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-细丝素相互作用及其在黏附信号和细胞迁移中的作用的研究。整合素是支持细胞黏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发挥着至关重要的作用。大多数整合素的功能需要连接到细胞内的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亚单位胞浆尾巴与各种细胞内蛋白质的调节相互作用来调节的。因此，对相互作用的蛋白质的作用、相互作用的功能后果、相互作用的分子基础、如何调控以及不同整合素2尾巴结合蛋白之间的相互作用进行表征，对于全面理解这类重要的黏附分子是至关重要的。丝蛋白是由一个N-末端肌动蛋白结合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，它与许多胞浆信号蛋白和跨膜受体相互作用，包括整合素。生化、细胞生物学和遗传学数据表明，Fln在细胞迁移、分化、信号和肌动蛋白细胞骨架中发挥作用。人类有3种细丝蛋白基因，丝蛋白基因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，我们已经在原子分辨率下表征了整合素-细丝蛋白相互作用，并鉴定了具有上调或下调亲和力的整合素和细丝蛋白突变体。我们已经确定了可能调节整合素-细丝素相互作用的机制，并揭示了细丝素在调节乳腺上皮细胞的整合素激活状态、收缩能力和小管形成中的作用。我们推测细丝蛋白控制着细胞的迁移和黏附信号，并且在整合素介导的生物机械力的传感和传递中起着重要的作用。为了验证这一点，我们的目标是：1)表征不同的细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细丝蛋白相互作用伙伴；2)评估细丝蛋白在调节整合素激活和信号传递中的作用；以及3)表征细丝蛋白在感知和传递生物机械力方面的作用。为此，我们将使用击倒或翻译后靶向技术产生丝胺缺乏的细胞系，并测试在特定相互作用中存在缺陷的不同丝胺异构体或突变体在一系列分析中逆转表型的能力，包括细胞迁移、整合素激活、Rho GTP酶激活、小管发生和细胞骨架连接。具有上调或下调丝胺结合的突变整合素将在类似的测试中进行测试，并使用结合分析、X射线结晶学、核磁共振和单分子力谱来表征丝素与整合素或其他调节剂的相互作用。公共卫生相关性：被称为整合素的LL表面受体介导细胞黏附，控制细胞迁移，并作为机械传感器提供有关细胞周围物理环境的信息，这些过程在发育、止血以及对损伤和感染的反应中是必不可少的，并在癌症、心血管疾病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的相互作用，如丝蛋白和丝蛋白的突变导致一系列发育障碍和缺陷神经元迁移。我们试图确定整合素-细丝素相互作用的特征，并确定它是如何控制细胞行为的；这应该有助于深入了解在健康和疾病中具有重要作用的基本分子，并可能确定新的治疗靶点。