GLUCOSE TRANSPORTER STRUCTURE AND FUNCTION

葡萄糖转运蛋白的结构和功能

• 批准号: 6198393
• 负责人: ANTHONY CARRUTHERS
• 金额: $ 19.5万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6198393
• 关键词: affinity chromatography; binding proteins; binding sites; electron microscopy; erythrocyte membrane; glucose transport; glucose transporter; homeostasis; intermolecular interaction; ligands; membrane transport proteins; mutant; protein purification; protein sequence; protein structure function; stop flow technique; tissue /cell culture; transfection

DESCRIPTION: Protein-mediated facilitative glucose transport is vital to cellular metabolism and organismal carbohydrate homeostasis. In spite of extensive study, the mechanism of glucose transport is unknown. The long-range goal of this laboratory is to understand the molecular mechanism of protein-mediated glucose transport. To achieve this goal, we propose to investigate the structure and function of the human glucose transport protein Glut1. This mammalian glucose transport protein is uniquely amenable to analysis being available as a purified protein from red blood cells and as a recombinant protein expressed in, and affinity-purified from Cos-7 cells. Specific Aims 1, 4, and 5 test the hypothesis that Glut1 functions as a cooperative homo tetramer in the cell membrane. Specific Aim 1 asks if tetrameric Glut1 is an artifact of isolation by investigating the effects of different detergents and lipids on transporter oligomeric size. Specific Aim 4 measures Glut1 cooperativity by stopped flow analysis of ligand binding to ask if perturbations of Glut1 oligomeric size by cysteine mutagenesis and by heterocomplex formation also affect Glut1 cooperativity. Specific Aim 5 exploits an unanticipated and recently discovered result of Glut1 cooperativity (transport stimulation by subsaturating levels of competitive inhibitors) to ask if altered Glut1 oligomeric size affects this cooperative behavior. Carbohydrate libraries will also be screened for potential "kinetic activators" of transport. Specific Aims 2 and 3 address Glut1 structure. Specific Aim 2 proposes tryptophan mutagenesis to identify Glut1 tryptophan residue(s) that mediate Glut1 tryptophan fluorescence quenching when ligands bind specifically at sugar uptake or sugar export sites. Specific Aim 3 maps Glut1 sugar uptake and export domains by use of site-specific photo-ligands, peptide mapping and peptide sequencing. Identified sites will be subjected to alanine scanning mutagenesis and the recombinant Glut1 will be remapped and tested for activity to confirm or refute loss of function.

描述：蛋白质介导的促进性葡萄糖转运对于 细胞代谢和有机体碳水化合物稳态。尽管 大量的研究表明，葡萄糖转运的机制尚不清楚。远距离的 该实验室的目标是了解其分子机制 蛋白质介导的葡萄糖转运。为了实现这一目标，我们建议 研究人类葡萄糖转运蛋白的结构和功能 过剩1。这种哺乳动物葡萄糖转运蛋白具有独特的作用 分析可作为从红细胞中纯化的蛋白质和作为 在 Cos-7 细胞中表达并亲和纯化的重组蛋白。 具体目标 1、4 和 5 检验 Glut1 充当 细胞膜中的协同同源四聚体。具体目标 1 询问是否 四聚体 Glut1 是通过研究以下因素的影响而分离出来的产物 不同的去垢剂和脂质对转运蛋白寡聚体大小的影响。具体目标 4 通过配体结合的停流分析来测量 Glut1 协同性 如果通过半胱氨酸诱变和通过 异源复合物的形成也会影响 Glut1 的协同作用。具体目标 5 利用 Glut1 协作性的一个意想不到的和最近发现的结果 （通过竞争性抑制剂的亚饱和水平来刺激运输） 询问改变的 Glut1 寡聚体大小是否会影响这种合作行为。 碳水化合物库还将筛选潜在的“动力学激活剂” 的运输。 具体目标 2 和 3 解决 Glut1 结构。具体目标 2 建议 色氨酸诱变以鉴定介导的 Glut1 色氨酸残基 当配体特异性结合糖时 Glut1 色氨酸荧光猝灭 摄取或糖出口地点。具体目标 3 绘制 Glut1 糖摄取和输出图 通过使用位点特异性光配体、肽图谱和肽来确定结构域 测序。确定的位点将进行丙氨酸扫描诱变 重组 Glut1 将被重新定位并测试活性以确认 或驳斥功能丧失。