MEMBRANE-MICROFILAMENT CONNECTIONS

膜-微丝连接

• 批准号: 3282057
• 负责人: JOHN R GLENNEY
• 金额: $ 9.98万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282057
• 关键词: binding proteins; calcium; calmodulin; cell adhesion; cell membrane; chickens; cytoskeleton; electron microscopy; fluorescence microscopy; guinea pigs; immunoprecipitation; laboratory mouse; laboratory rabbit; membrane proteins; microfilaments; monoclonal antibody; neoplastic transformation; phosphorylation; protein structure; proteolysis; radioimmunoassay

The goal of this project is the determination of the mechanism and result of connections between the microfilament system and the plasma membrane. Previously we had analyzed the system of non-erythroid spectrins as a model for this subcortical network. We now propose to extend our research to the analysis of 36,000 Mr tyrosine kinase substrates termed calpactins. These proteins which exist as monomers or as complexes with a 10,000 Mr light chain interact with the cytoskeletal proteins actin and spectrin and are modulated by Ca++ and phospholipid. We will identify other members of this family using a technique which relies on its ability to interact with actin and phospholipid, and a detailed comparison of the structure and function of these proteins will be made. A detailed analysis of Ca++-binding, including affinity modulators will be undertaken. We will analyze the functional relatedness of the light chain with the S-100 proteins. Monoclonal and polyclonal antibodies will be raised to defined regions (peptides) of calpactin for use in analysis of its function. The tissue and subcellular distribution using Western Blotting and immunofluorescence microscopy. The in vivo target of calpactins will be determined using cross-linking reagents followed by immunoprecipitation. The calpactin heavy and light chain genes will be expressed in bacteria and protein products will be purified and tested for biological activity. We will attempt to identify the region of calpactin needed for Ca++-binding. The effect of tyrosine phosphorylation of calpactin will be tested by determining whether there is any difference in the ability of phosphorylated calpactin to interact with phospholipid, Ca++ or spectrin. These studies will further our understanding of the subcortical filament network and its relationship to cell transformation.

这个项目的目标是确定机制 以及微丝系统和微丝之间的连接结果 质膜。在此之前，我们已经分析了 非红系血影蛋白作为这个皮质下网络的模型。 我们现在建议将我们的研究扩展到对36,000个 MR酪氨酸激酶底物称为calpactins。这些蛋白质 它们以单体或具有10,000 mR光的络合物的形式存在 链与细胞骨架蛋白肌动蛋白和幽灵蛋白相互作用 并受钙离子和磷脂的调节。我们将确定 这个家族的其他成员使用一种依赖于其 与肌动蛋白和磷脂相互作用的能力，以及详细的 将对这些蛋白质的结构和功能进行比较 制造。钙离子结合的详细分析，包括亲和力 将进行调制器。我们将分析功能 轻链与S-100蛋白的相关性。单克隆 多克隆抗体将被提升到特定的区域 用于分析其功能的钙调蛋白(多肽)。这个 免疫印迹和免疫印迹法检测组织和亚细胞分布 免疫荧光显微镜。Calpactins的体内靶点 将使用交联剂进行测定，然后 免疫沉淀。钙调蛋白重链和轻链基因 将在细菌中表达，蛋白质产品将被提纯 并进行了生物活性测试。我们将尝试确定 钙离子结合所需的钙化蛋白区域。的影响 Calpactin的酪氨酸磷酸化将通过 确定是否有任何不同的能力 磷酸化钙调蛋白与磷脂、钙或钙相互作用 幽灵。这些研究将进一步加深我们对 皮质下细丝网络及其与细胞的关系 转型。