SIGNALING CASCADE OF RHOA MEDIATED PLD ACTIVATION

在过去的十年中，信号通路的研究已经变得越来越复杂。 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细胞内组件的组织方式。因此，重要的是要了解这些区室化成分的调节机制。 我们处于一个很好的位置来研究这种机制，在一个系统中，α-凝血酶刺激RhoA的选择性易位到细胞核中，导致PLD活性的增加。 该建议的重点是这个信号级联的关键要素。目的通过确定哪种G蛋白将α-凝血酶受体与RhoA介导的核PLD激活偶联，确定受体后直接信号传导成分。 我们的策略是建立是否G α亚基或β γ二聚体参与这种激活，并确定G蛋白的同种型的反义消融或突变体α亚基的表达。 目的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PLD在其他膜（质膜和内质网）中的活性是否由α-凝血酶诱导并以类似的RhoA依赖性方式调节，以及这些活性的激活是否由Aim I中鉴定的相同异源三聚体G蛋白介导。 目标III的重点是确定RhoA中控制其靶向的结构域。 我们将检验RhoA的“高变C-末端”或N-末端/效应结构域参与RhoA的选择性定位的假设。 这将通过检查嵌合RhoA构建体的靶向和PLD活化能力来实现，其中N-末端或C-末端被其他小G蛋白中的相应区域替换。

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.