Direct binding of β-arrestins to two distinct intracellular domains of the δ opioid receptor

β-Arrestins regulate opioid receptor-mediated signal transduction and play an important role in opiate-induced analgesia and tolerance/dependence. This study was carried out to measure the direct interaction between β-arrestins and opioid receptor. Immunoprecipitation experiments demonstrated that β-arrestin 1 physically interacts with delta opioid receptor (DOR) co-expressed in human embryonic kidney 293 cells in an agonist-enhanced manner and truncation of the carboxyl terminus of DOR partially impairs the interaction. In vitro data from glutathione-S-transferase pull-down assay showed that the carboxyl terminus (CT) and the third intracellular loop (13L) of DOR are both capable of and either domain is sufficient for binding to β-arrestin 1 and 2. Surface plasmon resonance determination further revealed that binding of CT and 13L of DOR to β-arrestin is additive, suggesting these two domains bind at distinctly different sites on β-arrestin without considerable spatial hindrance. This study demonstrated for the first time the direct binding of β-arrestins to the two distinct domains, the carboxyl terminus and the third intracellular loop, of DOR.