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) family, triggers a cascade of events leading to gonadotropin release and stimulation of the reproductive system. An unusual feature of this receptor, observed in mice, rats, and humans, is the presence of Asn⁸⁷ in the second putative transmembrane helix at the location of a highly conserved asparate in the GPCR family and of Asp³¹⁸ in the putative seventh transmembrane helix where nearly all other GPCRs have asparagine. The possibility that these residues interact was suggested by this reciprocal pattern and by a three- dimensional model of the gonadotropin-releasing hormone receptor and was investigated by site-directed mutagenesis. Replacing Asn⁸⁷ in the second transmembrane domain by aspartate eliminated detectable ligand binding. A second mutation, generating the double-mutant receptor Asp⁸⁷ Asn³¹⁸, recreated the arrangement found in other GPCRs and re-established high affinity agonist and antagonist binding. The restoration of binding by a reciprocal mutation indicates that these two specific residues in helices 2 and 7 are adjacent in space and provides an empirical basis to refine the model of the transmembrane helix bundle of the receptor.