Functional inactivation of the nociceptin receptor by alanine substitution of glutamine 286 at the C terminus of transmembrane segment VI: evidence from a site-directed mutagenesis study of the ORL1 receptor transmembrane-binding domain.

A site-directed mutagenesis approach has been used to gain insight into the molecular events whereby the heptadecapeptide nociceptin binds and activates the opioid receptor-like 1 (ORL1) receptor, a G protein-coupled receptor. Alanine mutation, in the human ORL1 receptor, of transmembrane amino acid residues that are conserved in opioid receptors, Asp(130) and Tyr(131) in transmembrane segment (TM) III, Phe(220) and Phe(224) in TM V, and Trp(276) in TM VI, yields mutant receptors with reduced affinity, and proportionally decreased reactivity, toward nociceptin. Least to most deleterious in this respect are Ala substitutions of Phe(220) approximately W276A < Tyr(131) << Phe(224) 10,000 nM compared with 0.8 nM at the wild-type receptor). In all respects, this mutant receptor appears to be functionally inactive, indicating that residue Gln(286) may play a pivotal role in ORL1 receptor-mediated transduction of the nociceptin signal.