles in female and male reproduction. Expression of FSHR is developmentally regulated in relation to the stages of folliculogenesis and spermatogenesis by various endocrine/paracrine/autocrine factors, such as FSH and growth factors. Mutations of FSHR may disrupt structural organization of the receptor and may result in partial or total loss of fertility. Structure-function relationship of FSHR has been extensively studied in recent years by using molecular biological and biophysical techniques such as site-directed mutagenesis and crystallization, revealing complex ingenious molecular mechanism that regulates cellular response to FSH. Further understanding in the FSHR will benefit for developing agonists/antagonists for better management of human and animal reproductive health.
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