A single residue, aspartic acid 95, in the delta 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 delta, kappa, and mu. To investigate mechanisms by which agonists and antagonists interact with the delta opioid receptor, we have substituted aspartic acid 95 in the transmembrane segment 2 of the cloned mouse delta opioid receptor with an asparagine (D95N). The D95N mutant receptor had reduced affinity for delta 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 delta-selective non-peptide agonists was also reduced. In contrast, the delta 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 delta, kappa, and mu opioid receptors, showed no difference in binding to the wild-type and mutant delta 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 delta receptor and show that Asp-95 contributes to high affinity delta-selective agonist binding. The identification of a key residue involved in selective agonist binding to the delta opioid receptor will facilitate the development of novel therapeutic reagents that can be used for the treatment of chronic pain and other conditions.