? The secreted protein netrin is a prototypical guidance cue for projecting axons and migrating neurons. We have obtained evidence that it can also regulate the migration of glioma cells, indicating that studies of netrin function and signaling are relevant to glioma invasion and can potentially suggest novel approaches for treating glioma. This application proposes to investigate signal transduction mechanisms mediating neuronal responses to netrin. Although the functional roles of the netrins in multiple axons are well studied, little is known about intracellular components involved in netrin signaling. Our preliminary studies reveal that netrin-1 induces tyrosine phosphorylation and activates kinases including the Src family kinase Fyn, the focal adhesion kinase (FAK), and the adaptor protein p130CAS. Work by others has shown that Rac1, a small GTPase of the Rho family, is activated by netrin-1. We propose to test a hypothetic signal transduction pathway for netrin signaling in neuronal guidance, leading from the DCC receptor, to Fyn and FAK, p130CAS, DOCK180, Rac1 and actin. We will use molecular and biochemical approaches to study biochemical modification and activation of these intracellular signaling molecules after extracellular stimulation with netrin-1. The recent addition of the biophysical (imaging) approach to my lab makes it possible for us to study with high spatial and temporal resolution to examine the activities of signaling molecules in living cells, which will allow us to examine the activation of Rac1 in cell lines and primary neurons upon netrin-1 treatment. These studies will reveal relationship among the signaling molecules. We have established multiple functional assays in preliminary studies that provide powerful and complementary approaches to investigate the roles of specific intracellular molecules in axon outgrowth and attraction induced by netrin-1. Taken together, the proposed studies will further fundamental understanding of signal transduction mechanisms involved in axonal and neuronal guidance, providing a basis to study the behavior of brain tumor cells and to control their invasion. ? ?
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