NEURONAL MAP INTERACTIONS WITH MICROTUBULES

神经元图谱与微管的相互作用

• 批准号: 3304104
• 负责人: Daniel Lee Purich
• 金额: $ 15.26万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-27 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304104
• 关键词: dendrites; fluorescence spectrometry; laboratory mouse; laboratory rat; microinjections; microtubule associated protein; microtubules; molecular weight; phosphorylation; polymerase chain reaction; protein sequence; radiotracer; synthetic peptide; tau proteins; transfection; dendrites; fluorescence spectrometry; laboratory mouse; laboratory rat; microinjections; microtubule associated protein; microtubules; molecular weight; phosphorylation; polymerase chain reaction; protein sequence; radiotracer; synthetic peptide; tau proteins; transfection

MAP-2a and MAP-2b are high-molecular-weight neuronal microtubule- associated proteins that interact with dendritic microtubules (MTs), and MAP-2c is a corresponding lower-molecular-weight MAP abundant in developing axons. Both proteins share highly conserved N-terminal and C-terminal regions of about 150 and 310 amino acids, respectively, with the latter containing the three 18-amino-acid repeated sequences thought to constitute the MT-binding domain. We propose to further define microtubule binding interactions of the MT-binding fragment, relying on new procedures for preparing the fragment and building on our studies of the three non-identical repeated amino-acid regions. We also plan to characterize m2-peptide displacement of intact MAP-2 from assembled microtubules. To define the amino acid side-chains responsible for binding of peptide-m2 to microtubules and to assess the minimal sequence needed to promote tubule assembly and/or MAP-2 displacement. We plan to study microtubule length redistribution kinetics of microtubules assembled from pure tubulin in the absence or presence of the peptide-m2. We propose to complete the determination of the amino acid sequence of the bovine MT-binding fragment using PCR-derived bovine brain cDNA clones. In a parallel effort, we will investigate phosphorylation of tubule-binding fragment of bovine MAP-2, including: (a) evaluation of the affinity of phosphorylated-tubule-binding fragment for microtubules with the peptide displacement assay; (b) characterization of m2-peptide phosphorylation effects using direct synthesis of m2 analogues containing p-Ser and p-Thr residues; and (c) localization of phosphorylated sites in the MT-binding fragment using enzymatic and chemical cleavage, followed by the diagonal electrophoresis with intervening alkaline phosphatase treatment. Finally, we will carry out microinjection experiments using the MT-binding fragment and synthetic peptides to investigate their efficacy in displacing MAP-2 and tau proteins, and their action in altering cellular distribution of MAP-2 and tau, as well as any changes in cell morphology.

MAP-2A和MAP-2B是高分子量的神经元微管 与树突状微管（MT）相互作用的相关蛋白质和 MAP-2C是相应的下分子量的相应地图 发展轴突。 两种蛋白质共享高度保守的N末端和 C末端区域分别为150和310个氨基酸 后者包含三个18-氨基酸的重复序列思想 构成MT结合域。 我们建议进一步定义 MT结合片段的微管结合相互作用，依靠 在我们的研究中准备碎片和构建的新程序 三个非相同的重复氨基酸区域。 我们还计划 表征来自组装的完整MAP-2的M2肽位移 微管。 定义负责的氨基酸侧链 肽-M2与微管的结合并评估最小序列 需要促进小管组件和/或MAP-2位移。 我们计划 研究微管的微管重新分布动力学微管的动力学 在不存在或存在肽-M2的情况下从纯微管蛋白组装而成。 我们建议完成确定的氨基酸序列 使用PCR衍生的牛脑cDNA的牛MT结合片段 克隆。 我们将在平行的努力中研究 牛MAP-2的小管结合片段，包括：（a）评估 微管与微管的磷酸化小管结合片段的亲和力 肽位移测定； （b）M2肽的表征 使用含有M2类似物的直接合成的磷酸化作用 p-ser和p-th残留物； （c）磷酸化位点的定位 在使用酶和化学裂解的MT结合片段中， 其次是对角电泳与中间碱性的 磷酸酶处理。 最后，我们将进行显微注射 使用MT结合片段和合成肽进行实验 研究它们在移位MAP-2和TAU蛋白中的功效，以及 它们在改变MAP-2和TAU的细胞分布方面的作用 随着细胞形态的任何变化。