G-protein-coupled receptor agonists activate endogenous phospholipase Cε and phospholipase Cβ3 in a temporally distinct manner

Abstract

Phospholipase Cε (PLCε) 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 PLCβ isoforms through G q family G proteins and Gβγ 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 PLCε and the PLCβ isoform, PLCβ3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist- and isoform-specific profiles. PLCβ 3 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. PLCε, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLCε in knockdown cells dose-dependently increases sustained, but not acute, agonist-stimulated PI hydrolysis. Furthermore, combined knockdown of both PLCε and PLCβ3 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 PLCε or PLCβ3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLCε and PLCβ3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist-specific temporal profiles; however, PLCβ3 is predominantly involved in acute and PLCε in sustained PI hydrolysis. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.