Follicle stimulating hormone (FSH) plays a crucial role in the control of male and female reproduction. FSH is a heterodimeric glycoprotein consisting of an α-subunit non-covalently associated with a β-subunit. The α-subunit is shared with luteinizing hormone (LH), chorionic gonadotropin (CG) and thyroid-stimulating hormone (TSH), whereas the β chain is specific of each glycoprotein hormone (1). FSH is synthesized and secreted by the pituitary and binds to a plasma membrane receptor (FSHR) that belongs to the class A of the G protein-coupled receptor (GPCR) superfamily. The FSHR displays a high degree of tissue specificity as it is expressed in Sertoli and granulosa cells located in the male and female gonads, respectively (2). FSH is required for normal growth and maturation of ovarian follicles in women and for normal spermatogenesis in men (3). Female mice with FSHβ or FSHR gene knockout present an incomplete follicle development leading to infertility, whereas males display oligozoospermia and subfertility (4, 5). Consistently, women expressing non-functional variants of the FSHR are infertile while men are oligozoospermic, yet fertile (6). To date, only native forms of FSH, either purified from urine or by using recombinant technology, are being used in reproductive medicine with no other pharmacological agents being currently available in clinic (7–9). Novel classes of FSHR agonists with varying pharmacological profiles could potentially help improving the overall efficiency of assisted reproductive technology. On the other hand, FSHR antagonists could represent an avenue for non-steroidal approach to contraception (10). This paper offers an updated overview of the way FSHR signals and of how selective modulation of its signaling can be achieved.

For the vast majority of GPCRs, the orthosteric site (i.e., the region that binds the natural ligand), is located in a cavity defined by the transmembrane helices. This is not the case for the FSHR, which binds its natural ligand, FSH, through its characteristic large horse-shoe-shaped extracellular domain (ECD). Consequently, the orthosteric site spans over the nine leucine rich repeats (LRRs) of the ECD (Figure 1). The first crystal structure of FSH bound to part of the ECD came out in 2005 and led to a detailed understanding of the molecular basis leading to the specificity of hormone binding (11). The ECD of the receptor contains 12 LRRs linked to three disulfide bonds and two unstructured sequence motifs that define the hinge region connecting the ECD to transmembrane domains (TMD). However, the recombinant protein used for crystallization did not include amino acid residues in the hinge region. Therefore, the receptor activation mechanism remained poorly understood, until recently, when another crystal structure including the hinge region was reported (12). Interestingly, it revealed a two-step activation mechanism of the receptor: interaction of FSH with LRR1-9 reshapes hormone conformation, so that exposed residues located at the interface of the hormone α- and β-subunits form a binding pocket for sulfated Tyr335 of the hinge region, resulting in a conformational change of the latter (13, 14). This two-step interaction process not only stabilizes the FSH/FSHR interaction but also relieves the tethered inverse agonistic activity previously mapped within the hinge region (15). Since no structural data of gonadotropin receptor TMD are currently available, homology modeling with other GPCRs is necessary. The structure of human neuropeptide Y1 receptor that recently came out (PDB:5BZQ) displays the highest identity with FSHR (25%) and LHR (24%) TM domains. Prior to that, gonadotropin receptor TM domains have been successfully modeled using adenosine receptor crystal structure (16). This revealed the existence of two adjacent pockets that could accommodate small ligands. These sites have been assigned P1 and P2 (major and minor site, respectively). The P1 site is located between TMs III, IV, V, and VI, and P2 between TMs I, II, III, and VII (Figure 1A) (17). These putative TM domain allosteric sites have been confirmed in studies utilizing chimeric receptors and mutagenesis. Interestingly, it was found that a FSHR small-molecule agonist at high concentration specifically displaced the binding of radiolabelled adenosine A3 receptor (A3R) agonist on A3R (18). This suggests a similarity between glycoprotein receptor and A3R in the TMD region for the allosteric binding pocket (19). As suggested from studies on other GPCRs, allosteric sites distinct from P1 and P2 may also exist and affect FSHR activity (20).

By analogy with other GPCRs, it is reasonable to posit that FSH binding leads to conformational rearrangements within the transmembrane regions, thereby causing the recruitment and coupling of signal transducers (G proteins, β-arrestins) that ultimately trigger a complex intracellular signaling network (21, 22). The primary transduction effector described for the FSHR is Gαs that triggers the canonical adenylyl cyclase/cAMP/protein kinase A (PKA) signaling cascade. Once activated, PKA phosphorylates many proteins such as transcription factors of the cAMP response element-binding protein (CREB) family (23–31). cAMP action is also mediated by the Exchange Proteins directly Activated by cAMP (EPACs) (32–34). Upon cAMP binding, EPAC1/2 stimulate Ras-related protein (RAP1/2), small GTPases that lead to protein kinase B phosphorylation (PKB) (35, 36). In addition, the FSHR has been reported to interact with Gαi and Gαq. Gαi inhibits adenylate cyclase, blocking Gαs-induced cAMP production (37). The stimulation of Gαq requires in vitro stimulation with high FSH concentrations (>50 nM) (22, 38–40). This coupling leads to the production of inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG), increased intracellular calcium concentration and activation of protein kinase C (PKC). Pleiotropic coupling of FSHR to various heterotrimeric proteins suggests the co-existence of multiple active conformations of the receptor in the plasma membrane (41, 42).

Similarly to most GPCRs, the FSHR interacts with β-arrestins, scaffolding proteins that control receptor desensitization, internalization and recycling (24, 43–46). Classically, β-arrestins are recruited following (i) receptor activation and (ii) receptor phosphorylation by G protein-coupled receptor kinases (GRK). Due to steric hindrance, FSHR coupling to Gαs is impaired once β-arrestins are recruited (47, 48). In a model of rat primary Sertoli cells that express the FSHR endogenously, it has been demonstrated that agonist-induced cAMP levels decreased upon β-arrestin overexpression, consistently with its role in FSHR desensitization (49). In heterologous cells, the carboxyl tail of FSHR has been reported to be phosphorylated on several serine and threonine residues (43). In addition to these classical functions, it has become increasingly clear that β-arrestins can also initiate specific, G protein-independent signaling events leading to the activation of many pathways, amongst which the ERK (Extracellular signal-Regulated Kinase) MAP (Mitogen-Activated Protein) kinase pathway has been the most studied (50). Of note, ERK activation kinetics at the FSHR has been reported to vary in heterologous cells as a function of the upstream transduction mechanism involved: β-arrestin-mediated ERK activation is delayed but more sustained compared to Gαs-dependent ERK activation, which occurs early but is transient (43). Consistent with the concept of “phosphorylation barcode” which links particular GRK-mediated phosphorylation signatures at the receptor level to the activation of distinct β-arrestin-dependent functions (51, 52), a relationship has been found between the subtype of GRK involved in FSHR phosphorylation and the nature of β-arrestin-mediated actions. In particular, β-arrestins recruited to GRK2 or GRK3-phosphorylated FSHR favor receptor desensitization whereas GRK5 or GRK6-mediated phosphorylation of FSHR were involved in β-arrestin-dependent ERK activation (43, 53, 54). Recently, phosphorylation of Tyrosine383 in β-arrestin 2 has proved to be crucial for β-arrestin-mediated ERK activation by the FSHR and other GPCRs. More precisely, ligand-induced receptor activation provokes MEK (Mitogen-activated protein kinase kinase)-mediated phosphorylation of Tyr³⁸³, necessary for β-arrestin 2-mediated ERK recruitment and activation (55). β-arrestins also play a role in FSHR-induced translation, mediated by a β-arrestin/p70S6K/ribosomal S6 complex that assembles in heterologous and in primary Sertoli cells. Upon FSH stimulation, activation of G protein-dependent signaling enhances p70S6K activity within the β-arrestin/p70S6K/rpS6 preassembled complex, leading to the fast and robust translation of 5′ oligopyrimidine track (5′TOP) mRNA (56). In addition, the balance between FSHR-mediated proliferation vs apoptosis seems to be regulated by β-arrestins. In hGL5 human granulosa cells, silencing of β-arrestins leads to an increase in cAMP/PKA and a decrease in β-arrestin-mediated proliferative pathway, resulting in cell death (57). Evidence reported for other GPCRs demonstrated that the internalized receptor can form molecular complexes involving simultaneous interactions with Gαs to the core domain and β-arrestin to the C-tail of the receptor (58). These complexes, named “megaplexes,” are able to signal from the endosome by inducing a second wave of cAMP (58, 59). Based on structural evidence, a two-step mechanism for β-arrestin recruitment has been proposed (60). First, β-arrestins are recruited to the phosphorylated C-tail, resulting in a so-called “partially engaged” complex which the authors reported to be sufficient for ERK signaling and internalization. Interestingly, this conformation allows the receptor to simultaneously couple to G protein α subunit. Second, a conformational rearrangement of β-arrestins allows them to interact with the receptor core domain, forming a “fully engaged complex” incompatible with further G protein coupling (58, 60–62). More recently, a separate study uncovered another mechanism of β-arrestin activation that the authors called “catalytic activation.” Upon ligand-induced recruitment of inactivated β-arrestin to the receptor core domain, a conformational change in β-arrestin occurs that exposes a PIP2-binding motif and allows β-arrestin to bind membrane lipid rafts independently of the receptor. Interestingly, the authors noticed an accumulation of active β-arrestin in clathrin-coated endocytic structures in the absence of the receptor, revealing the existence of a receptor C-tail-independent β-arrestin activation mechanism (63). No evidence currently exist that the aforementioned mechanisms also apply to the FSHR. Further studies will be necessary to clarify the molecular mechanisms involved in β-arrestin recruitment and activation at the FSHR and to determine their possible peculiarities.

Beside G proteins, GRKs and β-arrestins, the signal is also transduced at the FSHR by other direct binding partners (44). For example, adaptor protein, phosphotyrosine interacting with the Adapter protein with PH domain, PTB domain, and leucine zipper 1 (APPL1) binds intracellular loop 1 of the FSHR (64). This protein has lately retained the greatest attention in the gonadotropin community for two main reasons. The first one is that this adapter protein links the FSHR directly to inositide phosphate metabolism and Ca²⁺ release in granulosa cells (65), hence it induces cAMP-independent signaling; the second is that, like β-arrestins, APPL1 recruitment plays a role in the subcellular routing of FSHR. This discovery had been heralded by the previously identified interaction between GAIP-interacting protein C-tail (GIPC) adaptor and the FSHR (or the LHR), presumably requiring the carboxyterminal end of the receptor. Interestingly, GIPC reroutes the internalized FSHR from Early Endosomes (EE) to recycling Very Early Endosomes (VEE), and by these means, enables sustained ERK phosphorylation (66). Likewise, in HEK293 cells, APPL1 has been shown to convey internalized FSHR, as well as LHR, to VEE for recycling, and PKA-dependent phosphorylation of APPL1 leads to endosomal cAMP signaling (67). These two sets of observations on ERK MAP kinases and cAMP suggest that spatially restricted FSH signaling may be generalized to several of its components. In addition, 14-3-3τ has been shown to interact directly with the second intracellular loop of the receptor FSHR (68, 69). The 14-3-3τ interaction site on the FSHR encompasses the ERW motif involved in G protein association (70), that is consistent with the observation that 14-3-3τ overexpression in HEK293 cells reduces FSH-induced cAMP response (68). The co-occurrence of these direct binding partners as well as G protein, GRK and β-arrestins, raises fundamental questions about their sequence/dynamics of interaction on a single FSHR or about the possibility that FSHR oligomers might cluster transduction assembly of variable composition at the plasma membrane and in intracellular compartments.

Compartmentalization of signaling is now viewed as an important feature for many signaling proteins and plays key roles in cellular responses. This is particularly the case for membrane receptors such as GPCRs since, in the past years, several examples have revealed connections between membrane compartmentalization, endocytic trafficking and signaling patterns. Originally thought to function solely at the plasma membrane, the multifunctional protein β-arrestin assemble signaling molecules (e.g., MAPK, Src, etc) that direct GPCRs to the endocytic pathway and regulate their post-endocytic fate, as mentioned above. For some GPCRs forming a stable interaction with β-arrestin, β-arrestin/receptor/signaling molecule complexes are found in endosomes, allowing prolonged signaling from these intracellular structures (71–73). The nature of the β-arrestin binding motifs, in particular serine/threonine clusters in the C-tail of the receptor, regulates the stability of this interaction. GPCRs that display high affinity binding to β-arrestin, are classified as class B (74, 75). In the FSHR, a cluster of 5 serines/threonines is involved in both internalization and binding of β-arrestin to the receptor and is consistent with the class B definition. Such interaction was confirmed by bioluminescence energy transfer experiments (BRET) and co-immunoprecipitation experiments, however no imaging data have confirmed the existence of a functional complex in endosomes (24, 43). In addition, it is unclear whether β-arrestin-mediated ERK signaling by FSHR requires β-arrestin localization in endosomes. Recently, both the FSHR and LHR were reported to predominantly localize to an atypical endosome denoted as VEE (Figure 2). Alteration of this endosomal trafficking by blocking internalization inhibits activation of ERK through the LHR, suggesting that VEE are a location for signaling (66). These particular endosomes are upstream of the classical endosomes and are devoid of typical early endosomes markers such as the Rab5 GTPase or the phosphatidylinositol-3-phosphate (PI(3)P) or the PI(3)P-bound EEA1 proteins. Morphologically, they are smaller (<400 nm) than conventional sorting EE but their exact nature remains to be defined. Interestingly, gonadotropin receptor localization in VEE requires an intact receptor C-tail and the GIPC PDZ-domain protein (66). PDZ motifs are found in several GPCRs to regulate their spatial localization or trafficking (76). The PDZ motif of the LHR directly binds GIPC that sequesters the receptor into VEE following agonist-induced internalization. In fact, in cells depleted in GIPC or expressing LHR lacking the distal PDZ motif in its C-tail, the receptors are rerouted and accumulated into the classical EE where they fail to recycle back to the plasma membrane. In addition, they were not able to signal to ERK MAP kinases (66), suggesting that endosomal ERK activity occurs from this specific compartment. It is worth noting that the FSHR does not display a known PDZ ligand in its C-tail and the exact mechanism on how GIPC controls FSHR fate remains to be determined. However, APPL1, a known FSHR binding partner, localizes to a subset of VEE and displays a PDZ motif previously shown to interact with GIPC (64, 65, 77). A possible scenario would be that FSHR, via its interaction with APPL1, connects with GIPC and is targeted to VEE where it activates ERK (78). Earlier work supports the idea that endosomal APPL1 defines a signaling platform upstream of the Rab5/PI(3)P endosomes. Disruption of EE leads to the accumulation of the EGFR Tyrosine kinase receptor in APPL1 vesicles, leading to a sustained activation of ERK from this compartment (79, 80). As shown for the LHR, the endosomal cAMP/PKA dependent phosphorylation of APPL1 on Ser410 is necessary for the recycling of the receptor (67).

This concept of endosomal signaling compartmentalization was further supported by the findings that GPCR can induce a second phase of G protein activation following their internalization (81–83). This allowed the advent of a new paradigm where some GPCRs do not only transduce and activate G proteins from the plasma membrane but also from endocytic compartments (the so-called “megaplex” mentioned above). Interestingly, the two other members of the glycoprotein hormone receptor family, TSHR and LHR, were both shown to transduce via Gαs and promote sustained cAMP production from endocytic compartments (59, 67, 84, 85). While the TSHR acts from EE and trans-Golgi compartments, the LHR signaling is restricted to the VEE. It has yet to be shown whether the FSHR also activates G proteins from the VEE but the fact that it shares several features with GPCRs known to signal from endosomes, including LHR, V2R or PTHR, supports this possibility. FSHR trafficking mimics the LHR as discussed above and it displays a phosphorylation code similar to those found in the C-tail of V2R and PTHR. More precisely, the formation of a “megaplex” has been shown to induce cAMP from the EE in response to PTHR activation (86, 87). That the FSHR could signal in endocytic compartments through G proteins in a similar way as the PTHR, but from VEE, is conceivable, but further studies are needed to demonstrate this possibility. Despite the identification of structural determinants in the FSHR C-tail that regulate its trafficking (88), very little is known about the mechanisms involved in the post-endocytic trafficking of this receptor.

The action of a given ligand on its cognate receptor has classically been characterized by its effect on downstream effectors (second messengers). Compared to the reference ligand (generally, the physiological ligand), a pharmacological agent can be either an agonist (it produces a biological response similar to that of the reference ligand), an antagonist (it blocks the biological response elicited by the reference ligand) or an inverse agonist (it produces an opposite biological response that leads to a decrease in the receptor basal activity). Importantly, it has long been thought that these characteristics hold irrespectively of effector measured (89). However, some ligands did not match with one of these categories, because they displayed both agonist and antagonist (or inverse agonist) activities at the same receptor, depending on the downstream pathway measured. For instance, carvedilol, a clinically used β-blocker, has a clear inverse agonist profile on Gαs-dependent activity at the β2 adrenergic receptor while being a weak partial agonist for β-arrestin-dependent ERK activation (90). To deal with these discrepancies, the concept of biased signaling or functional selectivity, recently came to the fore (89, 91, 92). According to this concept, a ligand is biased when it triggers imbalanced responses, compared to a reference ligand acting on the same receptor (classically the endogenous ligand). Importantly, a biased ligand can selectively activate only a subset of the biological responses or activate all of them but with different efficacies compared to the reference ligand. As these ideas hold profound implications and potential for the design of new therapeutics, biased signaling is a very active area of research in pharmacology (93, 94). Over the last decade, biased signaling has been evidenced in many different receptors, including gonadotropin receptors, as will be discussed in a forthcoming section. By analogy with a ligand bias, the notion of receptor bias has been proposed (95). Two receptors, diverging only by a mutation or a polymorphism, once activated by the same ligand may induce two signaling pathways with different relative efficacies. Importantly, biased signaling has been extended to allosteric ligands, which can modulate not only the efficacy of a given ligand-induced receptor signal but also select and bias the activation of the receptor toward a subset of the biological responses (93). Ligand bias and receptor bias must be set apart from system bias or observational bias (95). System bias refers to bias that are due to the particular biological system used (some transducer molecules, such as G proteins, may be expressed differentially in different cell types for instance). Observational bias refers to the modification or amplification of the signals inherent to the specific assays used for the measurements. Besides being supported by numerous experimental evidences, biased signaling is consistent with the receptor conformation theory, which views a receptor population as an ensemble of conformations that evolve dynamically, according to some energy landscape and subjected to external perturbations (96). In such theory, ligand-induced receptor activation is concomitant with a stabilization of some receptor conformations and a modification of the receptor conformation energy landscape, resulting from the interaction of the ligand with its receptor. Several studies have thus shown that receptor conformational equilibrium models, such as the extended ternary complex model, can satisfactorily explain ligand bias (97, 98). Several groups have attempted to address the problem of bias quantification. Considering a given receptor and two signaling pathways (A and B), the objective is to be able to classify ligand bias, as having for instance a low, a moderate or a high bias toward pathway A vs pathway B, when compared to a reference ligand. The most popular method to quantify ligand bias uses dose-response data and the so-called operational model (99, 100). The latter is widely used to perform regression on dose-response data, which have in many cases a sigmoid shape. The parameters of the operational model are derived from a simple chemical reaction scheme that takes into account ligand receptor association/dissociation reactions and that links the ligand-receptor concentration to the biological response thanks to a logistic function (similar to enzymatic reaction models). The usefulness of this model for the quantification of ligand bias is associated with the interpretation of its parameters. In particular, the two main parameters of the operational model are the ligand-receptor dissociation constant K_a and the intrinsic efficacy τ (which describes the ability of the ligand-receptor association to be converted into a response). With these two coefficients, a single transduction coefficient, given by log(τ/K_a), has been proposed to characterize the agonism of a ligand for a given signaling pathway (100). This coefficient can then be compared between two pathways and between two ligands, to ensure normalization. Hence

quantifies the activation of a pathway by ligand A, compared to ligand B. In addition,

evaluates the differences of activation between the two pathways. Finally, the bias is defined as

This procedure for bias quantification, together with its statistical significance, has been detailed as a step-by-step protocol using Prism (v6.0; GraphPad Software) (101–103). Other logistic regressions that lead to different quantifications and parameter interpretations have been proposed and compared (104). A notion of dose-dependent ligand bias, which may reveal subtle nonlinear effects of the ligand, has also been defined using logistic function (105). Overall, the statistical regression of dose-response data (sigmoid curves) can be ambiguous and lead to a misinterpretation of the results. Moreover, it has been shown experimentally that different procedures may exhibit discrepancies between each other, and may fail to detect ligand bias or lead to false positives, probably due to the presence of system or observational bias (104). A semi-quantitative method to classify ligand bias that would be more robust than quantitative methods has been proposed, based on logistic regression (104). However, the major concern of the operational approach to quantify ligand bias is that it disregards an important aspect of signaling pathways, namely the temporal activation of the different signaling processes (106). This has been revealed by the relatively simple observation that the bias value, as calculated with the operational model, could change as a function of the kinetics of response (107). Actually, the apparent bias can even be in an opposite direction for two different time points when the biological responses are measured. While part of the explanation of this phenomena resides in the different time scales at stake within a signal transduction pathway (binding kinetics, second messenger and effector kinetics), it also reveals the whole complexity of a receptor trafficking system (108), that can certainly not be condensed into a single number. Thus, methodological developments such as dynamical versions of the operational model and/or the extended ternary complex model (109, 110) must be developed to address this complexity and allow better characterization of the effect of a ligand on its cognate receptor.

To date, different classes of biases have been reported to elicit selective modulation at the FSHR (Figure 3). Ligand bias can be provoked by small molecule ligands, glycosylation variants of FSH or by antibodies acting at FSH or FSHR. Receptor bias due to mutations or single nucleotide polymorphisms (SNP) at the FSHR have also been reported.

The observation that FSHR transduction can be finely tuned by a variety of biased ligands, mutations or polymorphisms, further emphasizes the importance to better understand the complex signaling networks that are modulated (i.e., activated or inhibited) downstream of the FSHR. These novel biased ligands and receptor variants are great research tools that should really help us deciphering the molecular mechanisms involved in FSHR-associated physiopathology. In addition, a number of existing ligands and mutants have been characterized solely by measuring plasma membrane expression and/or cAMP response. Further characterization is required and may generate insightful findings. Biased ligands also open intriguing prospects in drug discovery. In particular, low molecular weight agonists of the FSHR could lead to the development of orally-active treatments. Such administration route would bypass the multiple injections of gonadotropin preparations that remain needed in the current protocols used in assisted reproduction. Moreover, it becomes possible to sort out the pathways leading to ovulation and those responsible for OHSS, and the availability of pathway-selective low molecular weight agonists at the FSHR could pave the way for the development of safer treatments, presenting reducing risks of OHSS. Modulation of relative FSH and LH activities could also open new avenues in the treatment of polycystic ovarian syndrome (PCOS).

On a more general note, the availability of allosteric compounds active at the FSHR, opens the unprecedented opportunity to enhance or dampen the transduction activities of the FSHR in vivo, while conserving the rhythmicity and biochemical diversity of endogenous FSH, a property that no orthosteric compound can match. The conditions of application of such treatments will obviously require extensive pre-clinical and clinical studies. Despite of these limitations, hampering any hope for short-term clinical use, the advent of biased and allosteric compounds certainly represents an important juncture in a field that has uniquely relied for so long on natural and recombinant gonadotropins to treat infertility. Finally, orally active low molecular weight FSHR antagonists may also lead to novel classes of oral contraceptives devoid of the side effects associated with current sex steroid-based contraceptives.

Each author wrote a section of the review. AP and LPP designed Figures 1–4 respectively. ER integrated all the contributions into the final manuscript. All authors edited the complete paper.