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J. Biol. Chem., Vol. 281, Issue 5, 2639-2648, February 3, 2006

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G-protein-coupled Receptor Agonists Activate Endogenous Phospholipase C and Phospholipase C3 in a Temporally Distinct Manner^*

Grant G. Kelley¹, Katherine A. Kaproth-Joslin, Sarah E. Reks, Alan V. Smrcka^¶, and Richard J. H. Wojcikiewicz

From the Departments of Medicine and Pharmacology, State University of New York Upstate Medical University, Syracuse, New York 13210 and the ^¶Department of Pharmacology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Phospholipase C (PLC) 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 PLC isoforms through G_q family G proteins and G 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 PLC and the PLC isoform, PLC3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist- and isoform-specific profiles. PLC3 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. PLC, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLC in knockdown cells dose-dependently increases sustained, but not acute, agonist-stimulated PI hydrolysis. Furthermore, combined knockdown of both PLC and PLC3 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 PLC or PLC3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLC and PLC3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist-specific temporal profiles; however, PLC3 is predominantly involved in acute and PLC in sustained PI hydrolysis.

Received for publication, July 15, 2005 , and in revised form, November 23, 2005.

^* This work was supported by National Institutes of Health Grants DK56294 (to G. G. K.) and DK49194 (to R. J. H. W.) and American Diabetes Association Physician Scientist Training Award 7-03-PS-01 (K. A. K.-J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Medicine, SUNY Upstate Medical University, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464-5725; Fax: 315-464-5797; E-mail: kelleyg{at}upstate.edu.

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