Functional Studies of Erythrocyte Dematin

公认的是，“血影蛋白-肌动蛋白连接”在功能上是必需的。 维持红细胞膜的稳定性和机械性能。现行 范例是“血影蛋白-肌动蛋白连接”通过一个连接到质膜上。 蛋白4.1和血型糖蛋白C之间的相互作用。根据我们最近的研究结果，我们提出了一个 “血影蛋白-肌动蛋白连接”与质膜垂直连接的替代模型。 我们目前的模型预测，dematin，与内收蛋白，组装一个新的， 血影蛋白多肽末端的细胞骨架复合物，连接血影蛋白-肌动蛋白 通过跨膜蛋白连接到质膜。在这次竞争激烈的续约中， 以下实验方法将用于验证我们的假设：（1）组合 小鼠dematin和adducin的缺失导致具有高度脆性红细胞的严重贫血。 为了阐明减弱血影蛋白-肌动蛋白连接到质膜的机制，我们 将研究肌动蛋白原丝的状态，拯救膜不稳定性缺陷，检查 双突变对已知垂直相互作用的影响，并测试dematin的直接结合 内收蛋白及其通过磷酸化和寡聚化的调节。(2)Dematin绑定到 红细胞质膜上的蛋白酶敏感位点。为了识别这种蛋白质，我们将 进行细胞表面标记、免疫沉淀、蛋白质下拉测定，以及 红细胞膜囊泡的蛋白质组学分析。将进行类似的分析， 内收蛋白这些实验很可能揭示新的跨膜蛋白， 连接复合物与质膜的连接。(3)根据目标2中概述的战略，我们 在dematin免疫沉淀物中检测到50 kDa的红细胞表面蛋白。进一步 分析表明，这种蛋白质是葡萄糖转运蛋白1（GLUT-1），一种主要的跨膜转运蛋白， 红细胞膜的受体。我们建议描述的生化性质， 人dematin和adducin与GLUT-1的相互作用，鉴定相互作用的结构域，和 证明了非红细胞系细胞中新桥的保守性。自从dematin，内收蛋白， 和GLUT-1广泛表达，包括大脑，拟议的研究可能是 这对于理解脑血管病患者血脑屏障葡萄糖转运的分子基础具有重要意义。 正常和病理情况下，如GLUT-1缺乏综合征。

It is well recognized that the "spectrin-actin junctions" are functionally essential for the maintenance of erythrocyte membrane stability and mechanical properties. The prevailing paradigm is that the "spectrin-actin junctions" are linked to the plasma membrane via an interaction between protein 4.1 and glycophorin C. Based on our recent findings, we propose an alternate model for the vertical linkage of "spectrin-actin junctions" to the plasma membrane. Our current model predicts that dematin, in conjunction with adducin, assembles a novel cytoskeletal complex at the tail ends of spectrin polypeptides that links the ¿spectrin-actin junctions¿ to the plasma membrane via a transmembrane protein. In this competitive renewal, the following experimental approaches will be used to validate our hypothesis: (1) Combined deletion of mouse dematin and adducin results in severe anemia with highly fragile erythrocytes. To elucidate the mechanism of weakened spectrin-actin linkage to the plasma membrane, we will investigate the status of actin protofilaments, rescue membrane instability defect, examine the effect of double mutation on known vertical interactions, and test direct binding of dematin with adducin and its regulation by phosphorylation and oligomerization. (2) Dematin binds to a protease-sensitive site on the erythrocyte plasma membrane. To identify this protein, we will perform surface labeling of cells, immunoprecipitations, protein pull-down assays, and proteomic analysis of erythrocyte membrane vesicles. A similar analysis will be performed for adducin. These experiments are likley to unveil new transmembrane proteins that would tether the junctional complex to the plasma membrane. (3) Following the strategy outlined in Aim 2, we detected a 50 kDa erythrocyte surface protein in the immunoprecipitates of dematin. Further analysis indicates that this protein is glucose transporter 1 (GLUT-1), a major transmembrane receptor of the erythrocyte membrane. We propose to characterize the biochemical nature of human dematin and adducin interactions with GLUT-1, identify the interacting domains, and demonstrate the conservation of the new bridge in non-erythroid cells. Since dematin, adducin, and GLUT-1 are widely expressed, including the brain, the proposed studies are potentially significant for understanding the molecular basis of blood-brain barrier glucose transport in normal and pathological settings such as GLUT-1 deficiency syndrome.