G 12/13- and Rho-Dependent Activation of Phospholipase C- by Lysophosphatidic Acid and Thrombin Receptors

Because phospholipase C (PLC) is activated by G 12/13 and Rho family GTPases, we investigated whether these G proteins contribute to the increased inositol lipid hydrolysis observed in COS-7 cells after activation of certain G protein-coupled receptors. Stimulation of inositol lipid hydrolysis by endogenous lysophosphatidic acid (LPA) or thrombin receptors was markedly enhanced by the expression of PLC. Expression of the LPA1 or PAR1 receptor increased inositol phosphate production in response to LPA or SFLLRN, respectively, and these agonist-stimulated responses were markedly enhanced by coexpression of PLC. Both LPA1 and PAR1 receptor-mediated activation of PLCwas inhibited by coexpression of the regulator of G protein signaling (RGS) domain of p115RhoGEF, a GTPase-activating protein for G 12/13 but not by expression of the RGS domain of GRK2, which inhibits G q signaling. In contrast, activation of the Gq-coupled M1 muscarinic or P2Y2 purinergic receptor was neither enhanced by coexpression with PLCnor inhibited by the RGS domain of p115RhoGEF but was blocked by expression of the RGS domain of GRK2. Expression of the Rho inhibitor C3 botulinum toxin did not affect LPAor SFLLRN-stimulated inositol lipid hydrolysis in the absence of PLCbut completely prevented the PLC-dependent increase in inositol phosphate accumulation. Likewise, C3 toxin blocked the PLC-dependent stimulatory effects of the LPA1, LPA2, LPA3, or PAR1 receptor but had no effect on the agonist-promoted inositol phosphate response of the M1 or P2Y2 receptor. Moreover, PLC-dependent stimulation of inositol phosphate accumulation by activation of the epidermal growth factor receptor, which involves Rasbut not Rho-mediated activation of the phospholipase, was unaffected by C3 toxin. These studies illustrate that specific LPA and thrombin receptors promote inositol lipid signaling via activation of G 12/13 and Rho. Many extracellular hormones, neurotransmitters, and growth factors exert their physiological effects by mechanisms that in part involve phospholipase C-catalyzed breakdown of phosphatidylinositol (4,5)P2 into the Ca 2 -mobilizing second-messenger inositol (1,4,5)P3 and the protein kinase C-activating second-messenger diacylglycerol (Irvine et al., 1987; Rhee, 2001). For example, extracellular stimuli that activate members of the large family of seven transmembrane-spanning heterotrimeric G protein-coupled receptors (GPCRs) activate PLCisozymes by the release of -subunits of the Gq family of G proteins (Smrcka et al., 1991; Taylor et al., 1991; Waldo et al., 1991) or by the release of G dimers from activated Gi (Blank et al., 1992; Boyer et al., 1992; Camps et al., 1992). In contrast, PLCenzymes are activated by tyrosine phosphorylation after activation of receptor and nonreceptor tyrosine kinases (Meisenhelder et al., 1989; Wahl et al., 1989). PLC, which possesses Ras-associating (RA) domains at its carboxyl terminus, was initially identified in Caenorhabditis elegans as a Ras-binding protein (Shibatohge et al., 1998). Mammalian PLCis activated by coexpression with Ras (Kelley et al., 2001; Song et al., 2001) and by activators of GEFs that in turn promote the formation of active Rap or Ras (Schmidt et al., 2001; Evellin et al., 2002; Keiper et al., 2004). For example, Gs-coupled GPCRs promote PLC-dependent inositol lipid signaling through activation of the This work was supported by grants GM38213, GM57391, and GM65533 from the National Institute of General Medical Sciences (National Institutes of Health). M.D.H. is a predoctoral fellow of the Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.017921. ABBREVIATIONS: GPCR, G protein-coupled receptor; DMEM, Dulbecco’s modified Eagle’s medium; LPA, lysophosphatidic acid; EGF, epidermal growth factor; PLC, phospholipase C; RGS, regulator of G protein signaling; GEF, guanine nucleotide exchange factor; C3 toxin, C3 botulinum toxin; p115-RGS, the RGS domain of p115 Rho guanine nucleotide exchange factor; GRK2-RGS, the RGS domain of G protein receptor kinase-2; RA, Ras-associating; PDZ, postsynaptic density-95/disc-large/zona occludens. 0026-895X/06/6906-2068–2075$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 6 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 17921/3117915 Mol Pharmacol 69:2068–2075, 2006 Printed in U.S.A. 2068 at A PE T Jornals on Jauary 2, 2018 m oharm .aspeurnals.org D ow nladed from cAMP-activated GEF, EPAC, which in turn activates Rap1A (Schmidt et al., 2001). Initial studies of mammalian PLCrevealed activation by G 12 and G 13 but not by G q (Lopez et al., 2001; Wing et al., 2001), and G also has been shown to activate this PLC isozyme (Wing et al., 2001). Coexpression of Rho family GTPases with PLCresults in marked stimulation of inositol lipid hydrolysis (Wing et al., 2003). PLCmutants that lack functional RA domains retain activation by Rho, indicating that Rho family GTPases regulate this PLC isozyme by a mechanism distinct from that used by Ras and Rap. Observation of GTP-dependent activation of purified PLCby purified RhoA illustrates that the stimulatory action of Rho in inositol lipid signaling is direct (Seifert et al., 2004). GEFs for Rho are downstream effectors of G 12/13 (Hart et al., 1998; Fukuhara et al., 1999; Booden et al., 2002; Suzuki et al., 2003; Dutt et al., 2004). Thus, the observation of Rho-dependent activation of PLCsuggests that GPCRs that activate G 12/13 promote inositol lipid signaling through the activation of Rho. With the goal of establishing whether receptor-mediated regulation of inositol lipid signaling occurs through a mechanism involving G 12/13, Rho, and PLC, we studied the regulation of PLC-promoted inositol lipid hydrolysis by endogenous and recombinant GPCRs expressed in COS-7 cells. The results of these studies are consistent with the idea that G 12/13and Rho-dependent activation of PLCoccurs downstream of both LPAand thrombin-activated receptors and that the regulation of PLCby G 12/13 occurs at least in part through activation of Rho. Materials and Methods Materials. Expression vectors (in pcDNA3.1) for the human M1 muscarinic cholinergic, LPA1, LPA2, and LPA3 receptors were purchased from the University of Missouri–Rolla cDNA Resource Center (Rolla, MO). An expression vector encoding the human P2Y2 receptor was described previously (Nicholas et al., 1996). The plasmid encoding wild-type epidermal growth factor (EGF) receptor is described by Carter and Sorkin (1998). A pCMV-Script vector encoding FLAGtagged rat PLCwas generously provided by Grant Kelley, State University of New York (Syracuse, NY). An expression vector for C3 botulinum toxin was obtained from Channing Der, University of North Carolina (Chapel Hill, NC). cDNA encoding the first 240 amino acids of human p115RhoGEF was subcloned in-frame with an N-terminal tandem hemagglutinin-epitope tag into a modified pcDNA3.1 vector (Hains et al., 2004). cDNA encoding amino acids 45 to 178 of bovine GRK2 (designated GRK2-RGS) in frame with an N-terminal hemagglutinin-epitope tag in pcDNA3 was kindly provided by Dr. Jeffrey Benovic (Thomas Jefferson University, Philadelphia, PA). 1-Oleoyl-L-lysophosphatidic acid sodium salt (LPA) was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in water containing 1.0% fatty acid-free bovine serum albumin. The PAR1 receptor agonist peptide SFLLRN was synthesized as the carboxyl amide and purified by reverse-phase high-pressure liquid chromatography (University of North Carolina Peptide Facility, Chapel Hill, NC). UTP, carbachol, and EGF were purchased from Sigma-Aldrich. All other reagents were from sources noted previously (Wing et al., 2001, 2003; Seifert et al., 2004). Cell Culture and Transfection of COS-7 Cells. COS-7 cells were plated in 12or 96-well culture dishes and maintained in DMEM supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin at 37°C in a 10% CO2/90% air atmosphere. The indicated DNA expression vectors were transfected into COS-7 cells using FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) at a ratio of 3:1 (FuGENE/DNA) following the manufacturer’s protocol. Empty-vector DNA was used as necessary to maintain a constant total amount