Gonadotropin-releasing hormone (GnRH) regulates reproduction by binding and activating GnRH receptors on pituitary gonadotrope cells, which synthesize and secrete the gonadotropins, LH, and FSH. The gonadotropins act on the gonads to stimulate gametogenesis, gonadal cell proliferation, and production of the gonadal steroids. GnRH secretion is suppressed during childhood and increases at puberty, when increased production of gonadotropins and gonadal steroids initiate sexual development. Disruption of GnRH receptor function disrupts reproduction and mutations of the GnRH receptor gene disrupt or delay pubertal development, resulting in congenital hypogonadotropic hypogonadism (cHH). This central role in regulation of reproduction has made the GnRH receptor a target for treatment of infertility and of sex steroid-dependent hyperplasias, including uterine fibroids, endometriosis and prostatic cancer, where gonadal steroid production is decreased by administration of GnRH antagonists or high doses of GnRH agonists, which downregulate receptor expression. Agonist binding to the GnRH receptor activates the G q/11 family of heterotrimeric G proteins. Activated GTP-bound Gα q/11 subunits activate phospholipase Cβ, which catalyzes production of the second messengers diacylglycerol and inositol trisphosphate, which initiate the cellular signaling pathways that culminate in gonadotropin synthesis and secretion. Although the GnRH receptor is also reported to transiently activate G s proteins in the LβT2 gonadotrope cell line and inhibit cell growth via the inhibitory G i proteins, no direct GnRH receptor activation of Gα i or Gα s could be shown in a range of cell lines and it has been proposed that GnRH-stimulated activation of G i or G s proteins may be downstream of activation of the G q/11 proteins. The mammalian (type 1) GnRH receptor does not activate β-arrestin-dependent signaling, suggesting that all effects of the GnRH receptor may be mediated by activation of G q/11 proteins.
The GnRH receptor belongs to the G protein-coupled receptor (GPCR) family, which constitutes the largest family of membrane proteins in the human genome. The GPCRs regulate physiological systems ranging from vision and olfaction through neurotransmission and immunology in addition to endocrine systems. Physiological ligands that activate GPCRs range from cations (Ca 2+), small molecule neurotransmitters and immune modulators to peptide and protein hormones, cytokines and even light, which changes the 11-cis-retinal prosthetic group of rhodopsin from a covalently bound inverse agonist (an antagonist that actively stabilizes inactive receptor conformations) to an agonist. In spite of their diverse physiological functions and ligands, all GPCRs share a common molecular function, which consists of transducing an extracellular signal across a biological membrane via a change in receptor protein conformation. This conserved function is supported by a conserved protein structure that consists of an extracellular amino-terminus, a bundle of seven membrane-spanning α-helical segments connected by three intracellular and three extracellular loops and a cytoplasmic carboxy-terminus. No crystal structure of the GnRH receptor has yet been reported, but much can be learned about its structure and how it conveys the extracellular GnRH-binding signal to intracellular signaling pathways by studying the structures of related GPCRs that have been crystallized and combining this with biochemical studies. This review will focus on understanding of the structure of the GnRH receptor and ligand binding that has arisen since the last major review with emphasis on the application of recently described GPCR structures and how these may inform mechanisms of GnRH receptor structure, activation and ligand binding.
Based on conserved amino acid sequence features, the GnRH receptor is a class A GPCR. Class A is the largest and best-studied class of GPCR proteins and includes rhodopsin, adrenergic and other monoamine neurotransmitter receptors and many peptide and protein-binding receptors. The membrane-spanning segments of GPCRs are most conserved, whereas the loops and termini are more variable. To facilitate comparison of amino acid residues of the GnRH receptor with equivalent residues of other class A GPCRs, the Ballesteros and Weinstein numbering system will be used. Residues are numbered relative to the most conserved residue in each transmembrane (TM) segment, which is designated .50, preceded by the TM segment number and followed, where relevant, by the amino acid sequence number in the receptor in parenthesis. For example Asp 319 of the human GnRH receptor is designated Asp 7.49(319), because it immediately precedes the most conserved residue in TM7, Pro 7.50(320). The equivalent residue of the mouse receptor is Asp 7.49(318).
Table 1. Highly conserved amino acid residues and motifs in class A GPCRs and equivalent residues in type 1 and type 2 GnRH receptors.
This review will focus on the mammalian type 1 GnRH receptor, which is characterized by absence of a cytoplasmic carboxy-terminal tail that accounts for the lack of arrestin-dependent desensitization, internalization, and signaling. Many systems of nomenclature have been used for GnRH receptor subtypes, largely because of the unclear relationship between the tailless mammalian receptors and the other GnRH receptors, all of which have carboxy-terminal tails. The discovery that some lower vertebrates have tailless GnRH receptors that are structurally and functionally similar to mammalian receptors has now provided some consensus. All of the tailless GnRH receptors are designated type 1 and all of the tailed GnRH receptors, type 2.
Human GnRH receptors have all of the highly conserved Ballesteros and Weinstein reference residues, except for the acidic Asp 2.50 in TM2, which is substituted with uncharged Asn. Mutation of Asn 2.50(87) to the normal Asp disrupted GnRH receptor expression, confirming the functional importance of the substitution. The type 1 GnRH receptors also have variations of the highly conserved amino acid sequence motifs. In TM7 the NPxxY motif (Asn 7.49-Pro 7.50-x-x-Tyr 7.53 where x represents any amino acid) is changed to DPxxY (Asp7.49-Pro 7.50-Leu 7.51-Ile 7.52-Tyr 7.53). Mutation of Asp 7.49 to Asn reversed the disruption of GnRH receptor expression caused by mutation of Asn 2.50 to Asp, suggesting these resides might be close to each other in the three-dimensional structures of class A GPCRs. The CWxPY motif in TM6 is preserved as Cys 6.47-Trp 6.48-Thr 6.49-Pro 6.50-Tyr 6.51, whereas the DRY motif at the cytosolic end of TM3 is DRS (Asp 3.49-Arg 3.50-Ser 3.51).
Type 1 GnRH receptors have a Glu 2.53(90) residue in TM2, which has risen to prominence because a cHH-associated Glu 2.53(90)Lys mutation disrupts membrane expression of the receptor, but treatment with a pharmacoperone [small-molecule membrane-permeable GnRH receptor antagonists that act as templates for folding of nascent receptor proteins] rescues expression of the mutant receptor, both in vitro and in knock-in transgenic mice. In other class A GPCRs the equivalent residue is mostly large and hydrophobic (Leu, Val, or Phe) and is Ile 2.53, Val 2.53, or Met 2.53 in type 2 GnRH receptors, suggesting that the carboxyl side chain of Glu 2.53(90) may not be required.
The functional importance of the highly conserved Tyr 5.58 residue was revealed by crystal structures of active rhodopsin. Type 1 GnRH receptors have Asn 5.58, but all tailed GnRH receptors have the conserved Tyr 5.58. In most class A GPCRs a conserved large aliphatic amino acid, Ile 3.40, forms part of a group of conserved residues referred to as the “core triad” or “transmission switch”, which changes configuration during receptor activation. GnRH receptors have a small Ala 3.40(129) residue, which is also present in type 2 GnRH receptors.
Ligands interact with the variable extracellular half of GPCR molecules. The membrane-spanning domain conveys the
