Roles of SH2-Domain Proteins in Repulsive Axon Guidance by A-Ephrins

SH2 结构域蛋白在 A-Ephrins 排斥性轴突引导中的作用

• 批准号: 0548561
• 负责人: Renping Zhou
• 金额: $ 36万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0548561&HistoricalAwards=false
• 关键词: Roles; SH2; Domain; Proteins; Repulsive

Nerve fibers (axons) are guided to make appropriate connections during development of the nervous system. The guidance factors can be either attractive or repulsive to the axons. The Ephrin family molecules and their Eph receptors often function as repulsive guidance cues and induce axon retraction. The molecular mechanisms underlying repulsive axon guidance remain largely unknown. The long-term objective of this project is to elucidate the molecular pathways underlying axon retraction, using Eph receptors as model systems. Eph receptors are tyrosine kinases, and transmit signals through phosphotyrosine (P-Tyr)-dependent or independent coupling of downstream molecules. However, roles of molecular cascades coupled through P-Tyr binding in axon retraction have not been well investigated. In the preliminary studies, a candidate signaling protein, a phosphotyrosine phosphatase, termed Shp2, which contain the Src homology 2-domain known to bind to P-Tyr, has been isolated. It is hypothesized that Shp2, coupled through P-Tyr-binding to activated EphA receptors, initiates critical signaling cascades important for axon retraction induced by the Ephrins. To test this hypothesis, experiments are proposed to (1) identify specific phosphotyrosine residues that are important for Shp2 binding and axon repulsion, using mutagenesis and biochemical analyses; (2) Test whether Shp2 is required for EphA receptor-mediated biochemical and biological responses. To further define functions of Shp2, the roles of Shp2 effector Src in Ephrin-induced repulsive guidance will be examined. Several approaches, including expression of dominant-negative and constitutively active mutants and assaying Ephrin functions in gene-deleted neurons, will be used to test roles of Shp2 in EphA receptor-mediated repulsive axon guidance. The intellectual merits of proposed experiments lie in addressing an important area of axon guidance mechanisms, namely, functions of the P-Tyr-docked pathways, which have not been well investigated in axon guidance. The proposed studies are likely to identify novel signaling mechanisms mediating Ephrin-induced repulsive axon guidance. Since tyrosine kinases and phosphatases have been shown to play important roles in other axon guidance systems and possibly axon guidance in general, these studies are likely to significantly enhance understanding of how axons are guided, and may provide insights in future interventions to reconnect damaged neural circuits. The proposed studies will also have a broader impact. These studies will help generate viral vectors carrying various signaling proteins, which will be made available to other investigators. In addition, the proposed studies will train students and postdoctoral fellows. The PI also participates in the American Chemical Society SEED Program. This program provides training opportunities for minority high school students. Three minority high school students have performed summer research in the PI's laboratory in the past three years. The proposed studies will allow continued exposure for minority students to research experience in a laboratory environment.

神经纤维（轴突）在神经系统发育过程中被引导进行适当的连接。 引导因子对轴突既可以是吸引的，也可以是排斥的。 Ephrin家族分子和它们的Eph受体通常作为排斥性引导线索起作用并诱导轴突收缩。排斥性轴突导向的分子机制在很大程度上仍然未知。这个项目的长期目标是阐明轴突回缩的分子途径，使用Eph受体作为模型系统。Eph受体是酪氨酸激酶，并通过磷酸酪氨酸（P-Tyr）依赖性或非依赖性的下游分子偶联来传递信号。然而，通过P-Tyr结合偶联的分子级联在轴突收缩中的作用尚未得到很好的研究。在初步的研究中，一个候选的信号蛋白，磷酸酪氨酸磷酸酶，称为Shp 2，其中包含已知的Src同源2-结构域结合P-Tyr，已被分离。假设Shp 2通过P-Tyr结合与活化的EphA受体偶联，启动对由肝配蛋白诱导的轴突收缩重要的关键信号级联。为了检验这一假设，提出了实验来（1）使用诱变和生物化学分析来鉴定对于Shp 2结合和轴突排斥重要的特定磷酸酪氨酸残基;（2）测试Shp 2是否是EphA受体介导的生物化学和生物学应答所需的。为了进一步定义Shp 2的功能，将检查Shp 2效应物Src在Ephrin诱导的排斥性引导中的作用。几种方法，包括显性阴性和组成型活性突变体的表达和基因缺失的神经元中的Ephrin功能测定，将被用来测试Shp 2在EphA受体介导的排斥性轴突引导中的作用。所提出的实验的智力优点在于解决轴突指导机制的一个重要领域，即，P-Tyr对接途径的功能，这在轴突指导中还没有得到很好的研究。拟议的研究有可能确定新的信号转导机制介导的Ephrin诱导的排斥性轴突的指导。由于酪氨酸激酶和磷酸酶已被证明在其他轴突导向系统中发挥重要作用，并可能在一般的轴突导向，这些研究可能会显着提高轴突是如何引导的理解，并可能在未来的干预措施重新连接受损的神经回路提供见解。拟议的研究也将产生更广泛的影响。这些研究将有助于产生携带各种信号蛋白的病毒载体，这些载体将提供给其他研究人员。此外，拟议的研究将培训学生和博士后研究员。PI还参与了美国化学学会SEED计划。该方案为少数民族高中生提供培训机会。在过去的三年里，三名少数民族高中生在PI的实验室进行了暑期研究。拟议的研究将使少数民族学生能够继续在实验室环境中获得研究经验。