Integrin-Filamin Interactions in Migration and Signaling

整合素-细丝蛋白在迁移和信号传导中的相互作用

• 批准号: 7931117
• 负责人: DAVID A CALDERWOOD
• 金额: $ 5.34万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931117
• 关键词: Actins; Adhesions; Affinity; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biomechanics; Breast; Cardiovascular system; Cell Adhesion; Cell Adhesion Molecules; Cell Line; Cell Surface Receptors; Cell physiology; Cells; Collagen; Complex; Cytoplasmic Tail; Cytoskeleton; Data; Development; Dimerization; Disease; Engineering; Epithelial Cells; Exhibits; Gel; Genes; Genetic; Health; Hemostatic function; Human; Immigration; Immunoglobulin Domain; In Vitro; Infection; Inflammatory; Integrin Binding; Integrin-mediated Cell Adhesion Pathway; Integrins; Investigation; Knockout Mice; Malignant Neoplasms; Mechanics; Mediating; Membrane; Muscle; Mutation; N-terminal; Neurons; Phenotype; Physical environment; Play; Property; Protein Isoforms; Proteins; Publishing; RNA Splicing; Resolution; Role; Signal Pathway; Signal Transduction; Signaling Protein; Spectrum Analysis; Speed; Structural Protein; Supporting Cell; Surface; Tail; Talin; Techniques; Testing; Variant; X-Ray Crystallography; adhesion receptor; base; cell behavior; cell motility; density; filamin; insight; loss of function mutation; migration; mutant; new therapeutic target; protein crosslink; public health relevance; receptor; response; response to injury; rho GTP-Binding Proteins; sensor; single molecule; transmission process

DESCRIPTION (provided by applicant): Continued support is requested for our investigation of integrin-filamin interactions and their roles in adhesion signaling and cell migration. Integrins are transmembrane receptors that support cell adhesion and migration. They play essential roles throughout development, during hemostasis and in the response to injury and infection. Most integrin functions require a connection to intracellular signaling and cytoskeletal networks and these connections are largely mediated through the regulated interactions of integrin 2 subunit cytoplasmic tails with a variety of intracellular proteins. Characterizing the roles of interacting proteins, the functional consequences of their interaction, the molecular basis for their interactions, how they are regulated, and the cross-talk between different integrin 2 tail binding proteins is therefore central to a complete understanding of this important class of adhesion molecules. Filamins are large actin-crosslinking proteins composed of an N- terminal actin-binding domain and an array of immunoglobulin domains which interact with numerous cytosolic signaling proteins and transmembrane receptors, including integrins. Biochemical, cell biological and genetic data point to roles for FLN in cell migration, differentiation, signaling and the actin cytoskeleton. There are 3 filamin genes in humans, and mutations in filamin result in a wide range of developmental abnormalities and defective neuronal migration. During the current period of support we have characterized integrin-filamin interactions at atomic resolution and identified integrin and filamin mutants with up- or down-regulated affinities. We have identified mechanisms potentially regulating integrin-filamin interactions and revealed a role for filamin in regulating integrin activation state, contractility and tubule formation by breast epithelial cells. We hypothesize that filamin controls migration and adhesion signaling and plays an important role in integrin-mediated sensing and transduction of biomechanical force. To test this we aim to: 1) Characterize the role of different filamin isoforms in cell migration and to identify specific filamin interaction partners important in cell migration; 2) Assess the roles of filamin in regulating integrin activation and signaling and 3) characterize the role of filamin in sensing and transducing biomechanical force. To do this we will generate filamin deficient cell lines using knockdown or post-translational targeting techniques and test the ability of different filamin isoforms or mutants defective in specific interactions to reverse phenotypes in an array of assays including cell migration, integrin activation, Rho GTPase activation, tubulogenesis, and cytoskeletal tethering. Mutant integrins with up- or down-regulated filamin binding will be tested in similar assays, and the interaction of filamin with integrin or other regulators characterized using binding assays, X-ray crystallography, NMR an single molecule force spectroscopy. PUBLIC HEALTH RELEVANCE: ll surface receptors called integrins mediate cell adhesion, control cell migration and act as mechano- sensors providing information about the physical environment around cells, these processes are essential during development, for hemostasis and in the response to injury and infection and are perturbed in cancer, cardiovascular and inflammatory diseases. Integrin function depends on interaction with intracellular signaling and structural proteins such as filamins and mutations in filamins result in a range of development disorders and defective neuronal migration. We seek to characterize the integrin-filamin interaction and determine how it controls cell behavior; this should provide insight into essential molecules with important roles in health and disease and may identify novel therapeutic targets.

描述（由申请人提供）：请求持续支持，以调查整联蛋白 - 纤维素相互作用及其在粘附信号传导和细胞迁移中的作用。整合素是跨膜受体，可支持细胞粘附和迁移。它们在整个发育，止血期间以及对伤害和感染的反应中扮演着重要的作用。大多数整合素功能都需要与细胞内信号传导和细胞骨架网络建立联系，并且这些连接在很大程度上是通过整合素2亚基细胞质尾巴与各种细胞内蛋白质的调节相互作用介导的。表征相互作用蛋白质的作用，相互作用的功能后果，其相互作用的分子基础，如何调节以及不同的整合素2尾巴结合蛋白之间的串扰是对这一重要类别的完全理解粘附分子。丝蛋白是由N-末端肌动蛋白结合结构域组成的大型肌动蛋白 - 交联蛋白和一系列免疫球蛋白结构域，它们与包括整合素在内的众多胞质信号蛋白和跨膜受体相互作用。生化，细胞生物学和遗传数据表明FLN在细胞迁移，分化，信号传导和肌动蛋白细胞骨架中的作用。人类中有3种丝蛋白基因，丝蛋白的突变导致多种发育异常和有缺陷的神经元迁移。在当前的支持期间，我们表征了在原子分辨率下的整联蛋白 - 纤维素相互作用，并确定了具有上调或下调亲和力的整联蛋白和丝蛋白突变体。我们已经确定了潜在调节整联蛋白 - 纤维素相互作用的机制，并揭示了丝蛋白在调节整合素激活状态，收缩力和小管形成中的作用。我们假设Filamin控制迁移和粘附信号，并在整联蛋白介导的生物力学力的传感和转导中起重要作用。为了测试这一点，我们的目的是：1）表征不同丝蛋白同工型在细胞迁移中的作用，并确定在细胞迁移中重要的特定丝蛋白相互作用伴侣； 2）评估丝蛋白在调节整联蛋白激活和信号传导中的作用，以及3）表征丝蛋白在传感和转导生物力学中的作用。为此，我们将使用敲低或翻译后的靶向技术生成丝蛋白缺陷细胞系，并测试不同丝蛋白同工型或突变体在特定相互作用中有缺陷在特定相互作用中反向表型在一系列测定中反向表型的能力，微管生成和细胞骨架链接。具有上调或下调的丝蛋白结合的突变蛋白将在类似的测定中进行测试，并将丝蛋白与整合素或其他使用结合测定，X射线晶体学，NMR，单个分子力光谱谱图进行表征的整合素或其他调节剂的相互作用。公共卫生相关性：称为整联蛋白的LL表面受体介导细胞粘附，控制细胞迁移并充当提供有关细胞周围物理环境的信息的机械传感器，这些过程在发育过程中至关重要在癌症，心血管和炎症性疾病中受到干扰。整合素功能取决于与细胞内信号传导和结构蛋白（例如丝蛋白和丝蛋白突变）的相互作用，导致一系列发育障碍和缺陷的神经元迁移。我们试图表征整联蛋白 - 纤维素的相互作用并确定其如何控制细胞行为。这应该提供对具有重要作用在健康和疾病中重要作用的基本分子的洞察力，并可以鉴定出新的治疗靶标。