A SINGLE RESIDUE, LYS108, OF THE DELTA-OPIOID RECEPTOR PREVENTS THE MU-OPIOID-SELECTIVE LIGAND [D-ALA(2),N-MEPHE(4),GLY-OL(5)]ENKEPHALIN FROM BINDING TO THE DELTA-OPIOID RECEPTOR

Previously, we found that replacement of the region around the first e xtracellular loop of the delta-opioid receptor (OPR) with the correspo nding region of the mu-OPR gives the high affinity for [D-Ala(2),N-MeP he(4),Gly-ol(5)]enkephalin (DAMGO), a mu-opioid-selective ligand, to t he resultant chimeric receptor, DMDD, suggesting that the difference i n the amino acid sequence within this region between the mu- and delta -OPRs is critical for the discrimination between these receptors by DA MGO. In the current study, we carried out systematic replacements of s even nonconserved residues in this region of the delta-OPR with the co rresponding amino acid found in the mu-OPR. Among the seven mutant rec eptors, only one mutant receptor, delta K108N, showed high affinity (K -i = 18.68 +/- 5.27 nM) for DAMGO, which was comparable to that of the DMDD receptor (K-i = 23.77 +/- 4.27 nM) and 75-fold higher than that of the wild-type delta-OPR (K-i = 1405 +/- 161 nM). Lys108 in the delt a-OPR was systematically replaced with 19 kinds of amino acids other t han lysine. Among the resultant mutant receptors, 14 mutants bound DAM GO with K-i values comparable to those of the DMDD receptor, ranging f rom 4.20 to 43.38 nM. These findings suggest that Lysl08 of the delta- OPR prevents DAMGO from binding to the delta-OPR rather than that the asparagine residue at the corresponding position in the mu-OPR is nece ssary for DAMGO binding. In addition, the replacement of Lysl08 of the delta-OPR with asparagine dramatically increased the affinity for oth er peptidic mu receptor-selective ligands, such as dermorphin and D-Pe n-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2.