Selectivity of mu-opioid receptor determined by interfacial residues near third extracellular loop

We hypothesized that the selectivity profile of the rat mu-opioid receptor for opioid receptor-selective ligands is determined by the nature of the am ino acid residues at highly divergent sites in the ligand-binding pocket. T o determine which characteristics of these residues contribute to opioid re ceptor ligand selectivity, we made various mutant receptors that replaced t he Lys(303) and Trp(315) residues near the extracellular interface of trans membrane domains VI and VII, respectively. Ligand binding determinations us ing transiently transfected monkey kidney epithelial (COS-1) cells show tha t Lys(303) mutations cause little change in the receptor binding profile, w hereas the Trp(318) mutant receptors have considerably lower affinity for m u-opioid receptor-selective ligands and greatly increased affinity for delt a-opioid receptor-selective ligands. The nature of these mutations show tha t this effect is not due to sterics or charge alone. [S-35]guanosine-5'-O-( 3-thio)-triphosphate ([S-35]GTP gamma S) activity assays show that these re sidues may influence functional, as well as binding selection. We conclude that a primary role for Trp(318) is to form a basis for ligand selectivity. (C) 2000 Elsevier Science B.V. All rights reserved.