Regulation of Integrin Signaling by the Membrane Skeleton

膜骨架对整合素信号传导的调节

• 批准号: 6853209
• 负责人: JOAN E.B. FOX
• 金额: $ 32.84万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6853209
• 关键词: RNA interference; biological signal transduction; calpain; cell adhesion; cell migration; cytoskeletal proteins; fluorescence resonance energy transfer; guanine nucleotide binding protein; human subject; integrins; muscle proteins; platelets; protein localization; protein protein interaction; protein structure function; time resolved data; vascular endothelium; video microscopy

Our work on platelets suggests that alpha-IIb-beta3 associates with the cytoskeleton in unstimulated cells and that this interaction regulates its ability to transmit signals. Ligand binding causes integrins to cluster and form complexes with cytoskeletal proteins: these complexes, known as focal complexes and focal adhesions, are necessary for activation of molecules that induce actin filament polymerization and reorganization. The complexes themselves are induced by cdc42, Rac (focal complexes) and RhoA (focal adhesions), presumably by activation of exchange factors such as vav and effectors such as WASP. The overall goal of our work is to understand how association of beta3-containing integrins with cytoskeletal proteins regulates ligand binding and events upstream of activation of cdc42/Rac/Rho. In previous work, we identified skelemin as a cytoskeletal protein that interacts with beta1- and beta3-containing integrins in intact cells. We identified mu-calpain as a signaling molecule associated with the membrane skeleton in unstimulated platelets and showed that it was activated by signaling across alpha-IIb-beta3 in platelets or across beta1- and beta3-containing integrins in cultured cells. Our studies on the function of calpain identified a new type of integrin cluster that is induced by calpain, prior to Rac activation. The clusters are distinct from focal complexes and focal adhesions in that they contain skelemin, calpain-cleaved spectrin and beta3-integrin, and active mu-calpain. Dominant-negative Rac accumulates in the integrin clusters and prevents further spreading. These findings have led us to propose a model in which skelemin and calpain are required for formation of integrin clusters that provide foci for recruitment of signaling molecules that in turn initiate activation of the cdc42/Rac/RhoA cascades that induce progression of cytoskeletal reorganizations in adherent cells. In Specific Aim 1, we will test the hypothesis that skelemin-integrin interactions are required for integrin affinity modulation, clustering, avidity modulation, or transmission of signals leading to calpain and cdc42/Rac activation. In Specific Aim 2, we will investigate the role of calpain-induced vav and WASP cleavage in initiation of cdc42/Rac activity in integrin clusters. In Specific Aim 3, we will use time-lapse video microscopy to track the movement of beta3-integrin, skelemin, calpain, vav, and WASP. We will use FRET technology to identify sites of Rac activation. These studies will allow us to directly test the idea that skelemin and calpain are involved in formation of integrin clusters that are the sites of Rac activation. They will also provide insights into spatial and temporal formation of different types of integrin clusters and integrin-induced signaling in living cells.

我们对血小板的研究表明，α - iib - β 3与未受刺激细胞的细胞骨架相关，这种相互作用调节了其传递信号的能力。配体结合导致整联素聚集并与细胞骨架蛋白形成复合物：这些复合物，称为焦点复合物和焦点粘附，是激活诱导肌动蛋白丝聚合和重组的分子所必需的。这些复合物本身由cdc42、Rac（局灶复合物）和RhoA（局灶黏附）诱导，可能是由vav等交换因子和WASP等效应物的激活引起的。我们工作的总体目标是了解含有β - 3的整合素与细胞骨架蛋白的关联如何调节配体结合和cdc42/Rac/Rho激活的上游事件。在之前的研究中，我们发现骨骼蛋白是一种细胞骨架蛋白，它与完整细胞中含有β a1-和β a3的整合素相互作用。我们发现mu-calpain是一种信号分子