Background and Purpose: β-arrestin2 plays an important role in opioid receptor signaling, but its involvement in morphine- and fentanyl-induced respiratory depression is widely debated. The aim of this study was to determine whether β-arrestin2 signaling is associated with fentanyl- and morphine-induced respiratory depression. Experimental Approach: This study investigated whether β-arrestin2 is involved in respiratory depression induced by fentanyl or morphine by inhibiting the upstream signaling molecule GRK2 and knocking out β-arrestin2 in mice, using whole-body plethysmography chambers to assess changes in respiratory function. Key Results: In the experiment of inhibiting GRK molecules, GRK inhibitors significantly improved the respiratory depression induced by morphine, but had no effect on fentanyl. In experiment of knocking out β-arrestin2, respiratory depression was significantly improved in the morphine group, but less affected in the fentanyl group. Conclusion and Implications: Our results suggested that inhibition of β-arrestin2 signaling alleviated morphine-induced respiratory depression but had little effect on fentanyl-induced respiratory depression in both models, suggesting differences in the respiratory depression mechanisms between fentanyl and morphine. This suggests that we may need to give a differentiated dosing regimen in clinical treatment of respiratory depression caused by the two drugs.

Background and Purpose The development of biased agonism provides a promising avenue to improve the pharmacological properties of fentanyl derivatives, but the molecular mechanism underlying ligand bias still remains ambiguous. Therefore, we sought to find out the critical sites of μ-receptor governing ligand bias and clarify corresponding molecular mechanism for designing and synthesizing effective analgesics with reduced adverse effects. Experimental Approach Critical sites governing ligand bias were identified both by computational prediction and cell assay-based bias analysis on wild-type and site-directed mutant μ-opioid receptor. Then molecular dynamics simulations of wild-type and mutant μ-opioid receptor were conducted to investigate the mechanism of bias activation. Key Results D3.32A and H6.52L mutation disrupted the binding of fentanyl derivatives with μ-opioid receptor. W6.48L mutation drove most fentanyl derivatives to β-arrestin-bias but promote sufentanil to cAMP signaling-bias. The result of molecular dynamics simulation showed that W6.48 and Y7.43 were paired activation switches of ligand bias at μ-opioid receptor. Conclusion and Implications D3.32 and H6.52 were critical residues in driving morphine and fentanyl derivatives to bind with μ-opioid receptor. W6.48 was a pivotal residue in governing the bias signaling and the interactions of ligands with W6.48 and Y7.43 were the structural determinants for the signaling bias of μ-opioid receptor, which will be conducive for better design and synthesis of effective opioid analgesics with the reduced adverse effects.

Background: While opioids play a crucial role in pain’s relief, chronic exposure results in tolerance and dependence. Efforts should be made to alleviate the side effect induced by opioids. Many proteins which functionally interact with MOR can regulate the effect of opioids. Our bacterial yeast two-hybrid experiment showed ABIN-1 could bind to MOR. Here, we studied the profile and mechanism of ABIN-1 on morphine tolerance and dependence. Experimental Approach: ABIN-1 in mouse brain was interfered by AAV virus. The tolerance and dependence induced by morphine were assessed in hotplate and conditioned place preference test. The regulation of β-arrestin signalling of MOR was observed in MOR-CHO cell lines after ABIN-1 overexpression. The interaction of proteins was detected by co-immunoprecipitation and immunofluorescence. The expression of proteins was tested by western blotting and immunohistochemistry. Key Results: Morphine tolerance and dependence were attenuated by overexpression of ABIN-1 in mouse brains. ABIN-1 in the hippocampus and nucleus accumbens participated in morphine tolerance and physical dependence. MOR phosphorylation and internalization were weakened by ABIN-1 after opioids treatment. Formation of ABIN-1-β-arrestin-2 complexes promoted the translocation of β-arrestin-2 to the plasma membrane and accelerated its ubiquitination and degradation. Furthermore, attenuation of morphine tolerance by ABIN-1 was abolished in β-arrestin-2 knockout mice. Conclusions and Implications: These findings indicate that ABIN-1 co-operates with β-arrestin2 and MOR to alleviate morphine tolerance and dependence. ABIN-1 may be a target to alleviate morphine tolerance.