Identification of beta 1 integrin activating proteins

β1 整合素激活蛋白的鉴定

• 批准号: 7449516
• 负责人: DAVID A CALDERWOOD
• 金额: $ 20.69万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7449516
• 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

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）：本探索性和开发性研究项目申请的目的是鉴定参与调节1整合素活化的蛋白质。整合素粘附受体是异源二聚体形成的？然后，每个亚基是具有大的胞外部分、单个跨膜结构域和短的胞质尾区的I型跨膜蛋白。整合素粘附受体经历活化（对其细胞外配体的亲和力的快速调节变化）的能力对于心血管系统的发育和功能是必不可少的。不适当的整联蛋白活化有助于血栓形成、动脉粥样硬化、心肌梗塞、中风和再灌注损伤，并且整联蛋白是这些病症的治疗调节以及抑制和刺激血管生成的靶标。因此，对控制整合素活化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。我们的长期目标是了解调节整合素活化的分子机制。我们以前表明，结合的细胞骨架蛋白talin的<$3亚基胞质尾是必要的，足以激活血小板整合素？第二部分第3节。我们的新数据表明，talin也是必需的，但不足以激活<$1整联蛋白，并表明其他<$1尾结合因子也是必需的。我们的目标是确定和描述这些额外的因素。具体而言，我们的目标是：1）使用表达克隆来鉴定与talin合作以激活<$1整联蛋白的蛋白质：2）使用siRNA筛选来寻找<$1整联蛋白激活所需的蛋白质：3）鉴定其与<$1胞质尾的结合被突变改变的蛋白质，我们建议该突变损害第二激活因子的结合;以及4）表征鉴定的蛋白质。为了实现这些目标，我们将依赖于我们在基于FACS的测定中评估活细胞中整合素活化的能力。我们将用talin和cDNA表达文库转染细胞，并收集其中α 1整联蛋白已被激活的细胞;将回收转染的文库cDNA，重新筛选并测序以鉴定与talin合作激活α 1整联蛋白的基因。还将用siRNA文库转导靶细胞群，并收集表达失活整联蛋白的细胞;将回收siRNA序列，并通过微阵列分析鉴定在失活细胞群中富集的序列。这将揭示维持整合素活化所必需的基因和蛋白质。我们已经确定了在<$1尾部的点突变，似乎抑制了<$1激活因子的结合。我们将应用蛋白质谱分析和鉴定技术来鉴定能够结合野生型但不能结合突变体尾部的蛋白质。最后，我们将鉴定蛋白质的生物化学特性，并试图将它们置于整合素激活途径。 细胞之间以及细胞与其周围的细胞外基质之间的粘附相互作用的严格控制对于心血管系统的发育和功能是必不可少的，并且不适当的细胞粘附有助于心血管疾病。称为整合素的细胞表面粘附受体介导许多细胞的粘附相互作用，并且它们的活性通常受到严格调节。我们的目的是确定和表征的蛋白质，调节整合素的活性，以期了解整合素功能调节的分子机制。