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
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
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DOI：
10.1074/jbc.m110.198200
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发表时间：
2011-01-14
影响因子：
4.8
通讯作者：
Seaman, William E.
中科院分区：
文献类型：
--
作者：
Rebres, Robert A.;Roach, Tamara I. A.;Seaman, William E.
Gα(I)和Gα(Q)连接的G蛋白偶联受体之间的串扰在各种细胞类型中产生协同的钙反应。先前的研究表明，巨噬细胞G蛋白偶联受体的协同钙反应主要依赖于磷脂酶Cβ3(PLCβ3)，可能与PLCβ2有关，而通过PLCβ4的信号转导干扰协同作用。我们在这里表明，协同作用可以通过结合Gβ伽马和Gα(Q)激活单个PLCβ亚型来诱导。在缺乏PLCβ2和PLCβ3的巨噬细胞中没有协同作用，但仅用PLCβ3转导后，协同作用完全重建。在几乎不表达PLCβ2的NIH-3T3细胞中，进一步探讨了PLCβ介导的协同作用的机制。RNAi介导的内源性PLCβS的下调表明，在这些细胞中的协同作用依赖于PLCβ3，而PLCβ1和PLCβ4不起作用，两种异构体的过表达都抑制了钙离子的协同作用。当协同作用被内源性PLCβ3的RNAi阻断时，它可以通过人PLCβ3或小鼠PLCβ2的表达来重建。相反，Y盒突变的人PLCβ3不能重建协同作用，而人PLCβ3的C端突变只能部分恢复协同作用，而人PLCβ3的C端突变则部分阻断Gα(Q)的激活。因此，Gβ伽马和Gα(Q)都有助于激活细胞内的PLCβ3以实现钙协同作用。我们的结论是，Gα(I)偶联受体和Gα(Q)偶联受体之间的钙协同作用需要Gβ伽马和Gα(Q)对PLCβ的直接作用，并且主要由PLCβ3介导，尽管PLCβ2也是有活性的。
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.