EFFECTS OF ASN(87) AND ASP(318) MUTATIONS ON LIGAND-BINDING AND SIGNAL-TRANSDUCTION IN THE RAT GNRH RECEPTOR

The gonadotrophin-releasing hormone (GnRH) receptor is unlike other G- protein coupled receptors in that the highly conserved amino acids, As p in the second transmembrane region and Asn in the seventh, are inter changed. Site-directed mutagenesis studies mutated these residues back to their normally conserved positions. Two single mutants Asn(87)Asp & Asp(318)Asn and one double mutant Asn(87)Asp Asp(318)Asn were transi ently expressed in COS-I cells and their effect on binding to GnRH and inositol phosphate production measured. The single mutant Asp(318)Asn had no effect on ligand binding but abolished GnRH-dependent inositol phosphate production, whereas mutations Asn(87)Asp and Asn(87)Asp Asp (318)Asn show a complete loss of GnRH binding and subsequent inactivat ion of its second messenger system.