Integrin-Filamin Interactions in Migration and Signaling

整合素-细丝蛋白在迁移和信号转导中的相互作用

基本信息

批准号：
8245831
负责人：
DAVID A CALDERWOOD
金额：
$ 33.25万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/8245831
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

项目摘要

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）：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需要持续的支持。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、止血以及对损伤和感染的反应中发挥着重要作用。大多数整合素功能需要与细胞内信号传导和细胞骨架网络连接，这些连接很大程度上是通过整合素 2 亚基胞质尾部与各种细胞内蛋白质的受调节相互作用来介导的。因此，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、它们的调节方式以及不同整合素 2 尾部结合蛋白之间的串扰对于全面了解这一类重要的蛋白质至关重要。粘附分子。细丝蛋白是大型肌动蛋白交联蛋白，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，这些结构域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。人类有 3 个细丝蛋白基因，细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。在当前的支持期间，我们以原子分辨率表征了整合素-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的整合素激活状态、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传导，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导中的作用，3) 表征细丝蛋白在传感和传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生成细丝蛋白缺陷细胞系，并测试不同细丝蛋白亚型或特定相互作用缺陷的突变体在一系列测定中逆转表型的能力，包括细胞迁移、整合素激活、Rho GTP酶激活、肾小管发生和细胞骨架束缚。将在类似的测定中测试具有上调或下调的细丝蛋白结合的突变整合素，并使用结合测定、X射线晶体学、NMR和单分子力谱表征细丝蛋白与整合素或其他调节剂的相互作用。公共健康相关性：称为整合素的所有表面受体介导细胞粘附、控制细胞迁移并充当机械传感器，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至关重要，并且患有癌症、心血管疾病和炎症性疾病。整合素功能取决于与细胞内信号传导和结构蛋白（例如细丝蛋白）的相互作用，细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；这应该可以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。