β-Arrestin2 and c-Src regulate the constitutive activity and recycling of μ opioid receptors in dorsal root ganglion neurons

beta-Arrestins bind to agonist-activated G-protein-coupled receptors regulating signaling events and initiating endocytosis. In beta-arrestin2(-/-) (beta arr2(-/-)) mice, a complex phenotype is observed that includes altered sensitivity to morphine. However, little is known of how beta-arrestin2 affects mu receptor signaling. We investigated the coupling of mu receptors to voltage-gated Ca2+ channels ( VGCCs) in beta-arr2(+)/(+) and beta arr2(-/-) dorsal root ganglion neurons. Alack of beta-arrestin2 reduced the maximum inhibition of VGCCs by morphine and DAMGO (D-Ala(2)-N-Me-Phe(4)-glycol(5)-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 mu receptors to VGCCs. Agonist-independent mu 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 mu receptor expression in mu beta arr2(-/-) compared with beta arr2(-/-) neurons. Such changes could not be explained by the classical role of beta-arrestins in agonist-induced endocytosis. The localization of the nonreceptor tyrosine kinase c-Src appeared disrupted in beta arr2(-/-) 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 beta arr2(-/-) cellular phenotype of reduced mu agonist efficacy, increased constitutive mu receptor activity, and reduced constitutive recycling. We propose that beta-arrestin2 is required to target c-Src to constitutively active mu receptors, resulting in their internalization, providing another dimension to the complex role of beta-arrestin2 and c-Src in G-protein-coupled receptor function.