IRREVERSIBLE BINDING OF CIS-(-3-METHYLFENTANYL ISOTHIOCYANATE TO THE DELTA-OPIOID RECEPTOR AND DETERMINATION OF ITS BINDING DOMAIN())

Binding of cis-(+)-3-methylfentanyl isothiocyanate (SUPERFIT) to clone d opioid receptors stably expressed in Chinese hamster ovary cells was characterized, SUPERFIT inhibited [H-3]diprenorphine binding with muc h higher affinity for the delta than the mu or kappa receptor, Pretrea tment with SUPERFIT followed by extensive washing reduced delta bindin g with an IC50 value of 7.1 nM, yet it did not affect mu and kappa bin ding up to 0.1 mu M. The reduction in delta binding by SUPERFIT pretre atment was due to a decrease in B-max with no change in K-d. These res ults indicate that SUPERFIT is a highly selective delta irreversible l igand, We then determined the region in the delta receptor that confer ed binding selectivity for SUPERFIT by examining its binding to six mu /delta chimeric receptors, SUPERFIT bound to delta, mu/delta 1 (amino acids mu 1-94/delta 76-372), delta/mu 3 (delta 1-134/mu 154-398), and delta/mu 4 (delta 1-187/mu 207-398) receptors with high affinity but t o mu, delta/mu 1 (delta 1-75/mu 95-398), delta/mu 3 (delta 1-153/delta 135-372), and mu/delta 4 (mu 1-206/delta 188-372) receptors with low affinity, Pretreatment with SUPERFIT potently inhibited [3H]diprenorph ine binding to delta, mu/delta 3, delta/mu 3, and delta/delta 4 but af fected binding to mu, delta/mu 1, delta/mu 3, and mu/delta 4 only at m uch higher concentrations, Thus, the segment from the beginning of the first intracellular loop to the middle of the third transmembrane hel ix of the delta receptor is important for selective binding of SUPERFI T.