title: G-protein-coupled receptor agonists activate endogenous phospholipase Cepsilon and phospholipase Cbeta3 in a temporally distinct manner.
abstract: Phospholipase Cepsilon (PLCepsilon) is one of the newest members of the phosphatidylinositol-specific phospholipase C (PLC) family. Previous studies have suggested that G-protein-coupled receptors (GPCRs) stimulate phosphoinositide (PI) hydrolysis by activating PLCbeta isoforms through G(q) family G proteins and Gbetagamma subunits. Using RNA interference to knock down PLC isoforms, we demonstrate that the GPCR agonists endothelin (ET-1), lysophosphatidic acid (LPA), and thrombin, acting through endogenous receptors, couple to both endogenous PLCepsilon and the PLCbeta isoform, PLCbeta3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist- and isoform-specific profiles. PLCbeta3 is activated acutely within the first minute of ET-1, LPA, or thrombin stimulation but does not contribute to sustained PI hydrolysis induced by LPA or thrombin and accounts for only part of ET-1 sustained stimulation. PLCepsilon, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLCepsilon in knockdown cells dose-dependently increases sustained, but not acute, agonist-stimulated PI hydrolysis. Furthermore, combined knockdown of both PLCepsilon and PLCbeta3 additively inhibits PI hydrolysis, suggesting independent regulation of each isoform. Importantly, ubiquitination of inositol 1,4,5-trisphosphate receptors correlates with sustained, but not acute, activation of PLCepsilon or PLCbeta3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLCepsilon and PLCbeta3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist-specific temporal profiles; however, PLCbeta3 is predominantly involved in acute and PLCepsilon in sustained PI hydrolysis.
authors.affiliation: Department of Medicine and Pharmacology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA. kelleyg@upstate.edu
authors.forename: Grant G
authors.initials: GG
authors.lastname: Kelley
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Kaproth-Joslin
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Reks
authors.forename: Alan V
authors.initials: AV
authors.lastname: Smrcka
authors.forename: Richard J H
authors.initials: RJ
authors.lastname: Wojcikiewicz
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008246:Lysophospholipids / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D054799:Phospholipase C beta; D020033:Protein Isoforms / Q000235:genetics; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000819:agonists*; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
chemical_list: D019332:Endothelin-1; D007527:Isoenzymes; D008246:Lysophospholipids; D018129:Phosphatidylinositol Phosphates; D020033:Protein Isoforms; D043562:Receptors, G-Protein-Coupled; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D054799:Phospholipase C beta; C423320:phospholipase C epsilon; D013917:Thrombin; C032881:lysophosphatidic acid