title: Inhibitory signaling by CB1 receptors in smooth muscle mediated by GRK5/β-arrestin activation of ERK1/2 and Src kinase.
abstract: We examined whether CB1 receptors in smooth muscle conform to the signaling pattern observed with other Gi-coupled receptors that stimulate contraction via two Gβγ-dependent pathways (PLC-β3 and phosphatidylinositol 3-kinase/integrin-linked kinase). Here we show that the anticipated Gβγ-dependent signaling was abrogated. Except for inhibition of adenylyl cyclase via Gαi, signaling resulted from Gβγ-independent phosphorylation of CB1 receptors by GRK5, recruitment of β-arrestin1/2, and activation of ERK1/2 and Src kinase. Neither uncoupling of CB1 receptors from Gi by pertussis toxin (PTx) or Gi minigene nor expression of a Gβγ-scavenging peptide had any effect on ERK1/2 activity. The latter was abolished in muscle cells expressing β-arrestin1/2 siRNA. CB1 receptor internalization and both ERK1/2 and Src kinase activities were abolished in cells expressing kinase-deficient GRK5(K215R). Activation of ERK1/2 and Src kinase endowed CB1 receptors with the ability to inhibit concurrent contractile activity. We identified a consensus sequence (102KSPSKLSP109) for phosphorylation of RGS4 by ERK1/2 and showed that expression of a RGS4 mutant lacking Ser103/Ser108 blocked the ability of anandamide to inhibit acetylcholine-mediated phosphoinositide hydrolysis or enhance Gαq:RGS4 association and inactivation of Gαq. Activation of Src kinase by anandamide enhanced both myosin phosphatase RhoA-interacting protein (M-RIP):RhoA and M-RIP:MYPT1 association and inhibited Rho kinase activity, leading to increase of myosin light chain (MLC) phosphatase activity and inhibition of sustained muscle contraction. Thus, unlike other Gi-coupled receptors in smooth muscle, CB1 receptors did not engage Gβγ but signaled via GRK5/β-arrestin activation of ERK1/2 and Src kinase: ERK1/2 accelerated inactivation of Gαq by RGS4, and Src kinase enhanced MLC phosphatase activity, leading to inhibition of ACh-stimulated contraction.
authors.affiliation: Department of Physiology and Biophysics, VCU Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Sunila
authors.initials: S
authors.lastname: Mahavadi
authors.forename: Wimolpak
authors.initials: W
authors.lastname: Sriwai
authors.forename: Jiean
authors.initials: J
authors.lastname: Huang
authors.forename: John R
authors.initials: JR
authors.lastname: Grider
authors.forename: Karnam S
authors.initials: KS
authors.lastname: Murthy
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D019390:Arrestins / Q000378:metabolism*; D063388:Endocannabinoids / Q000494:pharmacology; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D053284:Polyunsaturated Alkamides / Q000494:pharmacology; D011817:Rabbits; D043884:Receptor, Cannabinoid, CB1 / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D000071557:beta-Arrestins; D054460:rho-Associated Kinases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001095:Arachidonic Acids; D019390:Arrestins; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D043884:Receptor, Cannabinoid, CB1; D000071557:beta-Arrestins; D019061:src-Family Kinases; D054460:rho-Associated Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C078814:anandamide
keywords: CB1 receptors; ERK1/2; GRK5; Src kinase; beta arrestin