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

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Abstract
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, Asp130 and Tyr131 in transmembrane segment (TM) III, Phe220 and Phe224 in TM V, and Trp276 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 Phe220 ∼ W276A < Tyr131 ≪ Phe224 ≤ Asp130. The dramatic effects of the D130A mutation on nociceptin binding and activity are not reversed in the D130N mutant, whereas those of the Y131A mutation are totally suppressed in Y131F. This suggests that a negative charge at position 130, and a phenyl ring at position 131 in TM III, are critical for occupancy and/or activation of the receptor by nociceptin. Alanine replacement of glutamine 286, located at the C terminus of TM VI, yields a mutant receptor that binds nociceptin with nearly the same affinity as does the wild-type receptor (Kdvalues of 0.13 and 0.22 nM, respectively) but, unlike the latter, is unable to mediate nociceptin inhibition of forskolin-induced cAMP synthesis in recombinant Chinese hamster ovary cells (ED50 > 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 Gln286 may play a pivotal role in ORL1 receptor-mediated transduction of the nociceptin signal.