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

Abstract

Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G proteincoupled 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 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 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) ␤. Macrophages expressed transcripts for three PLC␤ isoforms (PLC␤2, PLC␤3, and PLC␤4), but GPCR ligands selectively used these isoforms in Ca 2؉ signaling. C5a predominantly used PLC␤3, whereas UDP used PLC␤3 but also PLC␤4. Neither ligand required PLC␤2. Synergy between C5a and UDP likewise depended primarily on PLC␤3. Importantly, the Ca 2؉ signaling deficiency observed in PLC␤3-deficient BMDM was reversed by re-constitution with PLC␤3. 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. PLC␤3 may thus provide a selective target for inhibiting Ca 2؉

Structural Insights into Phospholipase C-βFunction

“…Early evidence for synergistic activation of PLCb enzymes came from macrophages, where treatment with G i -and G q -coupled receptor agonists resulted in superadditive Ca 21 increases over what either agonist could induce alone. This synergistic Ca 21 release required the activity of PLCb3 (Roach et al, 2008). In the presence of excess activated Ga q and Gbg, PLCb3 activity is stimulated approximately 19-fold over what either Ga q or Gbg can induce, which appears to depend on the very low basal activity of PLCb3 (Philip et al, 2010).…”

“…On the basis of biochemical and structural data, it is clear that Ga q has a distinct binding site and activation mechanism from Gbg and the Rho GTPases. Indeed, for some isoforms, regulation by these molecules has been shown to be synergistic (Roach et al, 2008;Philip et al, 2010;Rebres et al, 2011). Below we discuss the current state of knowledge regarding the molecular basis of PLCb activation by four key regulators: the phospholipid bilayer, Ga q , the Gbg heterodimer, and Rho GTPases.…”

The PRKAA1/AMPKα1 pathway triggers autophagy during CSF1-induced human monocyte differentiation and is a potential target in CMML

“…PLCB3 has been reported as the specific PLC isoform that is activated downstream of P2RY6. 23 Individual silencing of both proteins induced a significant inhibition of PRKAA1 expression and phosphorylation on Thr172 in monocytes stimulated for one d with CSF1 (Fig. 5D).…”

Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes

“…To compare the basal fluorescence signal intensity between PMCA4-GCaMP2 and PMCA4 mut GCaMP2 we used a modification of the method described in [19] To test the effect of various calcium concentrations on the fluorescent intensity of the sensors, we varied the calcium chloride concentration in the F max solution to produce corresponding free calcium concentration in the medium ranging from 100 nM to 10 mM as calculated using an online software tool based on the Fabiato formula (http://www.stanford. edu/~cpatton/CaEGTA-NIST.htm).…”