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Published June 20, 2008 | Published + Accepted Version + Supplemental Material
Journal Article Open

Signaling and crosstalk by C5a and UDP in macrophages selectively use PLCbeta 3 to regulate intracellular free calcium


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 Gai-dependent, while the UDP, PAF, and LPA responses were Gaqdependent. 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) ß. Macrophages expressed transcripts for three PLCß isoforms (PLCß2, PLCß3, and PLCß4), but GPCR ligands selectively used these isoforms in Ca2+ signaling. C5a predominantly used PLCß3, while UDP used PLCß3 but also PLCß4. Neither ligand required PLCß2. Synergy between C5a and UDP likewise depended primarily on PLCß3. Importantly, the Ca2+ signaling deficiency observed in PLCß3-deficient BMDM was reversed by reconstitution with PLCß3. Neither PI-3 kinase nor PKC was required for synergy. In contrast to Ca2+, PI3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI3- kinase. PLCß3 may thus provide a selective target for inhibiting Ca2+ responses to mediators of inflammation, including C5a, UDP, PAF, and LPA.

Additional Information

© 2008 the American Society for Biochemistry and Molecular Biology. Submitted on February 4, 2008. Revised on April 9, 2008. Accepted on April 14, 2008. Papers In Press, published online ahead of print April 14, 2008. We thank K. Rose Finley, Michael McWay, Christina Moon and Amanda Norton at the San Francisco VA Medical Center and Joelle Zavzavadjian, Jamie Liu, Leah Santat, Lucas Cheadle and Estelle Wall at Caltech for excellent technical assistance. 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.

Attached Files

Published - ROAjbc08.pdf

Accepted Version - ROAjbc08pip.pdf

Supplemental Material - ROAjbc08supdata.pdf


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