The structure-function properties and signaling pathways of receptors for gonadotropin-releasing hormone (GnRH) were analyzed in pituitary gonadotrophs, hypothalamic neurons, and immortalized GnRH neurons (GT1 cells). The GnRH receptor is a unique member of the G protein-coupled receptor (GPCR) family with several unusual features, including the absence of a C-terminal cytoplasmic domain. Another is a variant (DRS) of the conserved DRY sequence that is located in the third transmembrane domain of most GPCRs. A mutational analysis of the DRS motif of the mouse GnRH receptor demonstrated that the conserved asparagine and arginine residues make important contributions to the structural integrity of the receptor, and also influence receptor expression, agonist-induced activation, and internalization of the receptor. A concomitant analysis of polar amino acid residues in the C-terminal region of the second intracellular loop showed that alanine substitutions caused no change in receptor signaling, but significantly increased receptor internalization. The cloned gene of the rat GnRH receptor was found to span about 20 kb and to contain three exons that encode the receptor protein and its flanking regions. The two introns are located in the middle of the fourth transmembrane domain and between the fifth and six transmembrane domains. The 1.8 kb 5' flanking sequence contains several transcriptional consensus elements, including SF-1, AP-1, CRE, and Pit-1 sites. Expression of reporter constructs in GnRH receptor-expressing cell lines revealed that cell-specific promoter activity was maximum in a 1.2 kb sequence of the 5' region, and was increased by activation of protein kinase A but not protein kinase C. Studies on GnRH-induced signaling in immortalized GnRH-secreting neurons revealed that phospholipase D participates in the mechanism whereby the electrical activity of the GnRH neurons promotes episodic neuropeptide release. The GnRH neuron was also found to be regulated by cholinergic agents which modulate GnRH release from hypothalamic and GT1 cells by acting on specific muscarinic receptor subtypes that are coupled to Gq and Gi proteins. These in turn influence the activities of phospholipase C, adenylyl cyclase, and ion channels, with consequent effects on neurosecretion. The endogenous GnRH receptors of the GnRH neuron are also coupled to Gs and Gi proteins, as well as Gq, and exert both stimulatory and inhibitory actions on adenylyl cyclase signaling according to the prevailing agonist concentration. A further analysis of the autocrine actions of ET-1 in ovarian carcinoma cells indicated that protein kinase C participates in ET-stimulated mitogenic responses. In addition, the rapid tyrosine phosphorylation of several proteins, including p125FAK and p42 MAP kinase, suggests that intracellular signaling between the ETA receptor and a yet unidentified tyrosine kinase is involved in the mitogenic response to ET-1.
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