MOLECULAR INTERACTIONS OF BRAIN SPECTRIN

本提案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 膜骨架整合蛋白是某些遗传性溶血性 人类和小鼠的贫血，最近的研究表明， 骨骼蛋白在神经退行性变过程中的作用 老年痴呆症然而，人们对它的结构知之甚少， 神经元细胞膜骨架的功能和调节过程 和它的主要成分脑血影蛋白。神经元区室化 脑血影蛋白亚型进入轴突和突触前末梢（非红细胞 血影蛋白）并进入细胞体和树突（红细胞血影蛋白）表明 脑血影蛋白亚型可能执行相关但不同的功能， 神经元细胞对血影蛋白四聚体的形成和 血影蛋白SH 3结构域应该为我们理解其功能提供基础， 脑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 骨架及其破坏影响血影蛋白-肌动蛋白相互作用， 体外我们建议分析不同脑组织的四聚体形成 使用重组α-和β-血影蛋白多肽的血影蛋白同种型 包含血影蛋白的区域最有可能参与 四聚体形成。将使用建立在 体外功能性结合测定和定点诱变以获得 功能性氨基酸序列中的缺失和/或单个氨基酸取代 重组多肽。血影蛋白亚基的特定区域 参与互动的人将被确定。我们还将讨论 我怀疑脑血影蛋白的不同亚型是否能够形成 异聚体复合物在体外。 已经提出SH 3结构域在信号传导中起作用。 膜骨架组装的转导和调节。最近 几种与酪氨酸SH 3结构域结合的蛋白质的鉴定 激酶，Grb 2和PLC-γ表明血影蛋白SH 3结构域也可能 由于SH 3配体- 结合位点在几种已知的蛋白质中是保守的。 或者，脑血影蛋白SH 3结构域可以结合新的蛋白质。到 解决脑血影蛋白SH 3结构域的功能，我们将确定和 克隆与红细胞和非红细胞α- 血影蛋白SH 3结构域的表达。 与血影蛋白SH 3结构域相互作用的蛋白质配体可能 与血影蛋白在体内细胞中形成复合物。为了识别这些潜在的 脑血影蛋白SH 3结构域的生理相互作用，我们将使用GST- 亲和琼脂糖和免疫沉淀法鉴定SH 3结合蛋白 在来自天然表达血影蛋白的神经元细胞系的细胞裂解物中， 从转染的细胞系。

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.