Regulation of Actin Assembly and Cell Motility by IQGAPs

IQGAP 对肌动蛋白组装和细胞运动的调节

• 批准号: 6914093
• 负责人: George S Bloom
• 金额: $ 33.07万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6914093
• 关键词: actins; binding sites; biological signal transduction; cell cycle proteins; cell morphology; cell motility; cell surface receptors; fluorescence resonance energy transfer; immunoprecipitation; in situ hybridization; laboratory mouse; light microscopy; mass spectrometry; microfilaments; molecular assembly /self assembly; polymerase chain reaction; protein binding; protein reconstitution; tissue /cell culture; yeast two hybrid system

DESCRIPTION (provided by applicant): The overall goal of this application is to determine how IQGAP1 and other mammalian IQGAPs regulate actin filament nucleation by the Arp2/3 complex, and by extension, cellular motility. In multicellular animals like humans, cell movements and shape changes underly biological process as diverse as organismal development, wound healing, tumor metastasis, and synaptic plasticity. Unraveling the molecular mechanisms that allow cells to move and change shape has therefore been a major goal. It now seems clear that many forms of animal cell motility and morphogenesis are caused by the polarized assembly of branched actin filaments nucleated by the Arp2/3 complex. This activity of Arp2/3 complex is not consitutive. Instead, it relies on activating factors, like N-WASP and other members of the WASP/WAVE family. Likewise, maximal stimulation of Arp2/3 complex by N-WASP requires additional factors that release N-WASP from an autoinhibited state. A few such factors have been discovered within the past few years, most notably activated Cdc42 and PIP2. Recently, the applicant's laboratory discovered that IQGAP1 can potently stimulate actin assembly in vitro by mechanisms that require both N-WASP and Arp2/3 complex. These seminal observations suggest that IQGAP1 is a major, previously unrecognized regulator of the Arp2/3 complex in vivo, and serves as the foundation of the present application. Three Specific Aims are proposed. 1) The molecular mechanisms by which IQGAP1 regulates actin assembly through Arp2/3 complex will be determined by in vitro reconstitution. 2) The hypothesis that mammalian IQGAPs other than IQGAP1 also regulate Arp2/3 complex will be tested. These include IQGAP2, which is known to exist at the protein level, and IQGAP3, which has been inferred from genomic DNA and expressed sequence tags (ESTs). Evidence for expression of IQGAP3 at the protein level will be sought using immunological methods, mass spectrometry, in situ hybridization, and RTPCR. 3) A test will be made of the hypothesis that stimulation of actin assembly by IQGAP1 in cells is triggered by binding of a targetting domain on IQGAP1 to the cytoplasmic tails of ligand-activated cell surface receptors. The regulation of actin assembly by IQGAP1, and the consequences for cell motility will be studied in cultured mammalian cells by light microscopic methods and immunoprecipitation of cellular protein complexes. Similar studies will be initiated for other mammalian IQGAPs shown by pursuit of Specific Aim 2 to regulate actin assembly through the Arp2/3 complex.

描述（由申请人提供）：本申请的总体目标是确定IQGAP 1和其他哺乳动物IQGAP如何通过Arp 2/3复合物调节肌动蛋白丝成核，并通过延伸调节细胞运动。在人类等多细胞动物中，细胞运动和形状变化是生物体发育、伤口愈合、肿瘤转移和突触可塑性等多种生物过程的基础。因此，解开允许细胞移动和改变形状的分子机制一直是一个主要目标。现在看来很清楚，许多形式的动物细胞运动和形态发生是由Arp 2/3复合物成核的分支肌动蛋白丝的极化组装引起的。Arp 2/3复合物的这种活性不是结构性的。相反，它依赖于激活因子，如N-WASP和WASP/WAVE家族的其他成员。同样，N-WASP对Arp 2/3复合物的最大刺激需要额外的因子将N-WASP从自抑制状态释放出来。在过去的几年中，已经发现了一些这样的因子，最值得注意的是激活的Cdc 42和PIP 2。最近，申请人的实验室发现IQGAP 1可以通过需要N-WASP和Arp 2/3复合物的机制在体外有效地刺激肌动蛋白组装。这些开创性的观察结果表明，IQGAP 1是体内Arp 2/3复合物的一种主要的、以前未被认识到的调节剂，并作为本申请的基础。提出了三个具体目标。1)IQGAP 1通过Arp 2/3复合物调节肌动蛋白组装的分子机制将通过体外重建来确定。2)将检验哺乳动物IQGAP而不是IQGAP 1也调节Arp 2/3复合物的假设。这些包括已知存在于蛋白质水平的IQGAP 2，以及从基因组DNA和表达序列标签（EST）推断的IQGAP 3。将使用免疫学方法、质谱、原位杂交和RTPCR寻找IQGAP 3在蛋白水平表达的证据。3)将对以下假设进行检验：IQGAP 1对细胞中肌动蛋白组装的刺激是通过IQGAP 1上的靶向结构域与配体激活的细胞表面受体的胞质尾结合而触发的。将在培养的哺乳动物细胞中通过光镜方法和细胞蛋白复合物的免疫沉淀来研究IQGAP 1对肌动蛋白组装的调节以及对细胞运动的后果。类似的研究将启动其他哺乳动物IQGAP的追求特定目标2通过Arp 2/3复合物调节肌动蛋白组装。