Signaling and Cross-talk by C5a and UDP in Macrophages Selectively Use PLC3 to Regulate Intracellular

Studies in fibroblasts, neurons, and platelets have demon-strated the integration of signals from different G protein-coupled receptors (GPCRs) in raising intracellular freeCa2. 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 acti-vating factor (PAF), or lysophosphatidic acid (LPA). The C5a response was Gi-dependent, whereas the UDP, PAF, and LPA responseswereGq-dependent. SynergybetweenC5a andUDP, mediated by theC5a andP2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca2 from intra-cellular stores as well as sustained Ca2 levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggest-ing synergy in activating phospholipase C (PLC) . Macro-phages expressed transcripts for three PLC isoforms (PLC2, PLC3, and PLC4), but GPCR ligands selectively used these isoforms in Ca2 signaling. C5a predominantly used PLC3