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组件在细胞内的组织方式。因此，重要的是要了解这些被划分的成分被调节的机制。我们处于一个很好的位置来研究这一机制，在一个系统中，α-凝血酶刺激RhoA选择性易位到细胞核，导致PLD活性增加。本提案侧重于这一信令级联的关键要素。目的我将通过确定哪种G蛋白将α-凝血酶受体与RhoA介导的核磷脂酶脱氢酶激活偶联来鉴定受体后的即刻信号成分。我们的策略是确定Galpha亚基或Betagamma二聚体是否参与这种激活，并通过反义消融或表达突变的Alpha亚基来鉴定G蛋白同型。目的II将确定导致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(ER)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中确定的相同异三聚体G蛋白介导。Aim III的重点是确定RhoA中控制其靶向的结构域。我们将检验这一假设，即RhoA的“高变量C-末端”或N-末端/效应域参与RhoA的选择性定位。这将通过检测嵌合RhoA结构的能力来实现，在嵌合RhoA结构中，N末端或C末端被其他小G蛋白中的相应区域所取代，对靶向和PLD激活的能力。