Signal Transduction of the Netrin Pathway

描述（由申请人提供）： 分泌蛋白netrin是投射轴突和迁移神经元的原型指导线索。我们已经获得的证据表明，它也可以调节胶质瘤细胞的迁移，这表明netrin功能和信号转导的研究与胶质瘤侵袭相关，并可能为治疗胶质瘤提供新的方法。本申请提出研究介导对netrin的神经元反应的信号转导机制。虽然netrin在多个轴突中的功能作用已经得到了很好的研究，但对netrin信号传导中涉及的细胞内成分知之甚少。我们的初步研究表明，netrin-1诱导酪氨酸磷酸化和激活激酶，包括Src家族激酶Fyn，粘着斑激酶（FAK），和衔接蛋白p130 CAS。其他人的工作表明，Rac 1是Rho家族的一个小GTdR，由netrin-1激活。我们建议测试一个假设的信号转导通路netrin信号在神经元的指导，导致从DCC受体，Fyn和FAK，p130 CAS，DOCK 180，Rac 1和肌动蛋白。我们将使用分子和生物化学方法来研究netrin-1细胞外刺激后这些细胞内信号分子的生物化学修饰和激活。我的实验室最近增加了生物物理（成像）方法，使我们能够以高空间和时间分辨率研究活细胞中信号分子的活性，这将使我们能够研究netrin-1治疗后细胞系和原代神经元中Rac 1的激活。这些研究将揭示信号分子之间的关系。我们在初步研究中建立了多种功能测定，提供了强大和互补的方法来研究特定细胞内分子在netrin-1诱导的轴突生长和吸引中的作用。总之，这些研究将进一步从根本上理解轴突和神经元引导的信号转导机制，为研究脑肿瘤细胞的行为和控制其侵袭提供基础。

DESCRIPTION (provided by applicant): 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.