FUNCTIONAL STUDIES OF RED BLOOD CELL DEMATIN

红细胞脱质蛋白的功能研究

• 批准号: 2609324
• 负责人: Athar H. Chishti
• 金额: $ 15.35万
• 依托单位: STEWARD ST. ELIZABETH'S MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2609324
• 关键词: SDS polyacrylamide gel electrophoresis; actins; affinity chromatography; band 3 protein; binding proteins; complementary DNA; crosslink; cytoskeletal proteins; erythrocyte membrane; erythrocytes; gel filtration chromatography; glycophorin; immunoprecipitation; intermolecular interaction; laboratory rabbit; membrane proteins; molecular site; phosphorylation; physiology; protein isoforms; protein structure function; protein tyrosine kinase; sedimentation equilibrium; spectrin; western blottings

The aim of these studies is to define the physiological function of dematin in mature erythrocytes. We wish to identify the proteins(s) to which dematin binds on eh erythroid plasma membrane and to understand how these interactions are regulated by phosphorylation. The identification of dematin binding protein(s) on the membrane, and the understanding of the regulation of dematin-membrane interactions by phosphorylation, may elucidate a new mechanism by which spectrin-actin complexes are attached to the plasma membrane. Since immunoreactive homologs of dematin exist in many non-erythroid cells, the results of the proposed studies may have general implication well beyond red cell biology. In order to achieve these goals: (1) We propose to determine the number and affinity of dematin binding protein(s) on human erythrocyte inside- out-vesicles, and to identify the individual protein(s) using chemical crosslinking, immunoprecipitation, affinity chromatography, and gel filtration techniques. Then using defined cDNA constructs expressed in vitro, we will test whether dematin binding sites include the cytoplasmic domains of band 3 and glycophorin. Finally, we will examine whether tyrosine phosphorylation of dematin regulates its interaction with the plasma membrane. (2) To test the hypothesis that dematin may function as an adaptor protein that links spectrin-actin complexes to the plasma membrane, we will carry out a detailed characterization of the dematin- spectrin interaction. The studies will include quantification of the dematin-spectrin interaction by sedimentation equilibrium assays, the mapping of dematin and beta-spectrin binding site using cDNA constructs, and the regulation of dematin-spectrin interaction by tyrosine phosphorylation.

这些研究的目的是定义 成熟的红细胞中的dematin。 我们希望将蛋白质识别为 哪种症状与EH红细胞质膜结合，并了解如何 这些相互作用受磷酸化调节。 标识 膜上脑膜蛋白结合蛋白的理解 通过磷酸化来调节椎蛋白 - 膜相互作用，可能 阐明一种新机制，通过该机制连接了光谱 - 肌动蛋白复合物 到质膜。 由于存在免疫反应性同源物 在许多非果蝇细胞中，拟议的研究的结果可能具有 一般意义远远超出了红细胞生物学。 为了实现这些目标：（1）我们建议确定数量 dematin结合蛋白在人内部人类红细胞上的亲和力 - 外壁，并使用化学物质鉴定单个蛋白质 交联，免疫沉淀，亲和色谱法和凝胶 过滤技术。 然后使用在 体外，我们将测试dematin结合位点是否包括细胞质 带3和糖蛋白的结构域。 最后，我们将研究是否 dematin的酪氨酸磷酸化调节其与 质膜。 （2）检验dematin可能起作用的假设 作为将光谱 - 肌动蛋白复合物与血浆联系起来的衔接蛋白 膜，我们将对dematin-进行详细的表征 光谱相互作用。 研究将包括定量 通过沉积平衡测定法，症状 - 谱蛋白相互作用， 使用cDNA构建体对磁蛋白和β-光谱结合位点的映射， 以及通过酪氨酸对椎蛋白 - 光谱相互作用的调节 磷酸化。