Altered adenylyl cyclase responsiveness subsequent to point mutations of Asp 128 in the third transmembrane domain of the δ-opioid receptor

δ-Opioid receptors belong to the superfamily of G protein-coupled receptors, characterized by seven putative transmembrane domains, and have been shown to interact with a host of effector systems. It has been suggested that the charge on the conserved aspartic acid residue at position 128 in transmembrane domain 3 of the δ-opioid receptor contributes to both the conformation of the receptor binding pocket and the molecular rearrangements which accompany the establishment of high-affinity states of the receptor. In light of this, we used site-directed mutagenesis to determine whether this residue participates in the transmission of signals to adenylyl cyclase, the effector with which opioid receptors have been classically associated. Substitution of this aspartic acid (D128) for the neutral amino acid alanine, or the protonated amino acids lysine and histidine, constitutively couples the receptor to adenylyl cyclase, as evidenced by a curtailed response to forskolin stimulation in transfected cells. In addition, this constitutive activity can be blocked by pretreatment of the transfected cells with pertussis toxin. Interestingly, naloxone blocks this effect in cells expressing the D128A mutant, but acts as an agonist at the D128K mutant.Our findings support the hypothesis that the interaction between agonist and receptor promotes conformational changes that may be mimicked, at least in part, by mutation of the aspartate residue at position 128. Furthermore, these changes appear to be involved not only in receptor activation, but also in the functional discrimination between agonists and antagonists. Copyright (C) 1999 IBRO.