Project 1 Structure and Function of kindlin-2 in vascular cells

项目1 血管细胞中kindlin-2的结构和功能

• 批准号: 8855093
• 负责人: EDWARD Franklin PLOW
• 金额: $ 43.74万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8855093
• 关键词: Actins; Activator Appliances; Adherens Junction; Adhesions; Adhesives; Agonist; Animal Model; Binding; Binding Sites; Biological; Biology; Blood; Blood Cells; Blood Platelets; Blood Vessels; Blood coagulation; C-terminal; Cell Line; Cell surface; Cells; Cellular Structures; Cellular biology; Clathrin; Collaborations; Cytoskeleton; Data; Dependence; Disabled Persons; Elements; Embryonic Development; Endothelial Cells; Enzymes; Exhibits; Funding; Genes; Hematopoietic; Hemorrhage; Hemostatic function; Human; Integrin Binding; Integrin beta3; Integrins; Investigation; Knock-in Mouse; Knowledge; Laboratories; Lead; Leukocytes; Location; Membrane; Membrane Protein Traffic; Membrane Proteins; Microfilaments; Molecular; Mouse Strains; Mus; Organism; Phenotype; Play; Process; Property; Proteins; Regulation; Role; Signal Transduction; Site; Small Interfering RNA; Specificity; Spectrometry; Structure; Structure-Activity Relationship; Tail; Testing; Tight Junctions; Time; Transmembrane Domain; Up-Regulation; Vascular Endothelial Growth Factors; Vascular Permeabilities; Whole Organism; abstracting; actin 2; angiogenesis; base; blood vessel development; cadherin 5; in vivo; matrigel; migration; mutant; novel; preference; programs; protein expression; response; trafficking; tumor

Project 1- Abstract Project 1 focuses on the regulation of the β3 integrins on cells exposed to blood. In the past funding period, evidence has been developed to demonstrate a previously underappreciated role of the kindlins in integrin function by showing that they are essential regulators of integrin activation. Kindlin-2 (K2) and kindlin-3 are known to play a particularly prominent role in controlling the functions of the two β3 integrins, αIIbβ3 and αVβ3, on platelets, endothelial cells and leukocytes. This project focuses on K2, and the preliminary data support the hypothesis that K2 regulates: 1) integrin inside-out responses as exemplified by its control of αVβ3 activation; 2) outside-in signaling via its regulation of cell spreading; and 3) integrin independent responses as exemplified by its regulation of membrane proteins expression. Consequently, K2 plays important and diverse roles in vascular biology, including the capacity to regulate: a) αVβ3 activation, which controls angiogenesis; b) outside-in signaling via a previously unrecognized and direct interaction with actin; c) integrin independent responses as demonstrated by its selective regulation of trafficking of a subset of membrane proteins in endothelial cells, which controls hemostasis, and d) interaction with proteins of adherens junctions, which controls vascular permeability. To test the elements of this hypothesis will require an in-depth understanding of K2 structure-function relationships, and animal models in which the functions of K2 can be interrogated in various biological settings. To probe the structure-function relationships of K2, mutational analyses driven by NMR studies will be performed on specific sites in the molecule that regulate its biological activities. For in vivo analyses, unique mouse strains developed in the laboratory, floxed K2 and mutant K2QW/AA knock-in mice which are unable to bind integrins, will be used. The specific aims of the project are: 1) To delineate the function of K2 in vivo in which K2 has been selectively deleted from endothelial cells, express the K2WQ/AA mutant or K2+/- mice, which has been used successively and extensively by us to identify previously unknown functions of K2. Angiogenesis, hemostasis, trafficking of membrane proteins and changes in vascular permeability will be examined in these mouse strains. 2) To resolve the molecular mechanisms, which allow K2 to orchestrate its integrin-dependent and independent functions. The basis for the preference of kindlins for the β3 integrins, the specificity of clathrin binding motif in K2, and the previously unappreciated role of the small C-terminal segment of K-2 in integrin activation will be determined. A particularly novel aspect of these studies will be to pursue the lead that K2 binds to a membrane proximal site to alter the transmembrane region of β3 and influence the activation process. 3) To determine the location, specificity and functional consequences of the direct interaction of K2 with actin. These studies will establish how K2 functions at a molecular and cellular level and how these roles manifest in the intact organism.

项目1-摘要 项目1的重点是调节暴露于血液的细胞上的β3整合素。在过去的资金期间， 已经开发了证据来证明Kindlins在整合素中的作用不足 通过证明它们是整联蛋白激活的基本调节剂的功能。 Kindlin-2（K2）和Kindlin-3是 已知在控制两个β3整联蛋白αIIBβ3和αVβ3的功能中起着特别重要的作用 在血小板，内皮细胞和白细胞上。该项目侧重于K2，初步数据支持 K2调节的假设：1）整合素内外反应，例如其对αVβ3的控制 激活; 2）通过调节细胞扩散的外部信号传导； 3）整合素独立 反应以膜蛋白表达的调节为例。因此，K2播放 重要和多样性的血管生物学作用，包括调节的能力：a）αvβ3激活，该激活 控制血管生成； b）通过先前未识别的与肌动蛋白的直接相互作用的外部信号传导； c） 整合素独立反应的选择性调节对一部分的贩运的选择性调节 内皮细胞中控制止血的膜蛋白，D）与粘附剂的蛋白质相互作用 连接，控制血管通透性。要测试该假设的元素将需要深入 了解K2结构功能关系以及K2功能的动物模型 在各种生物环境中进行询问。为了探测K2的结构功能关系，突变 由NMR研究驱动的分析将在调节其生物学的分子的特定位点进行 活动。对于体内分析，实验室中开发了独特的小鼠菌株，Floxed K2和突变体 将使用无法绑定整联蛋白的K2QW/AA敲入小鼠。该项目的具体目的是： 1）描述K2在体内的功能，其中K2从内皮细胞中选择性删除， 表达K2WQ/AA突变体或K2 +/-鼠标，我们已成功地使用了我们 确定以前未知的K2功能。血管生成，止血，膜蛋白的运输和 这些小鼠菌株将检查血管通透性的变化。 2）解决分子 机制，允许K2策划其整合素依赖性和独立功能。的基础 偏爱Kindlins对β3整合素，网格蛋白结合基序的特异性K2和以前 将确定K-2小的C末端片段在整联蛋白激活中的不批准的作用。一个 这些研究的特别新方面将是追求K2与膜近端部位结合的铅 改变β3的跨膜区域并影响激活过程。 3）确定位置， K2与肌动蛋白直接相互作用的特异性和功能后果。这些研究将确定 K2如何在分子和细胞水平上发挥作用，以及这些角色如何在完整的生物体中表现出来。