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.
Awara
W M
WM
Oregon Regional Primate Research Center, Beaverton 97006, USA.
Guo
C H
CH
Conn
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics*; D002064:Buserelin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation; D007295:Inositol Phosphates / Q000096:biosynthesis; D051379:Mice; D010911:Pituitary Neoplasms / Q000378:metabolism / Q000473:pathology; D017354:Point Mutation; D011388:Prolactin / Q000378:metabolism; D051381:Rats; D011966:Receptors, LHRH / Q000235:genetics* / Q000502:physiology*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D007295:Inositol Phosphates; D011966:Receptors, LHRH; D001224:Aspartic Acid; D001216:Asparagine; D011388:Prolactin; D000242:Cyclic AMP; D002064:Buserelin