title: Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1.
abstract: Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr(696)) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser(19)) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.
authors.affiliation: Department of Physiology, VCU Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia, USA.
authors.forename: Sayak
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Sunila
authors.initials: S
authors.lastname: Mahavadi
authors.forename: Othman
authors.initials: O
authors.lastname: Al-Shboul
authors.forename: Senthilkumar
authors.initials: S
authors.lastname: Rajagopal
authors.forename: John R
authors.initials: JR
authors.lastname: Grider
authors.forename: Karnam S
authors.initials: KS
authors.lastname: Murthy
mesh_terms: D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D005746:Gastric Emptying; D041981:Gastrointestinal Tract / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009119:Muscle Contraction / Q000187:drug effects*; D009130:Muscle, Smooth / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering; D011817:Rabbits; D043585:Receptor, Muscarinic M2 / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D051242:Caveolin 1; D018679:Cholinergic Agonists; D010716:Phosphatidylinositols; D034741:RNA, Small Interfering; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D043562:Receptors, G-Protein-Coupled; D002217:Carbachol; D000242:Cyclic AMP; D054460:rho-Associated Kinases; D020930:MAP Kinase Kinase Kinases; C090757:mitogen-activated protein kinase kinase kinase 12; D043263:Myosin-Light-Chain Phosphatase
keywords: caveolin-1; gastric emptying; internalization; muscarinic receptors; smooth muscle