PROTEIN ASSOCIATIONS OF BRAIN SPECTRIN AND ANKYRIN

这项提议的长期目标是详细定义蛋白质 红细胞膜结构蛋白亚型的相关性， 鬼影蛋白和锚蛋白，已被发现是黄连的一般成分 细胞质膜，并阐明这些蛋白的细胞功能 联系。我们的方法将是对 纯化的放射性标记脑影蛋白的分子间相互作用 Ankyrin，并定义了这些蛋白的蛋白水解区，与蛋白质 在膜和细胞质组分中。这些测量将提供a) 鉴定和纯化结合蛋白的分析方法；b)允许 对蛋白质相互作用的调节进行评估；c)用于筛选 针对扰乱特定蛋白质关联的毒素和突变。 具体目标如下：(1)蛋白质研究 脑内血影蛋白及其定义域与脑功能的关系 细胞膜和细胞质中的蛋白质。新发现的幽灵蛋白结合 蛋白质将被提纯和鉴定。规管 血影蛋白与钙离子、钙调蛋白和 不同的蛋白激酶。(2)脑强直蛋白。这一蛋白质将被提纯 并且特征在于通过以下方式修改 磷酸化/去磷酸化及其细胞定位。(3) 脑锚定蛋白的结合蛋白将被定义和结合蛋白 按照脑部血影蛋白的描述进行纯化。(4)积分函数 与锚定蛋白结合的膜蛋白。(5)蛋白质的细胞功能 上述关联性将通过a)使用定义的蛋白质进行评估 具有竞争性的结构域或针对这些结构域的抗体 活细胞中蛋白质结合的拮抗剂，b)筛选毒素 用于调节蛋白质关联，目的是识别一种 特异性抑制物，c)筛选突变小鼠的功能或 表达鬼影蛋白、锚蛋白或任何结合蛋白。

The long range goal of this proposal is to define in detail the protein associations of isoforms of erythrocyte membrane structural proteins, spectrin and ankyrin, that have been discovered to be general components of cell plasma membranes, and to elucidate cellular functions of these protein linkages. The approach will be to analyse quantitatively the intermolecular associations of purified, radiolabeled brain spectrin, brain ankyrin, and defined proteolytic domains of these proteins, with proteins in membrane and cytoplasmic fractions. These measurements will a) provide assays for identification and purification of binding proteins; b) allow evaluation of regulation of the protein interactions; c) be used to screen for toxins and mutations that perturb specific protein associations. Specific goals are in the following areas: (1) studies of protein associations of brain spectrin and defined domains of brain spectrin with proteins in membranes and the cytosol. Newly identified spectrin-binding proteins will be purified and characterized. Regulation of spectrin-protein association will be studied with Ca++, calmodulin, and various protein kinases. (2) Brain ankyrin. This protein will be purified and characterized regarding modification by phosphorylation/dephosphorylation and its cellular localization. (3) Protein associations of brain ankyrin will be defined and binding proteins purified as described with brain spectrin. (4) Functions of integral membrane proteins that bind to ankyrin. (5) Cellular functions of protein associations described above will be evaluated by a) using defined protein domains or antibody against against these domains as competitive antagonists of protein associations in living cells, b) screening toxins for modulation of protein associations with the goal of identifying a specific inhibitor, c) screening mutant mice for alteration in function or expression of spectrin, ankyrin or any binding proteins.