Composition and Functions of the Integrin Activation Complex

描述(申请人提供)：整合素激活是血栓形成和止血中的血小板聚集所必需的，并在内皮细胞的迁移和血管生成中发挥重要作用。我们之前的工作导致发现了一个整合素激活复合体(IAC)，它含有Rap相互作用的接头分子(RIAM)、talin和整合素。为了验证IAC包含一个特征蛋白质组的假设，我们将通过一种新的串联亲和标签方法来确定IAC的蛋白质组成，以纯化RIAM复合体中未占据的整合素，并利用质谱学来确定这些分离的复合体的蛋白质图谱(蛋白质组)。生物信息学将被用来将识别的蛋白质组投影到物理、遗传和途径的蛋白质-蛋白质相互作用网络上，以帮助选择识别的IAC组分，以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。为了验证不同组成的IAC存在于特定细胞隔间的假设，我们将设计一种新的双分子荧光互补方法来显示IAC，并将其位置与特定的细胞位置联系起来，例如片状脂质体或特定的囊泡种群。为了验证IAC组分在IAC的形成或行为以及整合素激活和细胞迁移中具有特定作用的假设，我们将检测IAC组分的消除对IAC运动、整合素激活、细胞迁移、肌动蛋白流动和突起形态动力学的影响。这些研究将建立生化和细胞生物学手段，以表征和研究控制整合素激活和整合素依赖的肌动蛋白动态调节和细胞迁移的蛋白质的模块化组装。因此，这项工作将为整合素激活参与动脉血栓形成和炎症的机制提供新的见解。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.