Synergistic Ca2+ responses by Gαi- and Gαq-coupled GPCRs require a single PLCβ isoform that is sensitive to both Gβγ and Gαq

Abstract Cross-talk between Gαi- and Gαq-linked G protein-coupled receptors (GPCRs) yields synergistic Ca2+ responses in a variety of cell types. Prior studies have shown that synergistic Ca2+ responses from macrophage GPCRs are primarily dependent on PLCβ3, with a possible contribution of PLCβ2, while signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gαq activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNA-interference (RNAi)-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca2+ synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C-terminus, which partly disrupted activation by Gαq. Thus both Gβγ and Gαq contribute to activation of PLCβ3 in cells for Ca2+ synergy. We conclude that Ca2+ synergy between Gαi-coupled and Gαq-coupled receptors requires the direct action of both Gβγ and Gαq on PLCβ, and is mediated primarily by PLCβ3, though PLCβ2 is also competent.