CREATING A FUNCTIONAL OPIOID ALKALOID BINDING-SITE IN THE ORPHANIN FQRECEPTOR THROUGH SITE-DIRECTED MUTAGENESIS

Although much has been learned about the mechanisms of ligand selectiv ity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The r ecently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid liga nds. In the current study, we adopt a ''gain of function'' approach ai med at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, severa l orphanin FQ receptor mutants can be labeled with the opiate alkaloid [H-3]naltrindole and show greatly increased affinities toward the opi ate antagonists naltrexone, nor-binaltrophine HCl, and (-)-bremazocine . These orphanin FQ receptor mutants also display stereospecificity si milar to that of opioid receptors. Furthermore, the orphanin FQ recept or mutant that has the best affinities toward the opioid alkaloids sho ws, in the presence of GTP and high salt concentration, an affinity-sh ift profile similar to that of the delta receptor. Most strikingly, th e same mutant exhibits naltrindole-sensitive etorphine-stimulated [S-3 5]guanosine-5'-O-(3-thio)triphosphate binding, whereas the effect of e torphine on GTP binding cannot be inhibited by naltrindole in the wild -type receptor. Our results indicate that 1) several residues in the o rphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues t o those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact wi th the mutant receptor. It is reasonable to hypothesize that the corre sponding 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.