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的（E-）和 非交换性（N-）位点和E-位点GTP水解是驱动力 动态不稳定力与微管骨架 细胞周期中的重排。大鼠PC 12嗜铬细胞瘤细胞使用 MT细胞骨架以实现形态学变化，最特别地 分支神经突的生长，对神经生长因子的反应 （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替代GTP在 在NGF存在下生长的PC 12细胞的MT的微管蛋白N位点。 NGF处理后约80- 85%的微管蛋白生物合成含有 dGTP，基于一对一结合化学计量。 本研究 该项目将测试以下总体假设：“NGF刺激 神经突生长、微管蛋白生物合成和dGTP掺入 微管蛋白;考虑GTP调节转导的中心作用 细胞增殖系统中，这一迄今未被认识到的联系， 细胞生长条件和微管蛋白N-位点核苷酸组成可 提供一种调节细胞GTP水平和隔离 dGTP。“研究计划有四个具体目标：1）确定 在NGF处理的PC 12细胞中dGTP池的变化的时间过程， 对NGF的反应; 2)以确定新合成的 微管蛋白同种型相对于N-位点dGTP的周转率; 3） 研究了从PC 12细胞分离的MTs在处理前后的dGTP含量 用能够仅结合高- 4）使用视频显微镜建立时间- 用NGF处理的细胞中神经突生长的过程，并探索 从细胞体分离的MT的组装/拆卸动力学， 富含dGTP的MT的基因。