OBJECTIVE To identify and characterize novel intracellular signal molecules which specially mediated the phosphorylation and desensitization of MOR.METHODS Using a bacterial two-hybrid screen, we observed that the carboxyl tail of the rat MOR associates with A20-binding inhibitor of nuclear factor (NF)-κB (ABIN-1).RESULTS This interaction was confirmed by direct protein-protein binding and the coimmunoprecipitation of MOR and ABIN-1 proteins from cell lysates.Saturation binding studies showed that ABIN-1 had no effect on MOR binding.However, D-ala2, N-Me-Phe4-Gly-ol5enkephalin (DAMGO)-induced MOR internalization, phosphorylation, and ubiquitination were decreased in Chinese hamster ovary cells that coexpressed MOR and ABIN-1.The interaction of ABIN-1 and MOR also inhibited the activation of G-protein and the suppression of forskolin-stimulated adenylyl cyclase induced by DAMGO.Furthermore, morphine-induced dependence was almost abolished by the coexpression of ABIN-1, based on observed calcium levels.Additionally, the protein and mRNA levels of ABIN-1 were increased in chronic morphine-treated rat brain and primary neurons of the rat brain prefrontal cortex.CONCLUSION These data suggest that ABIN-1 is a negative regulator of MOR activation,phosphorylation, desensitization, and opioid dependence.