GLUCOSE TRANSPORTER STRUCTURE AND FUNCTION

葡萄糖转运蛋白的结构和功能

• 批准号: 6634991
• 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/6634991
• 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：蛋白质介导的促进葡萄糖转运对于 细胞代谢和有机碳水化合物稳态。尽管 广泛的研究，葡萄糖转运的机制尚不清楚。远程 该实验室的目标是了解分子机制 蛋白质介导的葡萄糖转运。为了实现这一目标，我们建议 研究人葡萄糖转运蛋白的结构和功能 glut1。该哺乳动物葡萄糖转运蛋白与 分析可作为来自红细胞的纯化蛋白质，作为纯化的蛋白 重组蛋白在COS-7细胞中表达并亲和力纯化。 具体目的1、4和5检验了GLUT1充当的假设 细胞膜中的合作均匀四聚体。特定的目标1问是否是否 四聚体Glut1是通过研究 转运蛋白低聚大小的不同洗涤剂和脂质。具体目标4 通过停止配体结合的流量分析来衡量GLUT1合作性 如果半胱氨酸诱变和通过 杂点的形成也会影响GLUT1的合作。具体目标5 利用GLUT1合作的意外而最近发现的结果 （通过竞争抑制剂的饱和水平进行运输刺激） 询问GLUT1低聚大小是否会影响这种合作行为。 碳水化合物文库也将被筛选，以获取潜在的“动力激活剂” 运输。 具体目标2和3地址GLUT1结构。特定目标2提出 色氨酸诱变，以鉴定介导的GLUT1色氨酸残基 Glut1色氨酸荧光淬灭时配体在糖上特别结合时 吸收或糖出口地点。特定的目标3地图GLUT1糖吸收和导出 使用特定于位点的光合物，肽映射和肽的域 测序。确定的位点将进行丙氨酸扫描诱变 重组GLUT1将重新构造并测试活动以确认 或反驳功能丧失。