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 whi ch function as neurotransmitters, neuromodulators, and hormones and ar e involved in the perception of pain, modulation of behavior, and regu lation of autonomic and neuroendocrine function. Pharmacological studi es have defined three classes of opioid receptors, designated as delta , kappa, and mu. To investigate mechanisms by which agonists and antag onists interact with the delta opioid receptor, we have substituted as partic acid 95 in the transmembrane segment 2 of the cloned mouse delt a opioid receptor with an asparagine (D95N). The D95N mutant receptor had reduced affinity for delta receptor-selective agonists such as enk ephalin, [D-Pen2,D-Pen5]enkephalin and [D-Ser2,Leu5]enkephalin-Thr6 su ch that it did not bind these peptides even at micromolar concentratio ns. The binding of delta-selective non-peptide agonists was also reduc ed. In contrast, the delta receptor-selective antagonists, such as nal trindole, the benzofuran analog of naltrindole, and 7-benyllidenenaltr exone, bound equally well to the wild-type and mutant receptor. Simila rly, non-selective opioid agonists such as bremazocine and buprenorphi ne, which interact with delta, kappa, and mu opioid receptors, showed no difference in binding to the wild-type and mutant delta receptor. T he D95N mutant remained coupled to G proteins, and the receptor was fu nctionally active since it mediated agonist inhibition of cAMP accumul ation. These results indicate that selective agonists and antagonists bind differently to the delta receptor and show that Asp-95 contribute s to high affinity delta-selective agonist binding. The identification of a key residue involved in selective agonist binding to the delta o pioid receptor will facilitate the development of novel therapeutic re agents that can be used for the treatment of chronic pain and other co nditions.