IDENTIFICATION OF A NOVEL SIGNAL PATHWAY FOR NGF

NGF 新型信号通路的鉴定

• 批准号: 6490115
• 负责人: RAYMOND B BIRGE
• 金额: $ 26.84万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490115
• 关键词: PC12 cells; biological signal transduction; cell adhesion; cell growth regulation; confocal scanning microscopy; extracellular matrix; gene induction /repression; genetically modified animals; immunofluorescence technique; immunoprecipitation; laboratory mouse; metalloendopeptidases; neurogenesis; neurotrophic factors; paxillin; phosphorylation; protein binding; protein structure function; protein tyrosine kinase; receptor coupling; regulatory gene; tissue /cell culture; transfection; yeast two hybrid system

Nerve Growth Factor (NGF) is a polypeptide growth factor that plays a critical role in the differentiation of immature neuroblasts and the subsequent survival of a subpopulation of mature neurons in both the peripheral and central nervous systems. NGF initiates its effects by binding to TrkA, a receptor type tyrosine kinase that when activated, initiates a signal transduction cascade resulting in Ras activation and the induction of early response genes. NGF-induced neurite outgrowth is also accompanied by extensive re-organization of the neuronal cytoskeleton to initiate neurite extension of neurite processes. Indeed, while the shape of filopodia and lamella in growth cones is largely determined by the organization of the actin cytoskeleton, little is known about how NGF and TrkA signaling is linked to the cytoskeleton. Over the past few years, we have been studying the Crk adaptor protein and its role during NGF-induced neuronal differentiation. Upon NGF addition to PC12 cells or primary dorsal root ganglia neurons, c-Crk is rapidly tyrosine phosphorylated at tyrosine 222. Interestingly, overexpression of mutant c-Crk carrying a mutation at Y222 (c-CrkY222F) impairs NGF-mediated neurite outgrowth in PC12 cells and abolishes TrkA- induced tyrosine phosphorylation of the cytoskeletal protein Paxillin. Our results indicate that Crk is involved in a novel aspect of TrkA signaling to adhesion complexes, thereby regulating actin re- organization and cell adhesion. In the present application, we shall study the molecular mechanisms by which c-Crk couples the TrkA receptor to the cytoskeleton, with the long-term goal to understand how NGF promotes differentiation and survival. Specifically, we plan to: (i) determine whether c-CrkY222F is a dominant negative c-Crk protein by impairing NGF-induced cell adhesion, (ii) determine the circuitry involved in NGF-induced tyrosine phosphorylation of c-Crk, (iii) determine the relationship between NGF- and adhesion-induced tyrosine phosphorylation of the c-Crk effector protein Paxillin, (iv) determine the biological role of Paxillin during NGF-mediated responses, and (v) determine the role of c-Crk during neuronal development using transgene approaches.

神经生长因子（NGF）是一种多肽生长因子， 在未成熟成神经细胞分化中的关键作用， 随后的存活的亚群成熟神经元在这两个 外周和中枢神经系统。 NGF通过以下方式启动其作用： 与TrkA结合，TrkA是一种受体型酪氨酸激酶，当被激活时， 启动导致Ras激活的信号转导级联， 早期反应基因的诱导。 神经生长因子诱导的神经突起生长 也伴随着神经元的广泛重组， 细胞骨架启动神经突突起的神经突延伸。的确， 而生长锥中的丝状伪足和片层的形状主要是 由肌动蛋白细胞骨架的组织决定， 了解NGF和TrkA信号如何与细胞骨架联系。 在过去的几年里，我们一直在研究Crk衔接蛋白， 及其在NGF诱导的神经元分化过程中的作用。 NGF后 除了PC 12细胞或原代背根神经节神经元外，c-Crk还 在酪氨酸222处快速酪氨酸磷酸化。 有趣的是， 携带Y222突变的突变型c-Crk（c-CrkY 222 F）的过表达 在PC 12细胞中损害NGF介导的神经突生长，并消除TrkA- 诱导细胞骨架蛋白桩蛋白的酪氨酸磷酸化。 我们的研究结果表明Crk参与TrkA的一个新方面 信号传导到粘附复合物，从而调节肌动蛋白的再活化。 组织和细胞粘附。 在本申请中，我们将 研究c-Crk与TrkA受体偶联的分子机制 长期目标是了解神经生长因子 促进分化和存活。 具体而言，我们计划：（i） 确定c-CrkY 222 F是否是显性阴性c-Crk蛋白， 损害神经生长因子诱导的细胞粘附，（ii）确定电路 参与NGF诱导的c-Crk酪氨酸磷酸化，（iii） 确定NGF和粘附诱导的酪氨酸之间的关系 （iv）测定c-Crk效应蛋白桩蛋白的磷酸化， 桩蛋白在NGF介导的应答期间的生物学作用，和（v） 利用转基因技术确定c-Crk在神经元发育过程中的作用 接近。