Identification of beta 1 integrin activating proteins

β1 整合素激活蛋白的鉴定

基本信息

批准号：
7293763
负责人：
DAVID A CALDERWOOD
金额：
$ 24.75万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7293763
关键词：
Adhesives Affinity Area Atherosclerosis Binding Binding Proteins Biological Assay Blood Platelets Cardiovascular Diseases Cardiovascular system Cell Adhesion Cell Separation Cell surface Cells Collecting Cell Complement Condition Cytoplasmic Tail Cytoskeletal Proteins DNA Library Data Databases Development Dominant-Negative Mutation Elements Event Exhibits Expression Library Extracellular Matrix Family Funding Mechanisms Gene Proteins Genes Integral Membrane Protein Integrin Binding Integrins Lead Leukocyte Trafficking Libraries Life Ligands Mediating Methodology Methods Microarray Analysis Mining Molecular Mutation Myocardial Infarction Pathologic Neovascularization Pathway interactions Physiological Platelet Activation Platelet Factor 4 Point Mutation Population Positioning Attribute Proteins Proteomics Purpose Regulation Reperfusion Injury Research Project Grants Reverse Transcriptase Polymerase Chain Reaction Risk Screening procedure Series Signal Pathway Small Interfering RNA Specificity Stroke Tail Talin Techniques Thrombosis Thrombus Transmembrane Domain Variant Work adhesion receptor angiogenesis base cDNA Expression cDNA Library cell motility expression cloning extracellular insight mutant novel small hairpin RNA therapeutic target

项目摘要

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.

描述（由申请人提供）：该探索性和开发性研究项目申请的目的是鉴定参与调节的蛋白质 ¿ 1 整合素粘附受体是由 ? 和 ¿ 形成的异二聚体。亚基 - 每个亚基都是 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部。整合素粘附受体的激活能力（对其细胞外配体的亲和力的快速调节变化）至关重要。心血管系统的发育和功能不当会导致血栓形成、动脉粥样硬化、心肌梗塞、中风和再灌注损伤，因此，整合素是治疗调节这些疾病以及抑制和刺激血管生成的靶标，因此，对控制整合素激活的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。了解调节整合素激活的分子机制我们之前表明细胞骨架蛋白talin与¿ 3 亚基胞质尾对于血小板整合素 IIb 的激活是必要且充分的3. 我们的新数据表明，talin 也是必需的，但不足以实现 ¿ 1 整合素激活并表明其他 ¿还需要 1 个尾部结合因子，我们的目标是识别和表征这些附加因子，具体而言，我们的目标是：1) 使用表达克隆来识别与 talin 协同激活的蛋白质。 1 整合素：2) 找到 ¿ 所需的蛋白质1 使用 siRNA 筛选整合素激活：3) 识别与 ¿ 结合的蛋白质1 细胞质尾部因突变而改变，我们认为这会损害第二个激活因子的结合：以及 4) 表征已识别的蛋白质，我们将依靠我们在基于 FACS 的测定中评估活细胞中整合素激活的能力。细胞与talin和cDNA表达文库并收集细胞在¿ 1个整合素已被激活；转染的文库cDNA将被回收、重新筛选和测序，以鉴定与talin配合激活的基因¿ 1 整合素也将用 siRNA 文库转导，并收集表达失活整合素的细胞；并通过微阵列分析鉴定在失活细胞群中富集的序列。我们已经确定了 ¿ 的点突变。 1 尾部似乎抑制 ¿ 的结合1 激活因子。我们将应用蛋白质分析和鉴定技术来鉴定能够与野生型但不能与突变体结合的蛋白质。最后，我们将对已识别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它们周围的细胞外基质之间的粘附相互作用对于心血管系统和细胞的发育和功能至关重要。不适当的细胞粘附会导致心血管疾病。称为整合素的细胞表面粘附受体介导许多细胞粘附相互作用，并且它们的活性通常受到严格调节。对调节整合素功能的分子机制的理解。