Signaling and cross-talk by C5a and UDP in macrophages selectively use PLCbeta3 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 Ca(2+). To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca(2+) 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 Galpha(i)-dependent, whereas the UDP, PAF, and LPA responses were Galpha(q)-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 Ca(2+) levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) beta. Macrophages expressed transcripts for three PLCbeta isoforms (PLCbeta2, PLCbeta3, and PLCbeta4), but GPCR ligands selectively used these isoforms in Ca(2+) signaling. C5a predominantly used PLCbeta3, whereas UDP used PLCbeta3 but also PLCbeta4. Neither ligand required PLCbeta2. Synergy between C5a and UDP likewise depended primarily on PLCbeta3. Importantly, the Ca(2+) signaling deficiency observed in PLCbeta3-deficient BMDM was reversed by re-constitution with PLCbeta3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca(2+), PI 3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLCbeta3 may thus provide a selective target for inhibiting Ca(2+) responses to mediators of inflammation, including C5a, UDP, PAF, and LPA.
Roach
Tamara I A
TI
Alliance for Cellular Signaling, Northern California Institute for Research and Education, San Francisco, California 94121, USA. Tamara.Roach@ucsf.edu
Rebres
Robert A
RA
Fraser
Iain D C
ID
Decamp
Dianne L
DL
Lin
Keng-Mean
KM
Sternweis
Paul C
PC
Simon
Mel I
MI
Seaman
William E
WE
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015936:Complement C5a / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D054799:Phospholipase C beta / Q000378:metabolism*; D010873:Pinocytosis; D020033:Protein Isoforms; D015398:Signal Transduction; D014530:Uridine Diphosphate / Q000737:chemistry*
D008024:Ligands; D020033:Protein Isoforms; D014530:Uridine Diphosphate; D015936:Complement C5a; D019869:Phosphatidylinositol 3-Kinases; D054799:Phospholipase C beta; D002118:Calcium