Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q.

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cß3 (PLCß3), with a possible contribution of PLCß2, whereas signaling through PLCß4 interferes with synergy. We here show that synergy can be induced by the combination of Gßγ and Gα(q) activation of a single PLCß isoform. Synergy was absent in macrophages lacking both PLCß2 and PLCß3, but it was fully reconstituted following transduction with PLCß3 alone. Mechanisms of PLCß-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCß2. RNAi-mediated knockdown of endogenous PLCßs demonstrated that synergy in these cells was dependent on PLCß3, but PLCß1 and PLCß4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCß3, it could be reconstituted by expression of either human PLCß3 or mouse PLCß2. In contrast, it could not be reconstituted by human PLCß3 with a mutation of the Y box, which disrupted activation by Gßγ, and it was only partially restored by human PLCß3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gßγ and Gα(q) contribute to activation of PLCß3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gßγ and Gα(q) on PLCß and is mediated primarily by PLCß3, although PLCß2 is also competent.

Clostridium difficile toxin B differentially affects GPCR-stimulated Ca2+ responses in macrophages: independent roles for Rho and PLA2.

Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca(2+) responses to Galphai-linked receptors, including the C5aR, but reduced responses to Galphaq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca(2+) responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCbeta isoform-deficient BMDM, we found that ToxB inhibited Ca(2+) signaling through PLCbeta4 but enhanced signaling through PLCbeta3. Effects of ToxB on GPCR Ca(2+) responses correlated with GPCR use of PLCbeta3 versus PLCbeta4. ToxB inhibited UDP Ca(2+) signaling without reducing InsP3 production or the sensitivity of cellular Ca(2+) stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca(2+)coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca(2+) signaling by C5a was prevented by inhibition of PLA(2) or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca(2+) signaling by different GPCR-linked PLCbeta isoforms in macrophages.

A dual receptor crosstalk model of G-protein-coupled signal transduction.

Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Galphai-coupled C5a receptor and the Galphaq-coupled UDP receptor signaling systems that results in synergistic calcium release.

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.

Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.

Regulation of intracellular cyclic adenosine 3 ',5 '-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G(s)-dependent receptors for isoproterenol and prostaglandin E(2). Whereas two ligands, uridine 5 '-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G(q)/calcium and G(i), the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G(s)-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P(2) receptor and the heterotrimeric G(13) protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.

Novel CD47-dependent intercellular adhesion modulates cell migration.

CD47 is a ubiquitously expressed plasma membrane protein, also known as Integrin Associated Protein, that modulates cell adhesion both through alteration of the avidity of integrin binding and through interaction with its own ligands, the extracellular matrix protein thrombospondin (TSP) and the plasma membrane response regulator SIRPalpha1. We now show that CD47 expression on fibroblasts can induce intercellular adhesion resulting in cell aggregation in the absence of active integrins, SIRPalpha1 binding, and detectable TSP. CD47-expressing cells preferentially bind to other CD47-expressing cells, and intercellular adhesion requires stimulation by serum or a CD47-binding peptide from TSP. Cell-cell adhesion is inhibited by pertussis toxin and C. difficile toxin B, and both adherent and aggregating CD47-expressing fibroblasts have more rac in the GTP bound state than CD47-deficient cells. Spontaneous migration of Jurkat lymphocytes through a fibroblast monolayer is decreased by fibroblast expression of CD47, consistent with an increased barrier function of the CD47 expressing cells. The lymphocyte chemoattractant SDF-1alpha stimulates migration of Jurkat cells through this monolayer only if both the lymphocytes and fibroblasts express CD47, and the inhibition of migration by a CD47-interacting peptide from TSP similarly requires CD47 expression on both cell types. Thus, signaling dependent on both heterotrimeric and rho family GTPases can induce CD47 to participate in cell-cell interactions independent of known ligands that enhance intercellular adhesion and modulate cell migration.