A novel opioid for photoaffinity labeling opioid receptor complexes in natively‐expressing tissues

A novel radioiodinated opioid photoaffinity label bearing an aryl azide moiety was synthesized, permitting covalent attachment of the drug to its target following irradiation with UV light. In receptor binding assays using membranes from stably transfected cell lines, the drug demonstrated extremely high affinity for mu and kappa opioid receptors with high selectivity over delta receptors. The drug was readily incorporated into mu receptors, and autoradiography following denaturing polyacrylamide gel electrophoresis (SDS‐PAGE) revealed a specific band corresponding to the mature, glycosylated form of the receptor which was fully competed by levallorphan (1uM). Photoaffinity labeling of mouse brain and human neuroblastoma cell membranes followed by immunoprecipitation also revealed specific bands corresponding to the mature forms of the receptor in these tissues. The molecular weight of the receptor differed between mouse brain and cell line membranes, consistent with differing glycosylation state of the receptor. Photolabeling the receptor in natively‐expressing tissue also facilitated the optimization of a purification scheme under native conditions to isolate opioid receptor signaling complexes which were resolved using Blue Native‐PAGE and could be further separated by a second dimension of SDS‐PAGE.