【Background】The basic principle of G-protein-coupled receptor signal involves G-protein-dependent and -independent, beta-arrestin-dependent pathways. Recent progress in structural study of chimeric beta-adrenaline receptor and beta-arrestin 2 complex proposed that the interaction went through two sequential steps. However, such steps in living cells and the practical application have not been provided, because these steps are difficult to analyze separately. Here, we report V1b vasopressin receptor as a valuable model to tackle this question.【Methods】Bioluminescent resonance energy transfer (BRET) between receptor-nanoluciferase and Venus-beta-arrestin 2 was employed to monitor interaction. Receptor dimers were further monitored by connecting two parts of split nanoluciferase to the receptor carboxyl terminus.【Results and Discussion】We detected two-step increases of BRET signal in the V1b receptor-beta-arrestin 2 interaction. Non-stimulated V1b receptor associated with beta-arrestin 2 and vasopressin further increased BRET. Heteromer formation between beta-arrestin 2-V1b receptor and mu-opioid receptor promoted morphine-induced analgesic tolerance in mice and in reconstituted cellular model. Our data indicates two-step interactions between V1b receptor and beta-arrestin 2 are the molecular target for alleviating morphine tolerance.