Mammalian gonadotropin-releasing hormone (GnRH I: pGlu-His-TrpSer-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2) stimulates pituitary gonadotropin secretion, which in turn stimulates the gonads. Whereas a hypothalamic form of GnRH of variable structure (designated type I) had been shown to regulate reproduction through a cognate type I receptor, it has recently become evident that most vertebrates have one or two other forms of GnRH. One of these, designated type II GnRH (GnRH II: pGlu-His-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH 2), is conserved from fish to man and is widely distributed in the brain, suggesting important neuromodulatory functions such as regulating K ؉ channels and stimulating sexual arousal. We now report the cloning of a type II GnRH receptor from marmoset cDNA. The receptor has only 41% identity with the type I receptor and, unlike the type I receptor, has a carboxyl-terminal tail. The receptor is highly selective for GnRH II. As with the type I receptor, it couples to G ␣q/11 and also activates extracellular signal-regulated kinase (ERK1͞2) but differs in activating p38 mitogen activated protein (MAP) kinase. The type II receptor is more widely distributed than the type I receptor and is expressed throughout the brain, including areas associated with sexual arousal, and in diverse non-neural and reproductive tissues, suggesting a variety of functions. Surprisingly, the type II receptor is expressed in the majority of gonadotropes. The presence of two GnRH receptors in gonadotropes, together with the differences in their signaling, suggests different roles in gonadotrope functioning.

The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is the only G-protein-coupled receptor (GPCR) in which the intracellular C-terminal tail is completely absent. In contrast to other GPCRs, the GnRH-R does not show rapid desensitization of total inositol (IP) production, and the rates of internalization are exceptionally slow. We investigated whether the incorporation of a cytoplasmic tail into the C terminus of the GnRH-R affects desensitization events and receptor internalization rates. A GnRH-R/TRH-R chimera was created where the intracellular tail of the rat thyrotropin-releasing hormone receptor (TRH-R) was engineered into the C terminus of the rat GnRH-R. Three different rat GnRH-R cDNA stop codon mutations (one for each reading frame) were also made. The GnRHstimulated IP production of the wild-type rat GnRH-R expressed in either COS-7 or HEK 293 cells did not desensitize even after prolonged stimulation with GnRH. In contrast, the catfish GnRH-R (which does possess an intracellular tail) and the TRH-R rapidly (<10 min) desensitized following agonist stimulation. The GnRH-R/TRH-R chimera also desensitized following treatment with GnRH, resembling the pattern shown by the TRH-R and the catfish GnRH-R. Two of the stop codon mutants did not show desensitization of IP production, and the third mutant with the longest tail was not functional. Internalization experiments showed that the rat GnRH-R had the slowest endocytosis and recycling rates compared with the TRH-R, the catfish GnRH-R, and the chimeric GnRH/TRH-R. This study demonstrates that the addition of a functional intracellular C-terminal tail to the GnRH-R produces rapid desensitization of IP production and significantly increases internalization rates.Gonadotropin-releasing hormone (GnRH) 1 is a decapeptide released by the hypothalamus which acts on specific receptors in the anterior pituitary. GnRH and its receptor (GnRH-R) are key mediators of the regulation of the reproductive axis controlling the synthesis and release of the gonadotropins, luteinizing hormone, and follicle-stimulating hormone. The GnRH-R is a member of the large family of G-protein-coupled receptors (GPCRs) (1) and is preferentially coupled to phosphoinositidase C via the G q /G 11 family of G-proteins. Following stimulation by GnRH, the production of inositol phosphates (IPs) and diacylglycerol is induced resulting in the elevation of cytosolic calcium and the activation of protein kinase C (2-4). A number of GPCRs exhibit homologous desensitization upon prolonged stimulation with agonist. This involves uncoupling of G-proteins, rapid down-regulation of the receptor's effector systems such as total inositol phosphate (IP) production followed by receptor internalization, and presumably subsequent proteolytic degradation. In particular this has been demonstrated for the m3 muscarinic acetylcholine receptor (5) and the thyrotropin-releasing hormone receptor (TRH-R) (6 -9). The phosphorylation of specific amino acids in the cytoplasmic part of the C-terminal tail of s...

GnRH-II peptide hormone exhibits complete sequence conservation across vertebrate species, including man. Type-II GnRH receptor genes have been characterized recently in nonhuman primates, but the human receptor gene homolog contains a frameshift, a premature stop codon (UGA), and a 3' overlap of the RBM8A gene on chromosome 1q.12. A retrotransposed pseudogene, RBM8B, retains partial receptor sequence. In this study, bioinformatics show that the human receptor gene promoter overlaps the peroxisomal protein 11-beta gene promoter and the premature UGA is positionally conserved in chimpanzee. A CGA [arginine (Arg)] occurs in porcine DNA, but UGA is shifted one codon to the 5' direction in bovine DNA, suggesting independent evolution of premature stop codons. In contrast to marmoset tissue RNA, exon- and strand-specific probes are required to distinguish differently spliced human receptor gene transcripts in cell lines (HP75, IMR-32). RBM8B is not transcribed. Sequencing of cDNAs for spliced receptor mRNAs showed no evidence for alteration of the premature UGA by RNA editing, but alternative splicing circumvents the frameshift to encode a two-membrane-domain protein before this UGA. A stem-loop motif resembling a selenocysteine insertion sequence and a potential alternative translation initiation site might enable expression of further proteins involved in interactions within the GnRH system.