Beta-arrestin2 and c-Src regulate the constitutive activity and recycling of mu 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. A lack of beta-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 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 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.
Walwyn
Wendy
W
Department of Psychiatry and Biobehavioral Sciences, Hatos Center, University of California, Los Angeles, Los Angeles, California 90095, USA.
Evans
Christopher J
CJ
Hales
Tim G
TG
D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D000081247:CSK Tyrosine-Protein Kinase; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D005260:Female; D005727:Ganglia, Spinal / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009475:Neurons, Afferent / Q000187:drug effects / Q000378:metabolism*; D009619:Nociceptors / Q000187:drug effects / Q000378:metabolism; D010146:Pain / Q000378:metabolism / Q000503:physiopathology; D018408:Patch-Clamp Techniques; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D019061:src-Family Kinases
D000701:Analgesics, Opioid; C000605258:Arrb2 protein, mouse; D019390:Arrestins; D015220:Calcium Channels; D043562:Receptors, G-Protein-Coupled; D017450:Receptors, Opioid, mu; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases