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.
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