Composition and Functions of the Integrin Activation Complex

描述（由申请方提供）：血栓形成和止血中血小板聚集需要整合素活化，并且整合素活化在内皮细胞迁移和血管生成中起重要作用。我们以前的工作导致发现了一种整合素激活复合物（IAC），含有RAP相互作用衔接子分子（RIAM），talin和整合素。为了验证IAC包含特征性蛋白质群体的假设，我们将通过使用新的串联亲和标签方法纯化与RIAM复合的未被占据的整合素并利用质谱法来指定这些分离的复合物的蛋白质制图（蛋白质组），通过建立其蛋白质组成来详细表征IAC。生物信息学将用于将鉴定的蛋白质组投射到物理、遗传和途径蛋白质-蛋白质相互作用网络上，以帮助选择鉴定的IAC组分，通过直接免疫沉淀和纯化的蛋白质-蛋白质相互作用进行验证。为了检验这一假设，即不同组成的IAC存在于特定的细胞隔室，我们将设计一种新的双分子荧光互补方法来可视化IAC，并将其位置与特定的细胞区域，如板状体或特定的囊泡群体。为了验证IAC组分在IAC的形成或行为以及整合素活化和细胞迁移中具有特定作用的假设，我们将研究消除IAC组分对IAC运动、整合素活化、细胞迁移、肌动蛋白流动和突起形态动力学的影响。这些研究将建立生物化学和细胞生物学手段来表征和研究控制整合素活化和肌动蛋白动力学和细胞迁移的整合素依赖性调节的蛋白质的模块化组装。因此，这项工作将为动脉血栓形成和炎症中整合素激活机制提供新的见解。这些研究还将有助于深入了解控制血管细胞迁移的机制。

DESCRIPTION (provided by applicant): Integrin activation is required for platelet aggregation in thrombosis and hemostasis and plays an important role in the migration of endothelial cells and in angiogenesis. Our previous work led to the discovery of an Integrin Activation Complex (IAC) containing Rap-interacting Adaptor Molecule (RIAM), talin, and integrins. To test the hypothesis that the IAC contains a characteristic population of proteins, we will characterize the IAC in detail by establishing its protein composition by use of a novel tandem affinity tag approach to purify unoccupied integrins in complex with RIAM and utilize mass spectrometry to specify the protein cartography (proteome) of these isolated complexes. Bioinformatics will be used to project the identified proteome onto physical, genetic, and pathway protein-protein interaction networks to help select identified IAC components for validation by direct immunoprecipitation and purified protein-protein interactions. To test the hypothesis to that IACs of differing composition exist in specific cellular compartments, we will devise a novel bimolecular fluorescence complementation method to visualize the IAC and relate its position to specific cellular locales e.g. lamellipodium or specific vesicle populations. To test the hypothesi to that components of the IAC have specific roles in the formation or behavior of the IAC and in integrin activation and cell migration, we will examine the effects of elimination of IAC components on the movements of the IAC, integrin activation, cell migration, actin flow, and protrusion morphodynamics. These studies will establish biochemical and cell biological means to characterize and study modular assemblies of proteins that control integrin activation and the integrin-dependent regulation of actin dynamics and cell migration. The work will therefore provide new insights into the mechanisms of integrin activation involved in arterial thrombosis and inflammation. The studies will also contribute insights into mechanisms controlling the migration of vascular cells.