Continued support is requested for studies to analyze suppression of integrin activation, a process central to the control of blood and vascular cell migration and to platelet aggregation. The applicant found that activation of H-Ras GTPase leads to suppression of integrin activation through the activation of ERK1/2 at the plasma membrane. The discovery that talin binding to the integrin ]3 cytoplasmic domain is a final step in integrin activation enabled him to partially reconstruct integrin activation in a nucleated cell and ordered the pathway as Agonist ->PKC ->Rap ->Talin ->lntegrin. He proposes to test the hypothesis that Rap1 activates integrins by stimulating the interaction of talin with the integrin /? cytoplasmic domain through a Rap1 effector and that ERK1/2 blocks activation by regulating one or more of the elements in this pathway. He will further reconstruct agonist-mediated integrin orllb/?3 activation in CHO cells through the introduction of talin and other signaling molecules (e.g. Protein kinase C, R-Ras, Rap 1). He will use the reconstructed system to order the participants and to establish the step(s) at which Rap1 and ERK 1/2 regulate integrin ollb/?3 activation. Secondly, he found that PEA-15 regulates integrin activation and cell proliferation by binding to ERK1/2 and that PEA-15 is expressed in endothelial cells and arterial smooth muscle cells. He will test the hypothesis that PEA-15 expression and/or phosphorylation regulates the proliferation and survival of endothelial cells and smooth muscle cells in vitro and in vivo. This will be done by examining the role of PEA-15 in regulating tumor cell invasion, angiogenesis and re-stenosis and in endothelial cell and smooth muscle cell proliferation in vitro. Finally, the small GTPase, R-Ras, reverses suppression of integrin activation and stimulates cell spreading via an apparently novel effector. By use of a TAP-tagged R-Ras and high throughput proteomics, he developed an experimental database of candidate R-Ras interacting proteins and found that RLIP76 had the properties expected of an effector that regulates integrin function. He will test the hypothesis that RLIP76 is an R-Ras effector that mediates adhesion-dependent cell signaling by Use of siRNA-mediated knockdown and structure-function analysis of RLIP76. These studies will provide fundamental insights into pathways that control the activation of integrins in cells of the blood and vasculature and may serve to identify novel therapeutic targets for diseases of hemostasis and thrombosis.
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