HUMAN ERYTHROCYTE MEMBRANE CYTOSKELETON ASSOCIATIONS

人类红细胞膜细胞骨架协会

• 批准号: 3483522
• 负责人: G Vann Bennett
• 金额: $ 18.75万
• 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/3483522
• 关键词: RNA splicing; X ray crystallography; actins; adenosinetriphosphatase; 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; phospholipids; phosphorylation; potassium; protein kinase C; protein purification; protein structure; protein structure function; sodium; 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：（改编自研究者的摘要）在本提案中， 首席研究员打算进行详细的分子生物学研究 人红细胞锚蛋白和人红细胞的生化研究 内收蛋白。 对红细胞锚蛋白的研究将包括结构分析 89 kDa 结构域及其组成的 33 个残基重复序列。 定义 89 k Da 结构域的区域将在细菌中表达，并且最小 天然二级结构所需的重复次数 将测定圆二色性。 89 k Da 域的晶体将 为通过X射线衍射进行结构分析做好准备。 绑定 红细胞带 3 的位点位于该区域的 33 个残基重复区域内 将映射域以及最小重复次数和唯一重复次数 确定结合所需的。 微管蛋白和 Na/K 位点图谱 将在 89 k Da 结构域内进行 ATP 酶结合。 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. 线虫 lin12 基因产物）将被表达并用于 确定微管蛋白和其他物质可能的共同结合特异性 具有周期性结构的大分子（核酸、磷脂）。 红细胞和脑锚蛋白的血影蛋白结合位点将被绘制 以及能够表达血影蛋白结合的最小区域 活动确定。 这些锚蛋白内的区域提供 红细胞血影蛋白的特异性，以及脑血影蛋白的脑锚蛋白的特异性， 将被确定。 锚蛋白区域缺失的假设 蛋白质 2.2 是 mRNA 选择性剪接的产物，其功能为 将探索假底物和块的潜在结合位点。 肾微粒体中的蛋白质已被证明可以识别 将鉴定蛋白质2.2和非未剪接的锚蛋白。 第二专业 该研究的组成部分涉及人红细胞内收蛋白。 完全的 编码人类 aplha 和 beta 亚基的 cDNA 的克隆和测序 建议使用红细胞内收素。 两个亚基都将被表达并且 重组形成异二聚体和可能的同二聚体。 活跃站点 与钙调蛋白、肌动蛋白、血影蛋白/肌动蛋白、亚基相互作用 异二聚体和异二聚体之间的缔合形成四聚体和 更高的低聚物将使用作图技术来确定。 网站 蛋白激酶 A 和 C 的磷酸化也将被测定。