Cytoskeletal Regulation of Erythrocyte Glucose Transporter-1

描述(由申请人提供)：血影蛋白-肌动蛋白连接在维持红细胞形状和膜特性方面起着关键作用。因此，它们的组织和与细胞膜的连接对红细胞生理学具有重要意义。我们最近的证据表明，Dematin通过将血影蛋白-肌动蛋白连接到细胞膜上来发挥其功能。在这里，我们认为Dematin通过人红细胞中的葡萄糖转运蛋白GLUT1和小鼠红细胞中的相关转运蛋白将血影蛋白-肌动蛋白连接到细胞膜。我们将通过以下三个目标来检验这一假设。A1：人类红细胞中GLUT1与血影蛋白-肌动蛋白连接相互作用的特征。Dematin头盔结构域的缺失会导致小鼠出现代偿性贫血，并伴有小细胞增多和球状细胞增多。我们发现GLUT1是人红细胞内收蛋白和降钙素的主要膜受体。我们将确定它们的相互作用结构域和关键序列元件，并通过生化手段破坏GLUT1-细胞骨架桥，评估其对膜稳定性的功能影响，从而影响人类红细胞的形状。A2.小鼠红细胞和脂肪细胞中褪黑素和内收蛋白结合受体(S)的鉴定GLUT1受体不将血影蛋白-肌动蛋白连接连接到小鼠红细胞膜上。我们预测Dematin和Adducin与小鼠红细胞膜上的一种新的膜受体(S)结合。我们将使用一系列生化和蛋白质组学方法鉴定这种膜受体。我们将确定Dematin和Adducin是否与GLUT4结合，GLUT4是一种普遍存在于脂肪和肌肉细胞中的胰岛素敏感型葡萄糖转运体，并研究它们在脂肪细胞中GLUT4的定位和循环中的作用。这些实验将阐明连接小鼠红细胞和脂肪细胞中的血影蛋白-肌动蛋白连接的交替受体的作用。A3.确定完全性去甲素缺乏症的影响。由于dematin的核心区仍然在头盔缺失的小鼠中表达，因此携带全长dematin纯合子基因中断的小鼠仍有可能产生更深刻的血液学和代谢表型。我们建议建立后一种小鼠模型，并对血液学和代谢损伤进行全面分析，特别是关注GLUT4的运输和糖尿病的潜在发展。总之，这些研究将阐明Dematin的头盔和核心区在红细胞生成和中间代谢中的特殊作用(S)。与公共卫生相关：红细胞膜已成为许多非红系细胞中发现红细胞膜蛋白功能的范例。本项目将研究Dematin、内收蛋白和葡萄糖转运蛋白在一种新的膜-细胞骨架桥的形成中的作用，该桥具有溶血性贫血、糖尿病和肥胖症的功能。

DESCRIPTION (provided by applicant): Spectrin-actin junctions play critical roles in both the maintenance of red blood cell shape and membrane properties. Their organization and linkage to the cell membrane is therefore of fundamental importance to erythrocyte physiology. Our recent evidence indicates that dematin performs its function by linking spectrin-actin junctions to the cell membrane. Here, we propose that dematin links spectrin-actin junctions to the cell membrane via the glucose transporter, GLUT1, in human erythrocytes and via a related transporter in mouse erythrocytes. We will test this hypothesis with the following three aims. A1: Characterization of GLUT1 interactions with spectrin-actin junctions in human erythrocytes. Deletion of the headpiece domain of dematin results in a compensated anemia with microcytosis and spherocytosis in mice. We identified GLUT1 as the primary membrane receptor for both dematin and adducin in human erythrocytes. We will identify their interacting domains and the critical sequence elements and, through disruption of the GLUT1-cytoskeletal bridge by biochemical means, assess its functional impact on membrane stability and thus the shape of human erythrocytes. A2. Identification of dematin- and adducin-binding receptor(s) in mouse erythrocytes and adipocytes. The GLUT1 receptor does not link spectrin-actin junctions to the membrane in mouse erythrocytes. We predict that dematin and adducin bind to a novel membrane receptor(s) in mouse erythrocytes. We will identify this membrane receptor using a series of biochemical and proteomic approaches. We will determine whether dematin and adducin bind to GLUT4, the insulin-sensitive glucose transporter generally found in adipose and muscle cells, and investigate their role in localization and recycling of GLUT4 in adipocytes. These experiments will clarify the role of alternate receptors that link the spectrin-actin junctions in mouse erythrocytes and adipocytes. A3. Determination of the effects of complete dematin deficiency. As the core domain of dematin is still expressed in headpiece-null mice, there remains the possibility that more profound hematological and metabolic phenotypes develop in mice carrying a homozygous gene disruption of full-length dematin. We propose to generate the latter mouse model and conduct a comprehensive analysis of the hematological and metabolic lesions, focusing particularly on GLUT4 trafficking and the potential development of diabetes. Together, these studies will elucidate the specific role(s) of the headpiece and core domains of dematin in erythropoiesis and intermediary metabolism. PUBLIC HEALTH RELEVANCE: Erythrocyte membrane has served as a paradigm for discovering the function of its proteins in many non-erythroid cells. This project will investigate the role of dematin, adducin, and glucose transporters in the formation of a novel membrane-cytoskeletal bridge with functional implications in hemolytic anemia, diabetes, and obesity.