title: Membrane-induced allosteric control of phospholipase C-β isozymes.
abstract: All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β (PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gαq, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gβ1γ2 did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes.
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Charpentier
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Waldo
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Matthew O
authors.initials: MO
authors.lastname: Barrett
authors.affiliation: the Division of Chemical Biology and Medicinal Chemistry, University of North Carolina School of Pharmacy, Chapel Hill, North Carolina 27599.
authors.forename: Weigang
authors.initials: W
authors.lastname: Huang
authors.affiliation: the Division of Chemical Biology and Medicinal Chemistry, University of North Carolina School of Pharmacy, Chapel Hill, North Carolina 27599.
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: T Kendall
authors.initials: TK
authors.lastname: Harden
authors.affiliation: From the Departments of Pharmacology and Biochemistry and Biophysics and the Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 and sondek@med.unc.edu.
authors.forename: John
authors.initials: J
authors.lastname: Sondek
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D055162:Biocatalysis; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology*; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D017930:Genes, Reporter; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008954:Models, Biological; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D054799:Phospholipase C beta / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012995:Solubility
chemical_list: D005456:Fluorescent Dyes; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; C518039:PLCB3 protein, human; D054799:Phospholipase C beta; D020962:Heterotrimeric GTP-Binding Proteins
keywords: Fluorescence; G Protein; Membrane; Phospholipase C; Phospholipid