Signaling and cross-talk by C5a and UDP in macrophages selectively use PLCβ3 to regulate intracellular free calcium

Tamara I A Roach, Robert A. Rebres, Iain D C Fraser, Dianne L. DeCamp, Keng Mean Lin, Paul C. Sternweis, Mel I. Simon, William E. Seaman

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30 Scopus citations

Abstract

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 Gαi- dependent, whereas the UDP, PAF, and LPA responses were Gαq- dependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca 2+ 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.

Original languageEnglish (US)
Pages (from-to)17351-17361
Number of pages11
JournalJournal of Biological Chemistry
Volume283
Issue number25
DOIs
StatePublished - Jun 20 2008

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Roach, T. I. A., Rebres, R. A., Fraser, I. D. C., DeCamp, D. L., Lin, K. M., Sternweis, P. C., Simon, M. I., & Seaman, W. E. (2008). Signaling and cross-talk by C5a and UDP in macrophages selectively use PLCβ3 to regulate intracellular free calcium. Journal of Biological Chemistry, 283(25), 17351-17361. https://doi.org/10.1074/jbc.M800907200