Regulation of GPIIb-IIIa by the platelet cytoskeleton

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DESCRIPTION (Applicant's Description Verbatim): Members of the integrin family of receptors play a critical role in inducing adhesion and are involved in numerous events that require integrin-induccd changes in cell behavior. For example, signaling across integrins is involved in angiogenesis, development, metastasis, inflammation, wound healing, and hemostasis. The overall goal of our research is to understand the signaling pathways leading to integrin activation and to integrin-induced changes in cell behavior. Our work focuses on the importance of membrane skeleton proteins and associated signaling molecules in regulating the two-way signaling across integrins. Studies over the past funding period have provided evidence that a subpopulation of a11bB3 in platelets is associated with membrane skeleton complexes containing spectrin, talin, pp60c-src, and calpain. As platelets or cultured cells spread, these proteins are incorporated into integrin clusters by a mechanism that requires calpain activity. Rac is subsequently activated by a mechanism that can be inhibited by spectrin's SH3 domain. Our findings are consistent with a model in which integrin engagement causes activation of calpain. Calpain cleaves several proteins including spectrin and the beta3 integrin subunit, allowing integrin that is otherwise restrained by the cytoskeleton to cluster into focal complexes. Signaling transmitted across these complexes, by a mechanism involving spectrin, leads to activation of Rac. We have identified skelemin as a new protein that can interact with the beta3 cytoplasmic domain and shown that it is present in the integrin clusters. Experiments in the present proposal are designed to test the hypothesis that association of alIbbeta3 with the complexes of membrane skeleton proteins and signaling molecules plays a critical role in allowing two-way signaling across the integrin. The Specific Aims are 1) to test the hypothesis that calpain is involved in Rac activation; 2) to test the hypothesis that the SH3 domain of spectrin is involved in Rac activation; 3) to test the hypothesis that interaction of skelemin with integrin cytoplasmic domains regulates two-way signaling across integrins. It is anticipated that these studies will provide information on mechanisms involved in two-way signaling across integnns. Further, the studies may lead to an increased understanding of key mechanisms involved in integrin-mediated functions such as platelet adhesion and aggregation, wound healing, angiogenesis, inflammation, metastasis, and cell migration.