TUBULIN BOUND DEOXY GTP AND NGF TREATED NEURONS

微管蛋白结合脱氧 GTP 和 NGF 处理的神经元

• 批准号: 2270715
• 负责人: Daniel Lee Purich
• 金额: $ 17.56万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-01 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2270715
• 关键词: PC12 cells; growth factor receptors; guanosine triphosphate; high performance liquid chromatography; microtubules; neurogenesis; neuropharmacology; neurotrophic factors; protein biosynthesis; protein isoforms; tubulin; video microscopy

DESCRIPTION: (adapted from Applicant's Abstract) Tubulin aB- heterodimers bind GTP or GDP at their exchangeable (E-) and nonexchangeable (N-) sites, and E-site GTP hydrolysis is the driving force for dynamic instability and microtubule (MT) cytoskeletal rearrangements in the cell cycle. Rat PC12 pheochromocytoma cells use the MT cytoskeleton to achieve morphologic changes, most particularly outgrowth of branched neurites, in response to nerve growth factor (NGF). Using HPLC analysis for nucleotide content of assembled MTs, the investigators recently discovered that dGTP substitutes for GTP in the tubulin N-site of MTs from PC12 cells grown in the presence of NGF. About 80- 85% of tubulin biosynthesized after NGF treatment contained dGTP, based on a one-to-one binding stoichiometry. This research project will test the following overall hypothesis: "NGF stimulates neurite outgrowth, tubulin biosynthesis, and incorporation of dGTP into tubulin; considering the central role of GTP-regulatory transduction systems in cell proliferation, this hitherto unrecognized link between cell growth conditions and tubulin N-site nucleotide composition may provide a mechanism for adjusting cell GTP levels and sequestering dGTP." The research plan has four specific aims: 1) to determine the time-courses for changes in dGTP pools in NGF-treated PC12 cells in response to NGF; 2) to determine turnover rates of newly synthesized tubulin isotypes relative to the turnover rate of N-site dGTP; 3) to investigate dGTP content of MTs isolated from PC12 cells before and after treatment with a mutant of NGF capable of binding only to the high- affinity NGF receptor; 4) to use video microscopy to establish time- courses of neurite outgrowth in cells treated with NGF and to explore the assembly/disassembly dynamics of MTs isolated from cell bodies and of the dGTP-rich MTs from neurites.

描述：（改编自申请人的摘要）微管蛋白 aB- 异二聚体在其可交换 (E-) 处结合 GTP 或 GDP，并且 不可交换（N-）位点，E位点GTP水解是驱动 动态不稳定性和微管（MT）细胞骨架的力 细胞周期中的重排。大鼠PC12嗜铬细胞瘤细胞用途 MT细胞骨架以实现形态变化，尤其是 分支神经突的生长，响应神经生长因子 （NGF）。 使用 HPLC 分析组装的 MT 的核苷酸含量， 研究人员最近发现 dGTP 可以替代 GTP 来自在 NGF 存在下生长的 PC12 细胞的 MT 的微管蛋白 N 位点。 NGF 处理后生物合成的微管蛋白中约 80-85% 含有 dGTP，基于一对一结合化学计量。 这项研究 项目将测试以下总体假设：“NGF 刺激 神经突生长、微管蛋白生物合成以及 dGTP 掺入 微管蛋白； 考虑 GTP 调节转导的核心作用 细胞增殖系统中，这种迄今为止未被认识到的联系 细胞生长条件和微管蛋白 N 位核苷酸组成可能 提供调节细胞 GTP 水平和隔离的机制 dGTP。”该研究计划有四个具体目标：1）确定 NGF处理的PC12细胞中dGTP池变化的时间过程 对 NGF 的反应； 2）确定新合成的周转率 微管蛋白同种型与 N 位 dGTP 周转率的关系； 3）到 研究前后从 PC12 细胞中分离的 MT 的 dGTP 含量 使用仅能结合高浓度 NGF 突变体的治疗 亲和性NGF受体； 4) 使用视频显微镜确定时间- 用 NGF 处理的细胞中神经突生长的过程并探索 从细胞体中分离出的 MT 的组装/分解动力学 来自神经突的富含 dGTP 的 MT。