MOLECULAR INTERACTIONS OF BRAIN SPECTRIN

这项建议的长期目标是深入了解 人类疾病中的细胞膜骨架。红细胞缺陷 膜骨架整合蛋白是一些遗传性溶血的基础 人类和小鼠的贫血，以及最近的研究涉及神经细胞膜 骨骼蛋白在神经退行性变过程中的作用 阿尔茨海默氏症。然而，人们对这种结构知之甚少， 神经细胞膜骨架的功能及其调控过程 以及它的主要成分--大脑中的幽灵蛋白。大脑皮层的神经元区划 脑影蛋白亚型进入轴突和突触前终末(非红系 血影蛋白)进入细胞体和树突(红系血影蛋白)提示 大脑中的血影蛋白亚型可能执行相关但不同的功能 神经细胞。关于幽灵蛋白四聚体形成的拟议工作和 SPECTIN SH3结构域应该是我们理解功能的基础 脑部血影蛋白亚型之间的差异。 血影蛋白四聚体是膜的基本功能单位 骨骼及其破坏影响幽灵蛋白-肌动蛋白相互作用 体外培养。我们建议分析不同大脑的四聚体形成 利用重组α-和β-血影蛋白多肽制备血影蛋白异构体 它们包含最有可能参与的幽灵蛋白区域 四聚体形成。这种相互作用将使用在 体外功能结合试验和定点突变获得 功能基因中的缺失和/或单一氨基酸替换 重组多肽。幽灵蛋白亚基的特定区域 参与互动的情况将会确定。我们还将解决 质疑不同亚型的脑影蛋白是否能够形成 体外形成的杂多络合物。 已有研究提出SH3结构域在信号转导中起作用 膜骨架组装的转导和调节。近期 几种与酪氨酸SH3结构域结合的蛋白质的鉴定 Kases、Grb2和PLC-Gamma提示，血影蛋白SH3结构域也可能 通过与已知蛋白质(S)的结合发挥作用，因为SH3配体- 结合位点在已知的几种蛋白质中是保守的。 或者，大脑的光影蛋白SH3结构域可能结合了一种新的蛋白质。至 解决我们将识别的脑影蛋白SH3结构域的功能并 克隆一种与红系和非红系α结合的蛋白(S) 利用已建立的重组DNA技术获得了SPECTIN SH3结构域。 与光影蛋白SH3结构域相互作用的蛋白质配体很可能 在细胞内与血影蛋白形成复合体。要确定这些潜力 脑影蛋白SH3结构域的生理作用我们将使用GST- 亲和琼脂糖和免疫沉淀法鉴定SH3结合蛋白 在神经细胞系的细胞裂解物中自然表达血影蛋白和 从转基因的细胞系中分离出来。

The long range goal of this proposal is to gain insight into the role of the cell membrane skeleton in human diseases. Defects in the erythrocyte membrane skeleton integral proteins underlie some hereditary hemolytic anemias in humans and mice, and recent studies implicate neuronal membrane skeletal proteins in neurodegenerative processes in aging and in Alzheimer's disease. However, little is known about the structure, function and regulatory processes of the neuronal cell membrane skeleton and its major component - brain spectrin. Neuronal compartmentalization of brain spectrin isoforms into axons and presynaptic terminals (nonerythroid spectrin) and into cell body and dendrites (erythroid spectrin) suggests that brain spectrin isoforms may perform related but distinct functions in neuronal cells. The proposed work on spectrin tetramer formation and the spectrin SH3 domain should give us the basis to understand functional differences between brain spectrin isoforms. The spectrin tetramer is the basic functional unit of the membrane skeleton and its disruption affects the spectrin-actin interaction in vitro. We propose to analyze the tetramer formation of different brain spectrin isoforms using recombinant alpha- and beta-spectrin polypeptides that contain the regions of spectrin most likely to be involved in tetramer formation. The interaction will be studied using established in vitro functional binding assays and site-directed mutagenesis to obtain deletions and/or single amino acid substitutions in the functional recombinant polypeptides. The specific regions of spectrin subunits involved in the interaction will be determined. We will also address the question whether different isoforms of brain spectrin are able to form heteromeric complexes in vitro. It has been proposed that the SH3 domain plays a role in signal transduction and regulation of the membrane skeleton assembly. Recent identification of several proteins binding to the SH3 domain of tyrosine kinases, Grb2 and PLC-gamma suggests that the spectrin SH3 domain may also function through a binding to a known protein(s) since the SH3 ligand- binding site is conserved among several already known proteins. Alternatively, the brain spectrin SH3 domain may bind a novel protein. To address the function of the brain spectrin SH3 domain we will identify and clone a protein(s) which binds to the erythroid and nonerythroid alpha- spectrin SH3 domain using established recombinant DNA techniques. Protein ligands interacting with the spectrin SH3 domain are likely to form complexes with spectrin in vivo in cells. To identify these potential physiological interactions of brain spectrin SH3 domain we will use GST- affinity agarose and immunoprecipitation to identify SH3-binding proteins in cell lysates from neuronal cell lines naturally expressing spectrin and from transfected cell lines.