Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

Cross-talk between G alpha(i)- and G alpha(q)-linked G-protein-coupled receptors yields synergistic Ca2+ responses in a variety of cell types. Prior studies have shown that synergistic Ca2+ responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase C beta 3 (PLC beta 3), with a possible contribution of PLC beta 2, whereas signaling through PLC beta 4 interferes with synergy. We here show that synergy can be induced by the combination of G beta gamma and G alpha(q) activation of a single PLC beta isoform. Synergy was absent in macrophages lacking both PLC beta 2 and PLC beta 3, but it was fully reconstituted following transduction with PLC beta 3 alone. Mechanisms of PLC beta mediated synergy were further explored in NIH-3T3 cells, which express little if any PLC beta 2. RNAi-mediated knockdown of endogenous PLC beta s demonstrated that synergy in these cells was dependent on PLC beta 3, but PLC beta 1 and PLC beta 4 did not contribute, and overexpression of either isoform inhibited Ca2+ synergy. When synergy was blocked by RNAi of endogenous PLC beta 3, it could be reconstituted by expression of either human PLC beta 3 or mouse PLC beta 2. In contrast, it could not be reconstituted by human PLC beta 3 with a mutation of the Y box, which disrupted activation by G beta gamma, and it was only partially restored by human PLC beta 3 with a mutation of the C terminus, which partly disrupted activation by G alpha(q). Thus, both G beta gamma and G alpha(q) contribute to activation of PLC beta 3 in cells for Ca2+ synergy. We conclude that Ca2+ synergy between G alpha(i)-coupled and G alpha(q)-coupled receptors requires the direct action of both G beta gamma and G alpha(q) on PLC beta and is mediated primarily by PLC beta 3, although PLC beta 2 is also competent.