SIGNALING CASCADE OF RHOA MEDIATED PLD ACTIVATION

RHOA 介导的 PLD 激活的信号级联

• 批准号: 2829133
• 负责人: Daniel M. Raben
• 金额: $ 31.23万
• 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/2829133
• 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，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，即这些级联的输出取决于细胞内各成分的组织方式。因此，了解这些分隔成分的调节机制非常重要。 我们处于一个极好的位置，可以在一个系统中研究这一机制，α-凝血酶刺激 RhoA 选择性易位至细胞核，导致 PLD 活性增加。 该提案重点关注信号级联的关键要素。目标我将通过确定哪种 G 蛋白将 α-凝血酶受体与 RhoA 介导的核 PLD 激活偶联来识别紧接的受体后信号传导成分。 我们的策略是确定 Galpha 亚基或 betaamma 二聚体是否参与这种激活，并通过反义消融或突变 alpha 亚基的表达来鉴定 G 蛋白同种型。 目标 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 ER（内质网）中的 PLD 活性是否由 α-凝血酶诱导并以类似的 RhoA 依赖性方式进行调节，以及这些活性的激活是否由相同的异源三聚体 G 蛋白介导目标 I 中确定的目标。目标 III 的重点是确定 RhoA 中控制其目标的域。 我们将测试 RhoA 的“高变 C 末端”或 N 末端/效应结构域参与 RhoA 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 激活能力来实现，其中 N 末端或 C 末端被其他小 G 蛋白中的相应区域取代。