Vol. 53, Issue 4, 772-777, April 1998

Creating a Functional Opioid Alkaloid Binding Site in the Orphanin FQ Receptor through Site-Directed Mutagenesis

Fan Meng, Yasuko Ueda, Mary T. Hoversten, Larry P. Taylor, Rainer K. Reinscheid, Frederick J. Monsma, Stanley J. Watson, Olivier Civelli, and Huda Akil

Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109 (F.M., Y.U., M.T.H., L.P.T., S.J.W., H.A.) and Central Nervous System Research, Pharma Division, Hoffmann-La Roche AG, CH-4070 Basel, Switzerland (R.K.R., F.J.M., O.C.)

Although much has been learned about the mechanisms of ligand selectivity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The recently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid ligands. In the current study, we adopt a "gain of function" approach aimed at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, several orphanin FQ receptor mutants can be labeled with the opiate alkaloid [³H]naltrindole and show greatly increased affinities toward the opiate antagonists naltrexone, nor-binaltrophine HCl, and ()-bremazocine. These orphanin FQ receptor mutants also display stereospecificity similar to that of opioid receptors. Furthermore, the orphanin FQ receptor mutant that has the best affinities toward the opioid alkaloids shows, in the presence of GTP and high salt concentration, an affinity-shift profile similar to that of the  receptor. Most strikingly, the same mutant exhibits naltrindole-sensitive etorphine-stimulated [³⁵S]guanosine-5'-O-(3-thio)triphosphate binding, whereas the effect of etorphine on GTP binding cannot be inhibited by naltrindole in the wild-type receptor. Our results indicate that 1) several residues in the orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues to those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact with the mutant receptor. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a functional common binding pocket for opiate alkaloids. These findings may be helpful to medicinal chemists in designing ligands for the orphanin FQ receptor based on the structure of the opiate alkaloids.