Effects of Asn318 and Asp87Asn318 Mutations on Signal Transduction by the Gonadotropin-Releasing Hormone Receptor and Receptor Regulation

GnRH receptor (GnRH-R) contains Asn⁸⁷ and Asp³¹⁸ instead of the more frequently observed Asp⁸⁷ and Asn³¹⁸ found in other G protein-coupled receptors. In the present study, site-directed mutagenesis was used to introduce Asn³¹⁸ and Asp⁸⁷Asn³¹⁸ into GnRH-R. The effect on coupling and regulation of GnRH-R was studied by stable expression of wild and mutant mouse GnRH-R in the lactotropic GH₃ cells; these normally release PRL in response to TRH stimulation. The responses to Buserelin (a metabolically stable GnRH analog) in three different cell lines, M1, N8, and ND1 (expressing wild-type, Asn³¹⁸ mutant, and Asp⁸⁷Asn³¹⁸ mutant mouse GnRH-R, respectively) were compared with that observed in the previously characterized GGH₃-1′ cells, which stably express rat GnRH-R. The Asn³¹⁸ and Asp⁸⁷Asn³¹⁸ mutations had no measurable effect on ligand binding, but abolished the initial down-regulation of receptor that was observed in M1 and GGH₃-1′ cells, suggesting that the normal location of Asn⁸⁷ and Asp³¹⁸ in GnRH-R is involved in the regulation of GnRH-R. In N8 and ND1 cells, Buserelin-stimulated inositol phosphate (IP) production was attenuated, but the release of both cAMP and PRL was stimulated in a dose- and time-dependent manner. These mutations apparently impaired the coupling between GnRH-R and G proteins involved in IP production, but not those involved in cAMP release. In M1 cells, Buserelin stimulation produced a significant increase in IP production, but neither cAMP nor PRL release was significantly stimulated. These findings are consistent with the previous suggestion that GnRH-stimulated PRL release is mediated by a cAMP second messenger system in transfected GGH₃ cells.