Regulation of Actin Assembly and Cell Motility by IQGAPs

描述（由申请人提供）：本申请的总体目标是确定IQGAP1和其他哺乳动物iqgap如何通过Arp2/3复合物调节肌动蛋白丝成核，进而调节细胞运动。在像人类这样的多细胞动物中，细胞运动和形状变化是多种生物过程的基础，如生物体发育、伤口愈合、肿瘤转移和突触可塑性。因此，揭示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。现在似乎很清楚，许多形式的动物细胞运动和形态发生是由由Arp2/3复合物成核的支状肌动蛋白丝的极化组装引起的。Arp2/3复合物的这种活性不是构成性的。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Arp2/3复合物的最大刺激需要额外的因子将N-WASP从自抑制状态中释放出来。在过去的几年中，已经发现了一些这样的因子，最显著的是激活Cdc42和PIP2。最近，申请人的实验室发现IQGAP1可以通过需要N-WASP和Arp2/3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，IQGAP1是体内Arp2/3复合物的一个主要的、以前未被识别的调节因子，并为目前的应用奠定了基础。提出了三个具体目标。1) IQGAP1通过Arp2/3复合物调控肌动蛋白组装的分子机制将通过体外重构确定。2)验证哺乳动物除IQGAP1外的其他iqgap也能调控Arp2/3复合物的假说。其中包括已知存在于蛋白质水平的IQGAP2，以及从基因组DNA和表达序列标签（ESTs）推断出的IQGAP3。将使用免疫学方法、质谱法、原位杂交和RTPCR来寻找IQGAP3在蛋白质水平上表达的证据。3)通过IQGAP1上的靶结构域与配体激活的细胞表面受体的细胞质尾部结合，从而触发IQGAP1在细胞内刺激肌动蛋白组装的假设进行验证。IQGAP1对肌动蛋白组装的调控及其对细胞运动的影响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进行研究。类似的研究将开始于其他哺乳动物的iqgap，这些iqgap是通过寻求特异性靶蛋白2通过Arp2/3复合物调节肌动蛋白组装而显示的。

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.