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J. Biol. Chem., Vol. 283, Issue 25, 17351-17361, June 20, 2008

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Signaling and Cross-talk by C5a and UDP in Macrophages Selectively Use PLCβ3 to Regulate Intracellular Free Calcium^*

Tamara I. A. Roach¹², Robert A. Rebres¹³, Iain D. C. Fraser, Dianne L. DeCamp^¶, Keng-Mean Lin^¶, Paul C. Sternweis^¶, Mel I. Simon, and William E. Seaman^||4

From the Alliance for Cellular Signaling, Northern California Institute for Research and Education and the ^||University of California, Veterans Affairs Medical Center, San Francisco, California 94121, the Division of Biology, California Institute of Technology, Pasadena, California 91125, and the ^¶University of Texas Southwestern Medical Center, Dallas, Texas 75390

Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G protein-coupled receptors (GPCRs) in raising intracellular free Ca²⁺. To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca²⁺ response to GPCR ligands. We found a synergistic response to complement component 5a (C5a) in combination with uridine 5'-diphosphate (UDP), platelet activating factor (PAF), or lysophosphatidic acid (LPA). The C5a response was G_i-dependent, whereas the UDP, PAF, and LPA responses were G_q-dependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca²⁺ from intracellular stores as well as sustained Ca²⁺ levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) β. Macrophages expressed transcripts for three PLCβ isoforms (PLCβ2, PLCβ3, and PLCβ4), but GPCR ligands selectively used these isoforms in Ca²⁺ signaling. C5a predominantly used PLCβ3, whereas UDP used PLCβ3 but also PLCβ4. Neither ligand required PLCβ2. Synergy between C5a and UDP likewise depended primarily on PLCβ3. Importantly, the Ca²⁺ signaling deficiency observed in PLCβ3-deficient BMDM was reversed by re-constitution with PLCβ3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca²⁺, PI 3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLCβ3 may thus provide a selective target for inhibiting Ca²⁺ responses to mediators of inflammation, including C5a, UDP, PAF, and LPA.

Received for publication, February 4, 2008 , and in revised form, April 9, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM 62114. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ Both authors contributed equally to this manuscript.

² To whom correspondence may be addressed: VAMC 111R, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2104; Fax: 415-750-6920; E-mail: Tamara.Roach{at}ucsf.edu. ³ To whom correspondence may be addressed. E-mail: Robert.Rebres{at}ucsf.edu. ⁴ To whom correspondence may be addressed. E-mail: bseaman{at}medicine.ucsf.edu.

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