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: 8052740
• 负责人: DAVID A CALDERWOOD
• 金额: $ 44.99万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8052740
• 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; Cell-Cell Adhesion; 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; Intracellular Signaling Proteins; 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; 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）：要求继续支持我们对整合素-丝蛋白相互作用及其在粘附信号和细胞迁移中的作用的研究。整合素是支持细胞粘附和迁移的跨膜受体。它们在整个发育过程、止血过程以及对损伤和感染的反应中起着至关重要的作用。大多数整合素功能需要连接细胞内信号和细胞骨架网络，这些连接在很大程度上是通过整合素2亚基细胞质尾部与多种细胞内蛋白的调节相互作用介导的。表征相互作用蛋白的作用，它们相互作用的功能后果，它们相互作用的分子基础，它们是如何被调节的，以及不同整合素2尾部结合蛋白之间的串扰，因此是完整理解这类重要粘附分子的核心。丝蛋白是由N端肌动蛋白结合结构域和一系列免疫球蛋白结构域组成的大型肌动蛋白交联蛋白，可与许多胞质信号蛋白和跨膜受体（包括整合素）相互作用。生物化学、细胞生物学和遗传学数据指出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。人类有3种丝蛋白基因，丝蛋白的突变会导致广泛的发育异常和神经元迁移缺陷。在目前的支持期间，我们在原子分辨率上表征了整合素-丝蛋白的相互作用，并鉴定了整合素和丝蛋白突变体的亲和性上调或下调。我们已经确定了调节整合素-丝蛋白相互作用的潜在机制，并揭示了丝蛋白在调节乳房上皮细胞整合素激活状态、收缩性和小管形成中的作用。我们假设丝蛋白控制迁移和粘附信号，并在整合素介导的生物力学力的传感和转导中发挥重要作用。为了验证这一点，我们的目标是：1)表征不同丝蛋白异构体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伙伴；2)评估丝蛋白在调节整合素激活和信号传导中的作用；3)表征丝蛋白在生物力学力传感和转导中的作用。为此，我们将使用敲低或翻译后靶向技术生成丝蛋白缺陷细胞系，并测试不同丝蛋白异构体或在特定相互作用中有缺陷的突变体在一系列试验中逆转表型的能力，包括细胞迁移、整合素激活、Rho GTPase激活、小管形成和细胞骨架系固。将在类似的分析中测试具有上调或下调丝蛋白结合的突变整合素，并使用结合分析、x射线晶体学、核磁共振和单分子力谱来表征丝蛋白与整合素或其他调节因子的相互作用。公共卫生相关性：所有被称为整合素的表面受体介导细胞粘附，控制细胞迁移，并作为提供细胞周围物理环境信息的机械传感器，这些过程在发育，止血和对损伤和感染的反应中是必不可少的，并且在癌症，心血管和炎症性疾病中受到干扰。整合素的功能依赖于与细胞内信号和结构蛋白（如丝蛋白）的相互作用，而丝蛋白的突变会导致一系列发育障碍和神经元迁移缺陷。我们试图表征整合素-丝蛋白相互作用，并确定它如何控制细胞行为；这将有助于深入了解在健康和疾病中发挥重要作用的基本分子，并可能确定新的治疗靶点。