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.

ABSTRACT

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.