Cytoskeletal Regulation of Erythrocyte Glucose Transporter-1

摘要 红细胞血影蛋白-肌动蛋白连接在维持细胞形态和 膜特性。因此，它们的组织和膜连接是基本的 对红细胞生理学的重要性。我们最近的证据表明，Dematin执行一种 通过将这些连接连接到膜来发挥枢轴作用。在这里，我们建议戴蒙德 通过将血影蛋白-肌动蛋白连接到膜上来执行其生物学功能。 人红细胞葡萄糖转运蛋白-1(GLUT1)我们将在以下情况下测试这一假设 以下三个目标：A1：Glut1与人体内的血影蛋白-肌动蛋白连接的相互作用 红血球。我们已经确定GLUT1是Dematin的主要膜受体，并 人红细胞内收蛋白。我们建议确定GLUT1的相互作用结构域， 和内收蛋白，并通过生化手段破坏GLUT1-细胞骨架桥，从而 评估其对人红细胞膜稳定性和形状的影响。A2.明晰 和小鼠红细胞和脂肪细胞的内收蛋白结合受体(S)。我们建议 Dematin和Adducin与小鼠红细胞膜上一种新的膜受体(S)结合。我们会 用免疫沉淀、质谱学和化学交联法鉴定该受体 化验。我们将测试降钙素和内收蛋白与几种电位膜的结合情况。 在我们的小鼠红细胞膜囊泡蛋白质组学筛选中发现的蛋白质。我们会 此外，还研究了Dematin和Adducin与GLUT4的结合，并研究了它们的功能 对脂肪细胞GLUT4循环和葡萄糖摄取的影响。这些实验将澄清 连接血影蛋白-肌动蛋白连接的交替受体在小鼠红细胞中的作用 脂肪细胞。A3.完全性去甲素缺乏症的生理意义。最近，我们 在Dematin头盔区域缺失的小鼠中观察到肥胖表型。为了澄清 针对Dematin头盔结构域的具体作用，我们提出了一种新的小鼠模型 使用传统的基因打靶技术，完全缺乏降钙素。我们会 比较聚焦头盔和Dematin缺失小鼠的贫血和肥胖症表型 特别是针对受损的GLUT4贩运和糖尿病。总而言之，这些研究可能会 揭示Dematin、Adducin和葡萄糖转运蛋白在调节贫血中的新功能 和代谢紊乱，对其他头盔领域有更广泛的影响-包含 蛋白质。

SUMMARY Erythrocyte spectrin-actin junctions play a critical role in the maintenance of cell shape and membrane properties. Their organization and membrane linkage is therefore of fundamental importance to erythrocyte physiology. Our recent evidence indicates that dematin performs a pivotal function by linking these junctions to the membrane. Here, we propose that dematin performs its biological function by linking the spectrin-actin junctions to the membrane via glucose transporter-1 (GLUT1) in human erythrocytes. We will test this hypothesis under the following three aims: A1: GLUT1 interaction with the spectrin-actin junctions in human erythrocytes. We have identified GLUT1 as the primary membrane receptor for dematin and adducin in human erythrocytes. We propose to identify the interacting domains of GLUT1, dematin, and adducin, and disrupt the GLUT1-cytoskeletal bridge by biochemical means, thus assessing its impact on the membrane stability and shape of human erythrocytes. A2. Dematin and adducin binding receptor(s) in mouse erythrocytes and adipocytes. We propose that dematin and adducin bind to a novel membrane receptor(s) in mouse erythrocytes. We will identify this receptor using immunoprecipitation, mass spectrometry, and chemical crosslinking assays. We will test the binding of dematin and adducin with several potential membrane proteins identified in our proteomics screen of mouse erythrocyte membrane vesicles. We will also investigate the binding of dematin and adducin with GLUT4, and investigate their functional effects on GLUT4 recycling and glucose uptake in adipocytes. These experiments will clarify the role of alternate receptors that link the spectrin-actin junctions in mouse erythrocytes and adipocytes. A3. Physiological implications of complete dematin deficiency. Recently, we observed an obesity phenotype in the dematin headpiece domain null mice. To elucidate the specific role of the headpiece domain of dematin, we propose to generate a new mouse model with complete dematin deficiency using the conventional gene targeting techniques. We will compare the anemia and obesity phenotypes in the headpiece and dematin null mice focusing particularly on the impaired GLUT4 trafficking and diabetes. Together, these studies are likely to unveil a novel function of dematin, adducin, and glucose transporters in the regulation of anemia and metabolic disorders with broader implications for other headpiece domain-containing proteins.