Regulation of cytoskeletons by heterotrimeric GTP-binding proteins

本研究旨在探讨g - αq/11诱导表达m1毒蕈碱乙酰胆碱受体的HeLa细胞凋亡的分子机制。过表达组成型活性突变体Akt可抑制碳水化合物诱导的ROCK-I切割。胰岛素是许多细胞的主要存活因子，它强烈地增加Akt的磷酸化，而Akt的磷酸化完全被乙醇阻断。后一种效应被酪氨酸磷酸酶抑制剂正钒酸盐部分抑制。与这些观察结果相平行的是，碳二醇减弱了胰岛素刺激下胰岛素受体底物-1的酪氨酸磷酸化，这一效应被正钒酸盐消除了。另一方面，碳醇诱导内源性RhoA的快速刺激，RhoA组成型活性突变体的表达增加了ROCK-I的切割。正钒酸盐和RhoA显性负突变体部分或完全抑制了碳甾醇诱导的ROCK-I切割和细胞凋亡。这些结果进一步表明，Ga/11信号通过酪氨酸去磷酸化和激活RhoA，减少胰岛素刺激的Akt磷酸化，从而诱导HeLa细胞凋亡。为了阐明Gi2在胚胎脑心室区的作用，我们在E14.5时将百日咳毒素（PTX）注入小鼠脑侧脑室。E18.5检查显示，PTX减少了大脑皮层的总细胞和溴脱氧尿嘧啶阳性细胞，提示神经上皮细胞增殖受损。然后，我们培养了来自大鼠胚胎脑的神经祖细胞，并评估了激动剂对几种已知在心室区表达的gi偶联受体的有丝分裂作用。在所测试的激动剂中，内皮素通过内皮素- b受体在纤维连接蛋白存在的情况下最有效地刺激[^3H]胸苷的结合。这与ERK的磷酸化有关，PTX部分抑制内皮素刺激的DNA合成和ERK的磷酸化。这些发现表明，在发育中的大脑神经祖细胞中，Gi2介导来自内皮素- b受体等受体的信号传导，以维持有丝分裂活性。少

In this study, we investigate the molecular mechanisms of Gαq/11-induced apoptosis in m1 muscarinic acetylcholine receptor-expressing HeLa cells. Overexpression of the constitutively active mutant of Akt inhibited carbachol-induced ROCK-I cleavage. Insulin, a major survival factor in many cells, strongly increased phosphorylation of Akt, which was completely blocked by carbachol. This latter effect was partially inhibited by treatment with the tyrosine phosphatase inhibitor, orthovanadate. In parallel with these observations, carbachol attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1, an effect eliminated by orthovanadate. On the other hand, carbachol induced rapid stimulation of endogenous RhoA, and expression of a constitutively active mutant of RhoA increased ROCK-I cleavage. Orthovanadate and the dominant negative mutant of RhoA partially, and their combination completely, inhibited carbachol-induced ROCK-I cleavage and apoptosis. These results … More demonstrate that Ga/11 signaling induces apoptosis by reducing insulin-stimulated Akt phosphorylation through tyrosine dephosphorylation and activating RhoA in HeLa cells.In order to clarify the role of Gi2 in the ventricular zone of embryonic brains, we administered pertussis toxin (PTX) into the lateral ventricle of mouse brains at E14.5. Examination at E18.5 revealed that PTX decreased total cells and bromodeoxyuridine-positive cells in the cerebral cortices, suggesting impaired proliferation of neuroepithelial cells. Then we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors, which are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [^3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of ERK, and PTX partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of ERK. These findings indicated that Gi2 mediates signaling from receptors such as endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain. Less