NEURONAL MAP INTERACTIONS WITH MICROTUBULES

神经元图谱与微管的相互作用

• 批准号: 3304102
• 负责人: Daniel Lee Purich
• 金额: $ 16.69万
• 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/3304102
• 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

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 是高分子量神经元微管 与树突微管（MT）相互作用的相关蛋白，以及 MAP-2c 是一种相应的低分子量 MAP，富含 正在发育的轴突。 两种蛋白质都具有高度保守的 N 端和 C端区域分别含有约150个和310个氨基酸， 后者包含三个 18 个氨基酸重复序列 构成MT结合结构域。 我们建议进一步定义 MT 结合片段的微管结合相互作用，依赖于 准备片段的新程序并以我们的研究为基础 三个不相同的重复氨基酸区域。 我们还计划 表征组装后的完整 MAP-2 的 m2 肽置换 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 促进肾小管组装和/或 MAP-2 移位所需。 我们计划 研究微管的微管长度重新分布动力学 在不存在或存在肽-m2 的情况下由纯微管蛋白组装而成。 我们建议完成氨基酸序列的测定 使用 PCR 衍生的牛脑 cDNA 检测牛 MT 结合片段 克隆。 在并行的努力中，我们将研究磷酸化 牛 MAP-2 的肾小管结合片段，包括： (a) 评估 磷酸化管结合片段对微管的亲和力 肽置换测定； (b) m2-肽的表征 使用直接合成含有 m2 类似物的磷酸化效应 p-Ser 和 p-Thr 残基； (c) 磷酸化位点的定位 使用酶促和化学切割在 MT 结合片段中， 然后进行对角线电泳，并加入碱性物质 磷酸酶治疗。 最后，我们将进行显微注射 使用 MT 结合片段和合成肽进行的实验 研究它们在取代 MAP-2 和 tau 蛋白方面的功效，以及 它们在改变 MAP-2 和 tau 细胞分布方面的作用，以及 细胞形态的任何变化。