PROTEIN ASSOCIATIONS OF BRAIN SPECTRIN AND ANKYRIN

脑血影蛋白和锚蛋白的蛋白质组合

基本信息

批准号：
3284313
负责人：
G Vann Bennett
金额：
$ 6.23万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-09-30 至 1989-06-30
项目状态：
已结题

项目摘要

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.

该提案的长期目标是详细定义蛋白质红细胞膜结构蛋白亚型的关联，血影蛋白和锚蛋白，已被发现是细胞质膜，并阐明这些蛋白质的细胞功能联系。该方法将定量分析纯化的、放射性标记的脑血影蛋白、脑的分子间缔合锚蛋白，以及这些蛋白质的确定的蛋白水解域，以及蛋白质在膜和细胞质部分。这些测量将 a) 提供结合蛋白的鉴定和纯化测定； b) 允许评估蛋白质相互作用的调节； c) 用于筛选用于扰乱特定蛋白质关联的毒素和突变。具体目标有以下几个方面：（1）蛋白质研究脑血影蛋白和脑血影蛋白定义域的关联膜和细胞质中的蛋白质。新发现的血影蛋白结合蛋白质将被纯化和表征。监管将使用 Ca++、钙调蛋白和各种蛋白激酶。 (2)脑锚蛋白。该蛋白质将被纯化并以修改为特征磷酸化/去磷酸化及其细胞定位。 (3) 将定义脑锚蛋白的蛋白质关联并结合蛋白质按照脑血影蛋白的描述进行纯化。 (4) 积分函数与锚蛋白结合的膜蛋白。 (5)蛋白质的细胞功能上述关联将通过 a) 使用确定的蛋白质进行评估作为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关联的拮抗剂，b) 筛选毒素用于调节蛋白质关联，目的是确定特异性抑制剂，c) 筛选突变小鼠的功能改变或血影蛋白、锚蛋白或任何结合蛋白的表达。