A single residue, aspartic acid 95, in the δ opioid receptor specifies selective high affinity agonist binding

The enkephalins, dynorphins, and endorphins are endogenous opioids which function as neurotransmitters, neuromodulators, and hormones and are involved in the perception of pain, modulation of behavior, and regulation of autonomic and neuroendocrine function. Pharmacological studies have defined three classes of opioid receptors, designated as δ, κ, and μ. To investigate mechanisms by which agonists and antagonists interact with the δ opioid receptor, we have substituted aspartic acid 95 in the transmembrane segment 2 of the cloned mouse δ opioid receptor with an asparagine (D95N). The D95N mutant receptor had reduced affinity for δ receptor-selective agonists such as enkephalin, [D-Pen²,D-Pen⁵]enkephalin and [D- Ser²,Leu⁵]enkephalin-Thr⁶ such that it did not bind these peptides even at micromolar concentrations. The binding of δ-selective non-peptide agonists was also reduced. In contrast, the δ receptor-selective antagonists, such as naltrindole, the benzofuran analog of naltrindole, and 7- benyllidenenaltrexone, bound equally well to the wild-type and mutant receptor. Similarly, non-selective opioid agonists such as bremazocine and buprenorphine, which interact with δ, κ, and μ opioid receptors, showed no difference in binding to the wild-type and mutant δ receptor. The D95N mutant remained coupled to G proteins, and the receptor was functionally active since it mediated agonist inhibition of cAMP accumulation. These results indicate that selective agonists and antagonists bind differently to the δ receptor and show that Asp-95 contributes to high affinity δ- selective agonist binding. The identification of a key residue involved in selective agonist binding to the δ opioid receptor will facilitate the development of novel therapeutic reagents that can be used for the treatment of chronic pain and other conditions.