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-细丝蛋白相互作用及其在粘附信号传导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中，在止血过程中以及在对损伤和感染的反应中发挥重要作用。大多数整联蛋白功能需要连接到细胞内信号传导和细胞骨架网络，并且这些连接主要通过整联蛋白2亚基胞质尾与多种细胞内蛋白质的调节相互作用介导。表征相互作用的蛋白质的作用，其相互作用的功能后果，其相互作用的分子基础，它们是如何调节的，以及不同的整合素2尾结合蛋白之间的串扰，因此，对这类重要的粘附分子的完整理解至关重要。丝状蛋白是由N-末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肌动蛋白交联蛋白，其与许多胞质信号蛋白和跨膜受体（包括整联蛋白）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种细丝蛋白基因，细丝蛋白的突变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。我们已经确定了潜在的调节整合素-细丝蛋白相互作用的机制，并揭示了细丝蛋白在调节乳腺上皮细胞整合素活化状态、收缩性和小管形成中的作用。我们假设细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。为了测试这一点，我们的目标是：1）表征不同细丝蛋白同种型在细胞迁移中的作用并鉴定在细胞迁移中重要的特定细丝蛋白相互作用配偶体; 2）评估细丝蛋白在调节整合素活化和信号传导中的作用和3）表征细丝蛋白在感测和转导生物力学力中的作用。为此，我们将使用敲低或翻译后靶向技术产生细丝蛋白缺陷细胞系，并测试不同细丝蛋白同种型或特异性相互作用缺陷突变体在一系列测定中逆转表型的能力，包括细胞迁移、整联蛋白激活、Rho GT3激活、微管发生和细胞骨架束缚。将在类似的试验中测试细丝蛋白结合上调或下调的突变整合素，并使用结合试验、X射线晶体学、NMR和单分子力光谱法表征细丝蛋白与整合素或其他调节剂的相互作用。公共卫生关系：称为整联蛋白的所有表面受体介导细胞粘附、控制细胞迁移并作为机械传感器提供关于细胞周围物理环境的信息，这些过程在发育、止血和对损伤和感染的反应中是必需的，并且在癌症、心血管和炎性疾病中受到干扰。整联蛋白的功能依赖于与细胞内信号传导和结构蛋白如丝裂蛋白的相互作用，丝裂蛋白的突变导致一系列发育障碍和缺陷性神经元迁移。我们试图描述整合素-细丝蛋白相互作用的特征，并确定它如何控制细胞行为;这将提供对健康和疾病中具有重要作用的基本分子的深入了解，并可能确定新的治疗靶点。