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。 整合素是细胞表面的受体，调节细胞与环境之间的大量相互作用。 细胞外基质中整合素α-5-β-1与其配体纤维连接蛋白的结合在细胞内发挥基础作用 黏附和分化。在血细胞中，这些相互作用调节生物过程，包括 红细胞生成和单核细胞黏附，导致镰状细胞病、调节失调等病理变化。 包括造血、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”处伸展，并打开配基结合头盔。这些变化产生了一个整体 细胞表面三种相互转化的整合素构象状态：低亲和力Bent-Closed(BC)和 扩展闭合(EC)构象和高亲和力扩展开放(EO)构象。这项建议 利用上一个奖项下的开创性工作，在该奖项中，我们测量了自由能和本征能量 每种整合素α5β1状态的亲和力。使用与这些平衡测量相同的FAB工具 为了稳定扩展的、封闭的或开放的α5β1构象，我们现在提出三个目标。在目标1中，我们探索 锰离子、高镁离子和低钙离子如何激活整合素。我们的初步结果表明，Mn2+ 而高的镁离子既增加了EO态的布居，又增加了它与配体的内在亲和力 这些效应依赖于ADMIDAS的金属离子结合部位。检查细胞表面α5β1为何如此 在BC状态下稳定，我们检验了α和β亚单位TM结构域相互分离的假设 在欧共体和欧共体两个州。AIMS 2和3测量动力学以绘制整合素α5β1的激活轨迹， 即在休眠状态之间发生的配体结合和构象变化事件的序列， 当99.8%的未连接整合素α5β1处于BC状态时，最终的功能连接EO状态(EO·L) 一种与纤维连接蛋白结合的状态，通过肌动蛋白施加于整合素的张力而稳定 逆行流动，并被基质中的纤维连接蛋白抵抗。在目标2中，我们测量了本征配体结合 每种状态的动力学(Kon和Koff)。我们的初步数据令人惊讶地表明，低亲和力的BC和EC 状态比EO状态更快地与配体结合，这被&>10,000倍慢的关闭速率所补偿 EO状态的。在目标3中，我们使用单分子来测量整合素构象变化的动力学。 在存在或不存在构象的情况下测量延伸或打开步骤的FRET探头- 特定的Fabs和配体。未配位和非配位的BC、EC和EO态之间的所有跃迁的动力学 将测定纯化的、可溶性的胞外结构域和完整的配体结合的单个整合素分子 用高时间分辨率的TIRF显微镜观察血细胞上的整合素。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，随后是配体促进的构象 细胞骨架适配器(A)与EO·L·A态的结合与稳定 通过细胞骨架施加的力和细胞外配体的抵抗。