IDENTIFICATION OF A NOVEL SIGNAL PATHWAY FOR NGF

NGF 新型信号通路的鉴定

• 批准号: 6342942
• 负责人: RAYMOND B BIRGE
• 金额: $ 26.65万
• 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/6342942
• 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）是多肽生长因子 在不成熟神经细胞和分化中的关键作用 随后在这两个中成熟神经元亚群的生存 周围和中枢神经系统。 NGF通过 与TRKA结合，TRKA是一种受体型酪氨酸激酶，当激活时， 启动信号转导级联，导致RAS激活和 早期反应基因的诱导。 NGF诱导的神经突生长 还伴随着神经元的广泛重组 细胞骨架启动神经突过程的神经突扩展。的确， 虽然生长锥中丝状和薄片的形状在很大程度上是 由肌动蛋白细胞骨架的组织确定，几乎没有 知道NGF和TRKA信号如何与细胞骨架有关。 在过去的几年中，我们一直在研究CRK适配器蛋白 及其在NGF诱导的神经元分化中的作用。 在NGF上 除PC12细胞或原发性背根神经元外，C-CRK为 酪氨酸在酪氨酸222上迅速磷酸化。有趣的是， 在Y222（C-CRKY222F）处携带突变的突变体C-CRK的过表达 损害NGF介导的PC12细胞中的神经突生长，并废除TRKA- 诱导的细胞骨架蛋白pa​​xillin的酪氨酸磷酸化。 我们的结果表明，CRK参与了TRKA的新方面 向粘附复合物发出信号，从而调节肌动蛋白的重新 组织和细胞粘附。 在本申请中，我们将 研究C-CRK伴侣TRKA受体的分子机制 到细胞骨架，其长期目标是了解NGF 促进分化和生存。 具体来说，我们计划：（i） 确定C-CRKY222F是否是主要的负C-CRK蛋白 损害NGF诱导的细胞粘附，（ii）确定电路 参与NGF诱导的C-CRK的酪氨酸磷酸化，（III） 确定NGF-和粘附诱导的酪氨酸之间的关系 C-CRK效应蛋白帕西林（IV）的磷酸化确定 帕西林在NGF介导的反应中的生物学作用，（v） 使用转基因确定C-CRK在神经元发育中的作用 方法。