Identification of beta 1 integrin activating proteins

描述（由申请人提供）：本探索性和发展性研究项目申请的目的是鉴定参与整合素激活调节的蛋白质。整合素粘附受体是由？亚基-每一个都是I型跨膜蛋白，具有较大的细胞外部分，单个跨膜结构域和短的细胞质尾。整合素粘附受体的激活能力（对其细胞外配体亲和力的快速调节变化）对心血管系统的发育和功能至关重要。不适当的整合素激活会导致血栓形成、动脉粥样硬化、心肌梗死、中风和再灌注损伤，而整合素是这些疾病的治疗调节靶点，同时也抑制和刺激血管生成。因此，对控制整合素激活机制的详细了解与旨在理解和控制心血管疾病和中风的策略直接相关。我们的长期目标是了解调节整合素激活的分子机制。我们之前的研究表明，细胞骨架蛋白talin与3亚基细胞质尾部的结合是激活血小板整合素IIb¿3的必要和充分条件。我们的新数据表明，talin也是必需的，但不足以激活¿1整合素，并表明其他¿1尾巴结合因子也是必需的。我们的目标是识别和描述这些额外的因素。具体来说，我们的目标是：1)使用表达克隆来鉴定与talin合作激活¿1整合素的蛋白质；2)使用siRNA筛选找到激活¿1整合素所需的蛋白质；3)鉴定与¿1细胞质尾部结合被突变改变的蛋白质，我们认为这种突变损害了第二激活因子的结合；4)鉴定鉴定的蛋白质。为了实现这些目标，我们将依靠我们在基于FACS的检测中评估活细胞中整合素激活的能力。我们将用talin和cDNA表达文库转染细胞，收集有¿1整合素被激活的细胞；将转染的文库cDNA恢复，重新筛选和测序，以确定与talin合作激活¿1整合素的基因。靶细胞群也将用siRNA文库和收集的表达失活整合素的细胞进行转导；siRNA序列将被恢复，并在通过微阵列分析鉴定的失活细胞群中富集序列。这将揭示维持整合素激活所必需的基因和蛋白质。我们已经确定了1号染色体尾部的点突变，这似乎抑制了1号染色体激活因子的结合。我们将应用蛋白质分析和鉴定技术来鉴定能够与野生型而不是突变型尾巴结合的蛋白质。最后，我们将对鉴定的蛋白质进行生化表征，并尝试将它们置于整合素激活途径中。严格控制细胞之间以及细胞与周围细胞外基质之间的粘附相互作用对于心血管系统的发育和功能至关重要，而不适当的细胞粘附会导致心血管疾病。被称为整合素的细胞表面粘附受体介导了许多细胞的粘附相互作用，它们的活性通常受到严格调节。我们的目的是鉴定和表征调节整合素活性的蛋白质，以期了解调节整合素功能的分子机制。

DESCRIPTION (provided by applicant): The purpose of this exploratory and developmental research project application is to identify proteins involved in the regulation of ¿1 integrin activation. Integrin adhesion receptors are heterodimers formed from ? and ¿ subunits - each of which is a type I transmembrane protein with a large extracellular portion, a single transmembrane domain and a short cytoplasmic tail. The ability of integrin adhesion receptors to undergo activation (rapid regulated changes in affinity for their extracellular ligands) is essential for the development and functioning of the cardiovascular system. Inappropriate integrin activation contributes to thrombosis, atherosclerosis, myocardial infarction, stroke and reperfusion injury, and integrins are targets for therapeutic regulation of these conditions and for both inhibition and stimulation of angiogenesis. Detailed insight into the mechanisms controlling integrin activation is therefore directly relevant to strategies aimed at understanding and controlling cardiovascular disease and stroke. Our long-term aim is to understand the molecular mechanisms regulating integrin activation. We previously showed that binding of the cytoskeletal protein talin to the ¿3 subunit cytoplasmic tail is necessary and sufficient for activation of the platelet integrin ?IIb¿3. Our new data show that talin is also required but not sufficient for ¿1 integrin activation and indicate that other ¿1 tail binding factors are also required. We aim to identify and characterize those additional factors. Specifically we aim to: 1) Use expression cloning to identify proteins that cooperate with talin to activate ¿1 integrins: 2) Find proteins required for ¿1 integrin activation using a siRNA screen: 3) Identify proteins whose binding to ¿1 cytoplasmic tails is altered by mutations which we suggest impair binding of a 2nd activating factor: and 4) Characterize identified proteins. To achieve these aims we will rely on our ability to assess integrin activation in live cells in FACS based assays. We will transfect cells with talin and a cDNA expression library and collect cells where ¿1 integrins have become activated; the transfected library cDNA will be recovered, re- screened and sequenced to identify genes that cooperate with talin to activate ¿1 integrins. Target cell populations will also be transduced with siRNA libraries and cells expressing inactivated integrins collected; siRNA sequences will be recovered and sequences enriched in the inactivated cell population identified by microarray analysis. This should reveal genes, and so proteins, essential to maintain integrin activation. We have identified point mutations in the ¿1 tail which seem to inhibit binding of a ¿1 activating factor. We will apply protein profiling and identification techniques to identify proteins ca[able of binding to the wild-type but not mutant ¿ tails. Finally, we will characterize identified proteins biochemically and attempt to place them in integrin activation pathways. Tight control of the adhesive interactions between cells and between cells and the extracellular matrix which surrounds them is essential for the development and functioning of the cardiovascular system and inappropriate cell adhesion contributes to cardiovascular disease. Cell surface adhesion receptors called integrins mediate many of the cell's adhesive interactions and their activity is normally tightly regulated. We aim to identify and characterize the proteins that regulate integrin activity with a view towards understanding the molecular mechanisms regulating integrin function.