SIGNALING CASCADE OF RHOA MEDIATED PLD ACTIVATION

在过去的十年里，对信号通路的研究变得越来越复杂。这些研究得出了一个不可避免的结论，即这些级联的输出取决于这些成分在细胞内的组织方式。因此，了解这些被划分的成分是如何调控的机制是很重要的。我们在一个系统中很好地研究了这一机制，α -凝血酶刺激RhoA选择性易位到细胞核，导致PLD活性增加。本提案的重点是这个信号级联的关键要素。目的：我将通过确定哪种G蛋白将α -凝血酶受体偶联到RhoA介导的核PLD激活来识别直接受体后信号传导成分。我们的策略是确定Galpha亚基或β - γ二聚体是否参与这种激活，并通过反义消融或突变α亚基的表达确定G蛋白同型。Aim II将定义导致rhoa介导的核PLD激活的信号级联的特异性。我们将确定其他膜（质膜和内质网）中的PLD活性是否由α -凝血酶诱导并以类似的RhoA依赖方式调节，以及这些活性的激活是否由Aim i中鉴定的相同异三聚体G蛋白介导。Aim III的重点是鉴定RhoA中控制其靶向的结构域。我们将验证RhoA的“高变c端”或N端/效应域参与RhoA的选择性定位的假设。这将通过检查嵌合RhoA结构的能力来完成，其中n端或C端被其他小G蛋白的相应区域取代，用于靶向和PLD激活。

Over the past decade, studies of signaling pathways have become increasingly more sophisticated. These studies have lead to the inescapable conclusion that the output of these cascades is dependent on how the components are organized within the cell. Consequently, it is important to understand the mechanism by which these compartmentalized components are regulated. We are in an excellent position to study this mechanism in one system, the alpha-thrombin stimulated selective translocation of RhoA to the nucleus resulting in an increase in PLD activity. This proposal is focused on key elements of this signaling cascade. Aim I will identify the immediate post-receptor signaling component by determining which G protein couples the alpha-thrombin receptor to RhoA- mediated nuclear PLD activation. Our strategy is to establish whether Galpha subunits or betagamma dimers are involved in this activation and identify the G protein isotype by anti-sense ablation or expression of mutant alpha subunits. Aim II will define the specificity of the signaling cascade leading to the RhoA-mediated activation of nuclear PLD. We will determine whether PLD activity in other membranes, the plasma membrane and ER (endoplasmic reticulum), is induced by alpha- thrombin and regulated in a similar RhoA-dependent manner and whether the activation of these activities are mediated by the same heterotrimeric G protein identified in Aim I. Aim III is focused on identifying the domains in RhoA that govern it's targeting. We will test the hypothesis that either the "hypervariable C-terminus" or N- terminus/effector domain of RhoA is involved in the selective localization of RhoA. This will be accomplished by examining the ability of chimeric RhoA constructs, in which the N-terminus or C- terminus is replaced by the corresponding regions in other small G proteins, on targeting and PLD activation.