Cytoskeletal Regulation of Erythrocyte Glucose Transporter-1

描述（由申请人提供）：Spectrin-actin连接在维持红细胞形状和膜特性中起关键作用。因此，它们的组织和与细胞膜的联系对红细胞生理学具有根本的重要性。我们最近的证据表明，dematin通过连接谱蛋白-肌动蛋白连接到细胞膜来发挥其功能。在这里，我们提出在人红细胞中，通过葡萄糖转运蛋白GLUT1和在小鼠红细胞中通过相关转运蛋白将谱蛋白-肌动蛋白连接连接到细胞膜。我们将用以下三个目标来检验这一假设。A1：人红细胞中GLUT1与谱蛋白-肌动蛋白连接相互作用的表征。失活蛋白头部结构域的缺失导致小鼠代偿性贫血伴小细胞增多和球形细胞增多。我们发现GLUT1是人类红细胞中降脂素和内收蛋白的主要膜受体。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，从而评估人类红细胞的形状。A2。小鼠红细胞和脂肪细胞中退化素和内收素结合受体的鉴定。在小鼠红细胞中，GLUT1受体不能将谱蛋白-肌动蛋白连接连接到膜上。我们预测，在小鼠红细胞中，褪黑素和内收蛋白与一种新的膜受体结合。我们将使用一系列生化和蛋白质组学方法来鉴定这种膜受体。我们将确定脂质和内收蛋白是否与GLUT4结合，GLUT4是一种胰岛素敏感的葡萄糖转运蛋白，通常存在于脂肪和肌肉细胞中，并研究它们在脂肪细胞中GLUT4的定位和再循环中的作用。这些实验将阐明在小鼠红细胞和脂肪细胞中连接谱蛋白-肌动蛋白连接的替代受体的作用。A3。测定完全失钙的影响。由于痴呆蛋白的核心结构域在没有头套的小鼠中仍然表达，因此在携带全长痴呆蛋白纯合基因断裂的小鼠中，仍有可能出现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，并对血液学和代谢病变进行全面分析，特别关注GLUT4运输和糖尿病的潜在发展。总之，这些研究将阐明痴呆蛋白的头区和核心区在红细胞生成和中间代谢中的具体作用。公共卫生相关性：红细胞膜已成为发现其蛋白在许多非红细胞中的功能的范例。本项目将研究痴呆蛋白、内收蛋白和葡萄糖转运蛋白在溶血性贫血、糖尿病和肥胖中形成一种新型膜-细胞骨架桥的作用。

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.