ERYTHROCYTE MEMBRANE CYTOSKELETON ASSOCIATIONS

红细胞膜细胞骨架协会

• 批准号: 2138237
• 负责人: G Vann Bennett
• 金额: $ 20.12万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2138237
• 关键词: RNA splicing; X ray crystallography; actins; ankyrins; band 3 protein; binding proteins; calmodulin; circular dichroism; complementary DNA; cytoskeletal proteins; erythrocyte membrane; membrane proteins; membrane transport proteins; messenger RNA; molecular biology; molecular cloning; molecular site; nucleic acid sequence; phospholipids; phosphorylation; potassium; protein kinase C; protein purification; protein structure; protein structure function; sodium; sodium potassium exchanging ATPase; spectrin; tubulin

DESCRIPTION: (Adapted from investigator's abstract) In this proposal, the principal investigator intends to do detailed molecular biology and biochemical studies of human erythrocyte ankyrin and human erythrocyte adducin. Studies on erythrocyte ankyrin will include structural analysis of the 89 kDa domain and its constituient 33-residue repeats. Defined regions of the 89 k Da domain will be expressed in bacteria and the minimal number of repeats required for native secondary structure as monitored by circular dichroism will be determined. Crystals of the 89 k Da domain will be prepared for analysis of structure by X-ray diffraction. The binding sites for erythrocyte band 3 within the 33 residue repeat region of this domain will be mapped and the minimal number of repeats and unique repeats required for binding determined. Mapping for sites of tubulin and Na/K ATPase binding within the 89 k Da domain will be performed. Domains of other proteins with homologous 33 residue repeats (Drosophila Notch and C. elegans lin12 gene products) will be expressed and they will be used to identify possible common binding specificity for tubulin and other macromolecules with periodic structures (nucleic acids, phospholipids). The spectrin binding sites of red cell and brain ankyrins will be mapped and the minimum regions competent for expressing spectrin-binding activities determined. The regions within these ankyrins that provide specificity for red cell spectrin, and brain ankyrin for brain spectrin, will be determined. The hypothesis that the region of ankyrin deleted in protein 2.2, a product of alternatively spliced mRNA, functions as a pseudosubstrate and blocks potential binding sites will be explored. Proteins in kidney microsomes that have been demonstrated to recognize protein 2.2 and not unspliced ankyrin will be identified. The second major component of the study relates to human erythrocyte adducin. Complete cloning and sequencing of cDNA encoding aplha and beta subunits of human erythrocyte adducin are proposed. Boths subunits will be expressed and recombined to form heterodimers and possibly homodimers. Active sites for interaction with calmodulin, actin, spectrin/actin, subunit interactions to heterodimers and associations between heterodimers to form tetramers and higher oligomers will be determined using mapping techniques. Sites of phosphorylation by protein kinases A and C will also be determined.

描述：（根据调查员的摘要进行了改编）在此提案中 主要研究者打算进行详细的分子生物学和 人类红细胞的生化研究和人类红细胞 adducin。 关于红细胞伸蛋白的研究将包括结构分析 在89个kDa域及其构成33个保留重复序列中。 定义 89 K DA结构域的区域将在细菌中表达，最小 由本地二级结构所需的重复次数 将确定圆二色性。 89 K DA域的晶体将 准备通过X射线衍射分析结构。 结合 33个残基重复区域内红细胞带3的位点 域将被映射，重复的数量最少和独特的重复序列 确定结合所需。 小管蛋白和Na/K位的映射 将执行89 K DA结构域中的ATPase结合。 域的域 其他具有同源33个残基重复序列的蛋白质（果蝇Notch和C. 秀丽隐杆线lin12基因产品）将被表达，它们将用于 确定微管蛋白和其他可能的共同结合特异性 具有周期性结构（核酸，磷脂）的大分子。 将映射红细胞和脑藻蛋白的光谱结合位点 以及有能力表达光谱结合的最低区域 确定的活动。 这些伸道中的区域提供 红细胞光谱的特异性和脑谱蛋白的脑arnkyrin的特异性， 将确定。 Ankyrin区域删除的假设 蛋白2.2是一种剪接mRNA的产物，用作A 将探索伪基底和阻止潜在的结合位点。 肾脏微粒体中的蛋白质已被证明可以识别 将鉴定蛋白质2.2，而未斑nk骨会被鉴定出来。 第二大专业 研究的成分与人类红细胞添加剂有关。 完全的 编码APLHA和人类β亚基的cDNA的克隆和测序 提出了红细胞添加剂。 这两个亚基都将被表达并 重组以形成异二聚体和可能的同型二聚体。 活跃站点 与钙调蛋白，肌动蛋白，谱蛋白/肌动蛋白，亚基相互作用的相互作用 异二聚体和异二聚体之间形成四聚体和 将使用映射技术确定较高的低聚物。 站点 还将确定蛋白激酶A和C的磷酸化。