Activation trajectories of integrin α5β1

整合素 α5α1 的激活轨迹

• 批准号: 10545063
• 负责人: TIMOTHY A SPRINGER
• 金额: $ 73.43万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-20 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10545063
• 关键词: Acceleration; Actins; Adhesions; Affect; Affinity; Amino Acids; Atherosclerosis; Award; Binding; Binding Proteins; Binding Sites; Biological Process; Blood Cells; Cell Adhesion; Cell Communication; Cell Surface Proteins; Cell Surface Receptors; Cell surface; Cells; Compensation; Coupling; Cytoskeleton; Data; Disease; Disputes; Dissociation; Disulfides; Divalent Cations; Environment; Equilibrium; Erythropoiesis; Event; Extracellular Matrix; Fibronectins; Fibrosis; Fluorescence Resonance Energy Transfer; Free Energy; Grant; Hematopoiesis; Integrin Binding; Integrin alpha5beta1; Integrin beta Chains; Integrins; Ions; Kinetics; Knee; Ligand Binding; Ligands; Link; Malignant Neoplasms; Maps; Measurement; Measures; Mediating; Membrane; Metal Ion Binding; Metals; Molecular Conformation; Mutate; Mutation; Pathology; Play; Population; Positioning Attribute; Reaction; Reporting; Research; Rest; Role; Shapes; Sickle Cell Anemia; Site; Structure; Supporting Cell; Testing; Tissues; Titrations; Total Internal Reflection Fluorescent; Transmembrane Domain; Work; cell motility; conformational conversion; cyanine dye 5; drug development; experimental study; extracellular; fluorophore; insight; monocyte; rate of change; single molecule; temporal measurement; tool

Abstract. Integrins are cell surface receptors that mediate numerous interactions between cells and their environment. Binding of integrin α5β1 to its ligand fibronectin in the extracellular matrix plays fundamental roles in cellular adhesion and differentiation. In blood cells these interactions mediate biological processes including erythropoiesis and monocyte adhesion and contribute to pathologies such as sickle cell disease, dysregulation of hematopoiesis, atherosclerosis, and fibrosis. Integrin α5β1 undergoes two distinct conformational changes: extension at the ‘knees’ and opening of the ligand-binding headpiece. These changes give rise to an ensemble of three interconverting integrin conformational states on cell surfaces: low-affinity bent-closed (BC) and extended-closed (EC) conformations and a high-affinity extended-open (EO) conformation. This proposal leverages ground-breaking work under the previous award in which we measured free energy and intrinsic affinity of each integrin α5β1 state. Using the same Fab tools as used for these equilibrium measurements to stabilize the extended, closed, or open α5β1 conformations, we now propose three aims. In Aim 1, we explore how Mn2+, high Mg2+, and low Ca2+ concentrations activate integrins. Our preliminary results show that Mn2+ and high Mg2+ both increase the population of the EO state and increase its intrinsic affinity for ligand and that these effects are dependent on the ADMIDAS metal-ion binding site. To examine why cell surface α5β1 is so stable in the BC state, we test the hypothesis that the α and β-subunit TM domains separate from one another in both the EC and EO states. Aims 2 and 3 measure kinetics to map the activation trajectory of integrin α5β1, i.e. the sequence of ligand binding and conformational change events that occur between the resting state, when 99.8% of unliganded integrin α5β1 is in the BC state, and the final, functional liganded EO state (EO•L) state that is bound to fibronectin and is stabilized by tensile force that is applied to the integrin by actin retrograde flow and resisted by fibronectin in the matrix. In Aim 2, we measure the intrinsic ligand-binding kinetics of each state (kon and koff). Our preliminary data indicates, surprisingly, that the low-affinity BC and EC states bind more rapidly to ligand than the EO state, which is compensated by the >10,000-fold slower off-rate of the EO state. In Aim 3, we measure the kinetics of integrin conformational change using single-molecule FRET probes that measure either the extension or opening steps in the presence or absence of conformation- specific Fabs and ligand. Kinetics of all transitions between the BC, EC, and EO states for unliganded and ligand-bound single integrin molecules will be determined for both purified, soluble ectodomain and intact integrins on blood cells using TIRF microscopy with high temporal resolution. We expect to show an integrin activation trajectory in which ligand binds to the BC+EC states, followed by ligand-facilitated conformational conversion to the EO•L state, followed by cytoskeletal adaptor (A) binding and stabilization of the EO•L•A state by force applied by the cytoskeleton and resisted by extracellular ligand.

抽象的。 整联蛋白是细胞表面受体，可介导细胞及其环境之间的大量相互作用。 整联蛋白α5β1与其在细胞外基质中的配体纤连蛋白的结合在细胞中起基本作用 粘附和分化。在血细胞中，这些相互作用媒体生物过程包括 红细胞生成和单核细胞粘合剂并导致镰状细胞病，失调等病理 造血，动脉粥样硬化和纤维化。整合素α5β1经历了两个不同的构象变化： 在“膝盖”和配体结合头饰的打开处延伸。这些变化引起了合奏 在细胞表面上的三个相互转化的整联蛋白构象状态：低亲和力弯曲（BC）和 扩展闭合（EC）构象和高亲和力扩展（EO）构象。这个建议 在上一个奖项下，我们测量了自由能和内在的奖项，利用了上一项奖项的开创性工作 每个整合素α5β1状态的亲和力。使用与这些平衡测量相同的工具工具 稳定扩展，封闭或开放的α5β1构象，我们现在提出三个目标。在AIM 1中，我们探索 MN2+，高Mg2+和低Ca2+浓度如何激活整联蛋白。我们的初步结果表明MN2+ 高MG2+都增加了EO状态的人口，并增加了对配体的内在亲和力，并且 这些作用取决于Admidas Metal-Ion结合位点。检查为什么细胞表面α5β1如此 在卑诗省状态下，我们测试了α和β-亚基TM域彼此分开的假设 在EC和EO国家中。目标2和3测量动力学来绘制整合素α5β1的激活轨迹， 即配体结合和构象变化事件的序列，在静止状态之间发生， 当99.8％的非配体整联蛋白α5β1处于BC状态时，最终的功能性配体EO状态（EO•L） 与纤连蛋白结合并通过肌动蛋白应用于整联蛋白的拉伸力稳定的状态 逆行流动，并由基质中的纤连蛋白抵抗。在AIM 2中，我们测量固有的配体结合 每个州的动力学（Kon和Koff）。我们的初步数据表明，低亲和力BC和EC 与EO状态相比，状态与配体更快地结合，EO状态被速度慢10,000倍弥补 eo状态。在AIM 3中，我们使用单分子测量整联蛋白构象变化的动力学 在存在或不存在构象的情况下测量延伸或开放步骤的有点问题 - 特定的晶圆厂和配体。卑诗省，EC和EO国家之间所有过渡的动力学 将确定纯化的，可溶性外域和完整的结合配体单一整合素分子 使用具有高临时分辨率的TIRF显微镜在血细胞上的整合素。我们希望展示一个整合素 配体与BC+EC状态结合的激活轨迹，其次是配体辅助构象 转换为EO•L状态，然后转换为细胞骨架适配器（a）EO•l•a•a•a的结合和稳定 通过细胞骨架施加的力并由细胞外配体抵抗。