SIGNALING CASCADE OF RHOA MEDIATED PLD ACTIVATION

RHOA 介导的 PLD 激活的信号级联

• 批准号: 6386458
• 负责人: Daniel M. Raben
• 金额: $ 24.66万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386458
• 关键词: G protein; biological signal transduction; cell component structure /function; cell membrane; chimeric proteins; endoplasmic reticulum; enzyme activity; fluorescence microscopy; guanine nucleotide binding protein; immunologic assay /test; molecular cloning; nuclear membrane; phospholipase D; protein localization; protein sequence; protein structure function; radiotracer; thrombin; tissue /cell culture

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.

在过去的十年中，对信号通路的研究变得越来越复杂。 这些研究得出了不可避免的结论，即这些级联反应的输出取决于细胞内部的组件如何组织。因此，重要的是要了解这些分隔组件的调节机制。 我们在一个系统中研究这种机制的良好位置，α-凝血酶刺激了RhoA的选择性易位，从而导致PLD活性的增加。 该提案的重点是该信号级联的关键要素。目的，我将通过确定哪种G蛋白将α-凝血酶受体伴侣与RhoA介导的核PLD激活来确定直接的受体后信号传导成分。 我们的策略是确定Galpha亚基或Betagamma二聚体是否参与了这种激活，并通过突变α亚基的抗渗透消融或表达来鉴定G蛋白同种异体。 AIM II将定义信号级联的特异性，导致RhoA介导的核PLD激活。 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. 我们将检验以下假设：RhoA的“高变量C端”或N-末端/效应子域参与RhoA的选择性定位。 这将通过检查嵌合RhoA构建体的能力来实现，其中N末端或C末端被其他小G蛋白中的相应区域代替了靶向和PLD激活。