General

There are three glycoprotein hormone receptors (GpHRs) functioning, respectively, as the receptors for the pituitary hormones thyrotropin (TSH receptor), follitropin (FSH receptor) and lutropin (LH receptor). In primates and equidae the LH receptor is also the receptor for the placental hormone chorionic gonadotropin, hence its denomination LH/CG receptor.

Receptor structure

GpHRs and their respective hormones constitute an example of co-evolution. The hormones, TSH, FSH, and LH/CG are dimeric proteins of about 30kDa made of a common alpha subunit and specific beta subunits. The beta subunits of TSH, FSH, and LH are encoded by paralogous genes displaying substantial sequence similarity (about 40% in terms of encoded amino acids). Their corresponding receptors, FSHr, LH/CGr and TSHr, are members of the rhodopsin-like G protein-coupled receptor family. Their serpentine portion contains seven transmembrane helices with sequence signatures typical of this receptor family. In addition, they display a large (350-400 residues) amino terminal ectodomain responsible for the high affinity and selective binding of the corresponding hormones [2,10,44]. The presence in their ectodomain of nine leucine-rich repeats (LRRs) makes GpHRs belong to the larger group of LGRs (Leucine-rich containing G protein coupled Receptors) [20]. In mammals, LGR4, LGR5 and LGR6 are still orphan receptors, whereas LGR7 and LGR8 function as relaxin and InsL3 receptors, respectively [20-21,24]. The serpentine portions of the three GpHRs display high sequence identity (about 70%) when compared with their ectodomains (about 40%). This reflects the observation that recognition specificity is completely encoded within the LRRs. Isolated ectodomains are capable of binding the hormones with high affinity, as demonstrated by the co-crystallization of FSH with the ectodomain of the FSH receptor [15].

Whereas the mRNA for the FSH and LH/CG receptors are translated into mature single-chain proteins (which may di/oligomerize, see below), the TSH receptor undergoes a maturation step resulting in cleavage and removal of a short segment (50-60 amino acids) with no counterpart in the two other GpHRs. The result is a two-chain protein, held together by disulfide bonds [28,36,45]. The functional role of this peculiarity of the TSH receptor, if any, remains unknown.

In addition to hormone specific interactions involving identified residues of their leucine-rich repeats [15,42], high affinity binding of hormones to GpHRs requires that specific tyrosine residues, upstream of the first transmembrane helix of the receptors be sulfated [9].

A series of biophysical and functional studies have demonstrated that GpHRs are likely present at the cell surface as di/oligomers [15,22,25,33,38,46,48].

Receptor signalling

A current model for the intramolecular transduction of the signal, from the ectodomain to the serpentine portion of the receptor, holds it that, upon binding of the hormone, the ectodomain becomes an agonist of the serpentine portion [50]. According to this model, the true agonist of the rhodopsin-like serpentine portion is the hormone-ectodomain complex. This view fits with the observation that GpHRs can be fully activated by point mutation of a specific residue of their ectodomain (S281, S277, S73 in the TSH, LH, FSH receptors, respectively) [12,31], and that, in addition to TSH, the TSH receptor can be fully activated by a series of different monoclonal antibodies [8,39,39]. However recent crosslinking studies were not able to provide a biochemical basis for these two putative conformational states [23].

Two intramolecular locks have been identified within the serpentine domain of GpHRs. They involve TM3 and TM6, and TM6 and TM7, respectively (for references, see [40,50]). Upon breaking them, the receptors display strong increase in basal activity.

All three GpHRs are preferentially coupled to G_s, but the TSH and LH receptors do couple also to G_q, when stimulated by higher concentration of hormones [1,30]. In addition, the GpHRs have been shown to interact also with G_i/o, but the physiological meaning of these observations is not clear [6,18,26]. While it is clear that FSH receptor couples through G_sα to activate adenylate cyclase there is less evidence that it couples through G_i and G_q11 pathways like the LH receptor [6]. In any case, downstream effectors of cAMP include cyclic nucleotide exchange factors, channels, the well studied Protein Kinase A which is directly activated by cAMP binding [6], and the less studied Protein Kinase B whose activation is not yet clear [52]. It should be noted that if FSH acts on G_iα it may be age-dependent, and absent in mature animals [14].

An interesting observation with respect to coupling of the LH receptor to G_q has been made with a mutation in the 6^th transmembrane segment at position D578. In Leydig cell adenoma that cause precocious puberty in boys this amino acid residue is mutated to H, only in a somatic, non-hereditary pattern. LH receptors that carry the D578H mutation show constitutive full activation of the G_q coupled PI turnover [27]. In addition this mutant receptor has been shown to constitutively activate the MAPK pathway [19].

Physiology/Physiopathology

The TSH receptor is the main regulator of the thyroid gland. It controls both the function and the growth of the gland [11]. Activation of the receptor stimulates thyroid hormone synthesis, thyroid hormone secretion and thyroid growth. Basal stimulation of the receptor is required to maintain the differentiation state of the gland, which involves maintenance of transcription of thyroid-specific genes (thyroglobulin, thyroperoxidase, Sodium-Iodide symporter). Most of these effects, including stimulation of growth, are mediated by cAMP. In man, activation of thyroid hormone synthesis, which involves generation of hydrogen peroxide, is a G_q-dependent effect [7]. Control of the thyroid gland by the pituitary functions as a relatively straightforward chemostat, with the level of circulating thyroid hormones controlling negatively the production of TSH by the pituitary thyrotropes.

The TSH receptor has been shown to function also as the receptor of another, recently identified glycoprotein hormone, thyrostimulin. This novel hormone is made of two cystine knot subunits (α2 and β5) and is found to be expressed in the pituitary. Although it displays high affinity for the TSH receptor, its role is still largely unknown [32].

In females, the FSH receptor is found in ovarian granulosa cells, which line developing follicles that harbor the female gamete. In males, the FSH receptor is expressed in testicular Sertoli cells, which line the seminiferous tubule and provide tight interactions with the male germ cells as well as establishing a blood-testis barrier. In females, FSH is required for growth and development of the follicle, in part due to proliferation of granulosa cells, and for proper maturation of a viable, fertilizable egg [4]. In males, FSH is not strictly required for spermatogenesis, but rather is required for both quality and quantity of male gamete production [17].

Together with FSH, the LH/CG receptor is the main regulator of gonadal function. In the male LH action (hCG action in the fetal period) is essential for the production of testosterone, the hormone responsible for the differentiation and maintenance of the male sex characteristics. In women, LH regulates the androgen production by the theca cells that are part of the follicular wall (there does not appear to be an action of hCG in female fetuses). These androgens are subsequently converted to estrogens in the follicular granulosa cells. A second essential role of LH is the triggering of ovulation of the Graafian follicle [47].

Activating mutations rendering the TSH or LH/CG receptors constitutively active have been described. When germline, these mutations cause autosomal dominant hyperthyroidism or pseudoprecocious puberty of the male, respectively [13,41]. Somatic activating mutations of the TSH receptor constitute the main cause of autonomous thyroid adenomas [35], whereas somatic activating mutations of the LH/CG receptor cause Leydig cell tumors [27].

In higher primates, chorionic gonadotropin (with a beta subunit presenting 80% identity with beta LH) represents a challenge to the recognition specificity of GpHRs. The concentration of hCG achieved during the first trimester of pregnancy may be responsible for a "spillover" phenomenon. In most women, hCG concentrations reached around 12 weeks of pregnancy are enough to exert a slight thyrotropic effect [16]. In a single family a mutant TSH receptor displaying increased sensitivity to hCG has been demonstrated to be the cause of severe pregnancy-limited hyperthyroidism [37]. For a similar effect to manifest on the FSH receptor, exceptionally high concentrations of hCG must be achieved (as in trophoblastic disease or twin pregnancies), or the FSH receptor must become more promiscuous, secondary to activating mutations [43,51].

Pharmacology

The pharmacology of GpHRs is still very limited.

For the TSH receptor, monoclonal antibodies with stimulating or blocking activities have been produced in mouse (22;48-50), and a potent stimulating monoclonal has been isolated from a patient with Graves' disease [39]. Small non-peptide molecules with agonist or antagonist properties have just beginning to become available (Neumann S. et al 2005; Jaeschke H. et al 2005. Proc. XIII International Thyroid Conference, Buenos Aires. Abstracts 20 & 55). They derive from agents developed initially as agonists of the LH/CG receptor [49]. Preliminary data indicate that their site of action lies within the serpentine portion of the receptor, which makes it likely that they function as allosteric modulators.

The new, non-peptide, low molecular weight agonists for the LH receptor were shown to be active in cells transfected with the LH receptor, in gonadal cell models, and to be orally active in rats [49]. Low molecular weight LH receptor antagonists have not been reported yet.

The dimeric nature of the GpHRs has recently been associated with their displaying negative cooperativity [48]. All three members show strong acceleration of dissociation of a bound tracer hormone, upon dilution in the presence of excess cold hormone. This observation was already made in the 70's, before the structures of the receptors were known. The functional correlates of the allosteric behavior of GpHRs remain to be established, in the novel context of the possibility of heterodimerization, within the family (for FSH and LH/CG receptors), or with non GpHRs.

As revealed by the structure of hormone-ectodomain complex, the FSH receptor extracellular domain functions in a one to one stoichiometry of binding with hormone [15]. It is not yet known if the FSH receptor exists as a dimer in cell membranes. It is also not understood how the occupied exodomain activates the receptor transmembrane domains to undergo a conformation change which results in signaling. There are splice variants of the FSHR that have been reported and have unique (non-G-protein coupled) signaling properties [3]. Human FSH ligand must be heterodimeric to bind to FSH receptor, and its pharmacology is affected greatly by the carbohydrate complement [5]. Although most of this effect is concerned with circulatory half-life and heterodimer stability, carbohydrate also has a role in signaling at the level of the receptor [29]. Currently efforts are underway to develop small molecule non-steroidal drugs which act at the FSHR [34].

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