β-Arrestin2 and c-Src Regulate the Constitutive Activity and Recycling of μ Opioid Receptors in Dorsal Root Ganglion Neurons

摘要

β-Arrestins bind to agonist-activated G-protein-coupled receptors regulating signaling events and initiating endocytosis. In β-arrestin2^−/− (βarr2^−/−) mice, a complex phenotype is observed that includes altered sensitivity to morphine. However, little is known of how β-arrestin2 affects μ receptor signaling. We investigated the coupling of μ receptors to voltage-gated Ca²⁺ channels (VGCCs) in βarr2^+/+ and βarr2^−/− dorsal root ganglion neurons. A lack of β-arrestin2 reduced the maximum inhibition of VGCCs by morphine and DAMGO (d-Ala²-N-Me-Phe⁴-glycol⁵-enkephalin) without affecting agonist potency, the onset of receptor desensitization, or the functional contribution of N-type VGCCs. The reduction in inhibition was accompanied by increased naltrexone-sensitive constitutive inhibitory coupling of μ receptors to VGCCs. Agonist-independent μ receptor inhibitory coupling was insensitive to CTAP (Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2), a neutral antagonist that inhibited the inverse agonist action of naltrexone. These functional changes were accompanied by diminished constitutive recycling and increased cell-surface μ receptor expression in βarr2^−/− compared with βarr2^+/+ neurons. Such changes could not be explained by the classical role of β-arrestins in agonist-induced endocytosis. The localization of the nonreceptor tyrosine kinase c-Src appeared disrupted in βarr2^−/− neurons, and there was reduced activation of c-Src by DAMGO. Using the Src inhibitor PP2 [4-amino-5-(4-chlorophenyl)-(t-butyl)pyrazolo[3,4-d]pyrimidine], we demonstrated that defective Src signaling mimics the βarr2^−/− cellular phenotype of reduced μ agonist efficacy, increased constitutive μ receptor activity, and reduced constitutive recycling. We propose that β-arrestin2 is required to target c-Src to constitutively active μ receptors, resulting in their internalization, providing another dimension to the complex role of β-arrestin2 and c-Src in G-protein-coupled receptor function.