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: 2431223
• 负责人: Daniel Lee Purich
• 金额: $ 16.39万
• 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/2431223
• 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：（改编自申请人摘要）微管蛋白aB-