Gao Zhan-Guo, Gao Ray R, Meyer Clayton K, Jacobson Kenneth A
NIDDK, National Institutes of Health.
Res Sq. 2024 Dec 13:rs.3.rs-5442142. doi: 10.21203/rs.3.rs-5442142/v1.
Activation of PLCβ enzymes by G and G proteins is a common mechanism to trigger cytosolic Ca increase. We and others reported that G inhibitor FR900358 (FR) can inhibit both and G- and, surprisingly, G-mediated intracellular Ca mobilization. Thus, the G-G-PLCβ-Ca signaling axis depends entirely on the presence of active G, which reasonably explained FR-inhibited G-induced Ca release. However, the conclusion that G signaling is controlled by G derives mostly from HEK293 cells. Here we show that indeed in HEK293 cells both G siRNA and G inhibitors diminished Ca increase triggered by native G-coupled P2Y receptors, or by transfected G-coupled A-or G-coupled A adenosine receptors (ARs). However, in T24 bladder cancer cells, G inhibitor PTX, but not G inhibitors, FR, YM254890 (YM) or G siRNA, inhibited Ca increase triggered by native AAR activation. Simultaneous inactivation of G and G further suppressed AAR-triggered Ca increase in T24 cells. The G inhibitor YM fully and partially inhibited endogenous P2Y- and β-adrenergic receptor-induced Ca increase in T24 cells, respectively. PKC activator PMA partially diminished AAR-triggered but completely diminished β-adrenergic receptor-triggered Ca increase in T24 cells. Neither β-arrestin1 nor β-arrestin2 siRNA affected AAR-mediated Ca increase. Unlike in T24 cells, YM inhibited native AAR-triggered calcium mobilization in MDA-MB-231 breast cancer cells. Thus, G is vital for Ca increase in some cell types, but G-mediated Ca signaling can be Gα-dependent or independent based on cell type and receptor activated. Besides G proteins, PKC also modulates cytosolic Ca increase depending on cell type and receptor.
G蛋白和G蛋白激活PLCβ酶是触发胞质Ca升高的常见机制。我们和其他人报道，G抑制剂FR900358（FR）可以抑制G蛋白和G蛋白介导的细胞内Ca动员，令人惊讶的是，它还能抑制G蛋白介导的细胞内Ca动员。因此，G蛋白-G蛋白-PLCβ-Ca信号轴完全依赖于活性G蛋白的存在，这合理地解释了FR抑制G蛋白诱导的Ca释放。然而，G蛋白信号由G蛋白控制这一结论主要来自HEK293细胞。在这里我们表明，在HEK293细胞中，G蛋白小干扰RNA（siRNA）和G蛋白抑制剂确实减少了由天然G蛋白偶联的P2Y受体，或转染的G蛋白偶联的A2A或G蛋白偶联的A1腺苷受体（ARs）触发的Ca升高。然而，在T24膀胱癌细胞中，G蛋白抑制剂百日咳毒素（PTX），而不是G蛋白抑制剂FR、YM254890（YM）或G蛋白siRNA，抑制了由天然A2AAR激活触发的Ca升高。同时使G蛋白和G蛋白失活进一步抑制了T24细胞中A2AAR触发的Ca升高。G蛋白抑制剂YM分别完全和部分抑制了T24细胞中内源性P2Y受体和β-肾上腺素能受体诱导的Ca升高。蛋白激酶C（PKC）激活剂佛波酯（PMA）部分减少了T24细胞中A2AAR触发的Ca升高，但完全减少了β-肾上腺素能受体触发的Ca升高。β-抑制蛋白1和β-抑制蛋白2的siRNA均不影响A2AAR介导的Ca升高。与T24细胞不同，YM抑制了MDA-MB-231乳腺癌细胞中天然A2AAR触发的钙动员。因此，G蛋白在某些细胞类型中对Ca升高至关重要，但基于细胞类型和激活的受体，G蛋白介导的Ca信号传导可以是Gα依赖性或非依赖性的。除了G蛋白外，PKC也根据细胞类型和受体调节胞质Ca升高。
