A RECIPROCAL MUTATION SUPPORTS HELIX-2 AND HELIX-7 PROXIMITY IN THE GONADOTROPIN-RELEASING-HORMONE RECEPTOR

Activation of the pituitary gonadotropin-releasing hormone receptor, a member of the seven-transmembrane G protein-coupled receptor (GPCR) f amily, triggers a cascade of events leading to gonadotropin release an d stimulation of the reproductive system. An unusual feature of this r eceptor, observed in mice, rats, and humans, is the presence of Asn(87 ) in the second putative transmembrane helix at the location of a high ly conserved asparate in the GPCR family and of Asp(318) in the putati ve seventh transmembrane helix where nearly all other GPCRs have aspar agine. The possibility that these residues interact was suggested by t his reciprocal pattern and by a three-dimensional model of the gonadot ropin-releasing hormone receptor and was investigated by site-directed mutagenesis. Replacing Asn(87) in the second transmembrane domain by aspartate eliminated detectable ligand binding. A second mutation, gen erating the double-mutant receptor Asp(87) Asn(318), recreated the arr angement found in other GPCRs and re-established high affinity agonist and antagonist binding. The restoration of binding by a reciprocal mu tation indicates that these two specific residues in helices 2 and 7 a re adjacent in space and provides an empirical basis to refine the mod el of the transmembrane helix bundle of the receptor.