Gutknecht Eric, Van der Linden Ilse, Van Kolen Kristof, Verhoeven Kim F C, Vauquelin Georges, Dautzenberg Frank M
Johnson and Johnson Research and Development, CNS Research, Beerse, Belgium.
Mol Pharmacol. 2009 Mar;75(3):648-57. doi: 10.1124/mol.108.050427. Epub 2008 Dec 19.
The molecular mechanisms governing calcium signal transduction of corticotropin-releasing factor (CRF) receptors CRF(1) and CRF(2(a)) stably expressed in human embryonic kidney (HEK) 293 cells were investigated. Calcium signaling strictly depended on intracellular calcium sources, and this is the first study to establish a prominent contribution of the three major G-protein families to CRF receptor-mediated calcium signaling. Overexpression of Galpha(q/11) and Galpha(16) led to leftward shifts of the agonist concentration-response curves. Blockade of Galpha(q/11) proteins by the small interfering RNA (siRNA) technology partially reduced agonist-mediated calcium responses in CRF(1)- and CRF(2(a))-expressing HEK293 cells, thereby proving a contribution of the G(q) protein family. A small but significant inhibition of calcium signaling was recorded by pharmacological inhibition of G(i/o) proteins with pertussis toxin treatment. This effect was mediated by direct binding of Gbetagamma subunits to phospholipase C. G(i/o) inhibition also elevated cAMP responses in CRF receptor-overexpressing HEK293 cells and in Y79 retinoblastoma cells endogenously expressing human CRF(1) and CRF(2(a)) receptors, thereby demonstrating natural coupling of G(i) proteins to both CRF receptors. The strongest reduction of CRF receptor-mediated calcium mobilization was noted when blocking the G(s) signaling protein either by cholera toxin or by siRNA. It is noteworthy that simultaneous inhibition of two G-proteins shed light on the additive effects of G(s) and G(q) on the calcium signaling and, hence, that they act in parallel. On the other hand, G(i) coupling required prior G(s) activation.
研究了在人胚肾（HEK）293细胞中稳定表达的促肾上腺皮质激素释放因子（CRF）受体CRF(1)和CRF(2(a))的钙信号转导分子机制。钙信号严格依赖于细胞内钙源，这是第一项证实三大G蛋白家族对CRF受体介导的钙信号有显著贡献的研究。Galpha(q/11)和Galpha(16)的过表达导致激动剂浓度-反应曲线向左移动。通过小干扰RNA（siRNA）技术阻断Galpha(q/11)蛋白可部分降低表达CRF(1)和CRF(2(a))的HEK293细胞中激动剂介导的钙反应，从而证明G(q)蛋白家族的作用。用百日咳毒素处理对G(i/o)蛋白进行药理抑制可记录到对钙信号的轻微但显著的抑制。这种效应是由Gbetagamma亚基与磷脂酶C的直接结合介导的。G(i/o)抑制还提高了过表达CRF受体的HEK293细胞以及内源性表达人CRF(1)和CRF(2(a))受体的Y79视网膜母细胞瘤细胞中的cAMP反应，从而证明G(i)蛋白与两种CRF受体均存在天然偶联。当通过霍乱毒素或siRNA阻断G(s)信号蛋白时，观察到CRF受体介导的钙动员的最大程度降低。值得注意的是，同时抑制两种G蛋白揭示了G(s)和G(q)对钙信号的累加效应，因此它们是并行起作用的。另一方面，G(i)偶联需要先激活G(s)。
