title: M3 muscarinic receptor interaction with phospholipase C β3 determines its signaling efficiency.
abstract: Phospholipase Cβ (PLCβ) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Gαβγ heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCβ3. Both expressed and endogenous M3R interacted with PLCβ in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLCβ3. Deletion of the PLCβ3 C terminus or deletion of the PLCβ3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLCβ3 plasma membrane localization. Purified PLCβ3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLCβ3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated Gαq-dependent but not Gβγ-dependent PLCβ3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLCβ3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCβ3 signaling beyond its canonical role as a guanine nucleotide exchange factor for Gα.
authors.affiliation: Departments of Pharmacology and Physiology and University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
authors.forename: Wei
authors.initials: W
authors.lastname: Kan
authors.affiliation: Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.
authors.forename: Merel
authors.initials: M
authors.lastname: Adjobo-Hermans
authors.affiliation: Departments of Pharmacology and Physiology and University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
authors.forename: Michael
authors.initials: M
authors.lastname: Burroughs
authors.affiliation: Departments of Pharmacology and Physiology and University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
authors.forename: Guy
authors.initials: G
authors.lastname: Faibis
authors.affiliation: Departments of Pharmacology and Physiology and University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
authors.forename: Sundeep
authors.initials: S
authors.lastname: Malik
authors.affiliation: Departments of Pharmacology and Physiology and University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Tall
authors.affiliation: Departments of Pharmacology and Physiology and University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; Biochemistry and Biophysics and University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; Aab Institute of Cardiovascular Research, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 and. Electronic address: alan_smrcka@urmc.rochester.edu.
authors.forename: Alan V
authors.initials: AV
authors.lastname: Smrcka
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D054799:Phospholipase C beta / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D043587:Receptor, Muscarinic M3; C518039:PLCB3 protein, human; D054799:Phospholipase C beta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11
keywords: G Protein-coupled Receptors (GPCR); G Proteins; Phosphatidylinositol Signaling; Phospholipase C; Protein-Protein Interactions; Scaffolding