Signal Transduction of the Netrin Pathway

描述(由申请人提供)： 分泌蛋白netrin是投射轴突和神经元迁移的典型指导信号。我们已经获得的证据表明，它还可以调节胶质瘤细胞的迁移，表明对Netrin功能和信号转导的研究与胶质瘤的侵袭有关，并可能为胶质瘤的治疗提供新的方法。这项应用建议研究介导神经元对netrin反应的信号转导机制。尽管Netrins在多个轴突中的功能作用已被很好地研究，但对参与Netrin信号转导的细胞内成分知之甚少。我们的初步研究表明，netrin-1诱导酪氨酸磷酸化并激活包括Src家族激酶Fyn、粘着斑激酶(FAK)和适配蛋白p130Cas在内的各种激酶。其他人的研究表明，Rho家族的一个小GTP酶rac1被netrin-1激活。我们建议测试一条假设的Netrin信号转导通路，从DCC受体引导到Fyn和FAK，p130Cas，DOCK180，rac1和肌动蛋白。我们将使用分子和生化方法来研究这些细胞内信号分子在Netrin-1细胞外刺激后的生化修饰和激活。最近我的实验室增加了生物物理(成像)方法，使我们能够以高空间和时间分辨率进行研究，以检查活细胞中信号分子的活动，这将使我们能够检查netrin-1处理后细胞系和原代神经元中rac1的激活。这些研究将揭示信号分子之间的关系。我们已经在初步研究中建立了多种功能分析方法，为研究特定的细胞内分子在Netrin-1诱导的轴突生长和吸引中的作用提供了强大和互补的方法。综上所述，这些研究将进一步从根本上了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.