Identification of beta 1 integrin activating proteins

描述(申请人提供)：这项探索性和发展性研究项目申请的目的是鉴定参与调节整合素激活的蛋白质。整合素黏附受体是由？和亚基--每个亚基都是一种I型跨膜蛋白，具有较大的胞外部分、单一的跨膜结构域和较短的细胞质尾巴。整合素黏附受体的激活能力(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是必不可少的。不适当的整合素激活会导致血栓、动脉粥样硬化、心肌梗死、中风和再灌注损伤，整合素是治疗这些疾病的靶点，也是抑制和刺激血管生成的靶点。因此，对整合素激活控制机制的详细了解与旨在了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。我们以前证明了细胞骨架蛋白talin与3亚单位胞浆尾巴的结合是激活血小板整合素IIb的必要条件和充分条件。我们的新数据表明talin也是整合素激活所必需的，但不是充分的，并表明其他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。具体地说，我们的目标是：1)使用表达克隆来识别与talin合作激活整合素的蛋白质：2)使用siRNA筛选找到整合素激活所需的蛋白质：3)鉴定其与1细胞质尾巴的结合因突变而改变的蛋白质，我们认为这种突变会损害第二激活因子的结合：以及4)鉴定已鉴定的蛋白质。为了实现这些目标，我们将依赖于我们在基于FACS的检测中评估活细胞整合素激活的能力。我们将用talin和一个cdna表达文库转染细胞，并收集1整合素激活的细胞；我们将回收、重新筛选和测序导入的文库，以确定与talin协同激活1整合素的基因。还将利用收集的siRNA文库和表达失活整合素的细胞对靶细胞群体进行转导；将恢复siRNA序列，并通过微阵列分析鉴定失活细胞群体中的序列。这应该会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个尾部的点突变，这些突变似乎抑制了1个激活因子的结合。我们将应用蛋白质图谱和鉴定技术来鉴定能够与野生型尾巴结合但不能与突变尾巴结合的蛋白质。最后，我们将对识别出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其周围的细胞外基质之间的黏附相互作用对于心血管系统的发育和功能是至关重要的，而不适当的细胞黏附会导致心血管疾病。被称为整合素的细胞表面黏附受体介导了细胞的许多黏附相互作用，它们的活动通常受到严格的调控。我们的目标是鉴定和鉴定调控整合素活性的蛋白质，以期了解调控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.