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.
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