INVOLVEMENT OF TRP-284, VAL-296, AND VAL-297 OF THE HUMAN DELTA-OPIOID RECEPTOR IN BINDING OF DELTA-SELECTIVE LIGANDS

Given the high homology in amino acid sequence between the delta-opioi d receptor and the two other types (mu and kappa), distinct residues i n this receptor may confer its selectivity to some ligands, In order t o identify molecular determinants in the human delta receptor responsi ble for the selectivity of delta-selective ligands, two different delt a/mu chimeras were constructed, In the first one, the delta sequence f rom the top of transmembrane 5 to the C terminus was replaced by the e quivalent Ir sequence, and in the second one, 13 consecutive residues in the third extracellular loop region of the delta receptor were repl aced by the mu counterpart, These two chimeras retained the ability to bind the nonselective bremazocine but completely lost the ability to bind different delta-selective ligands, These results suggested that t he region of the third extracellular loop of the delta receptor is cru cial for the type selectivity, Furthermore, an alanine scan was perfor med by site-directed mutagenesis of 20 amino acids located in or proxi mal to the third extracellular loop, Among all the point mutations, on ly mutations of Trp-284, Val-296, or Val-297 significantly decreased t he binding of delta-selective ligands tested, Moreover, combined mutat ion of Trp-284, Val-296, and Val-297 considerably decreased the affini ties of the receptor for delta-selective ligands compared with the sin gle point mutations, These findings suggest that Trp-284, Val-296, and Val-297 are crucial residues involved in the delta receptor type sele ctivity.