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 cloned opioid receptors stably expressed in Chinese hamster ovary cells was characterized, SUPERFIT inhibited [H-3]diprenorphine binding with much higher affinity for the delta than the mu or kappa receptor, Pretreatment with SUPERFIT followed by extensive washing reduced delta binding with an IC50 value of 7.1 nM, yet it did not affect mu and kappa binding up to 0.1 mu M. The reduction in delta binding by SUPERFIT pretreatment was due to a decrease in B-max with no change in K-d. These results indicate that SUPERFIT is a highly selective delta irreversible ligand, We then determined the region in the delta receptor that confered 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 to 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]diprenorphine binding to delta, mu/delta 3, delta/mu 3, and delta/delta 4 but affected binding to mu, delta/mu 1, delta/mu 3, and mu/delta 4 only at much higher concentrations, Thus, the segment from the beginning of the first intracellular loop to the middle of the third transmembrane helix of the delta receptor is important for selective binding of SUPERFIT.