GLUCOSE TRANSPORTER STRUCTURE AND FUNCTION

描述：蛋白质介导的促进性葡萄糖转运对 细胞新陈代谢和机体碳水化合物动态平衡。尽管 广泛的研究表明，葡萄糖转运的机制尚不清楚。长距离的 本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。为达致这个目标，我们建议 研究人葡萄糖转运蛋白的结构和功能 Glut1.这种哺乳动物的葡萄糖转运蛋白是唯一一种服从于 分析可以作为从红细胞中纯化的蛋白质和作为 重组蛋白在Cos-7细胞中表达和亲和纯化。 特定目标1、4和5测试了Glut1作为 细胞膜上的协同性四聚体。特定目标1询问是否 四聚体Glut1是一种分离的人工制品，通过研究 不同的洗涤剂和脂类对转运蛋白低聚物大小的影响。具体目标4 用配体与ASK结合的停流分析测定Glut1的协同作用 如果半胱氨酸突变和半胱氨酸突变对Glut1寡聚体大小的扰动 异源复合体的形成也影响Glut1的协同作用。具体目标5 利用最近发现的Glut1协作性的意外结果 (通过亚饱和水平的竞争性抑制物来促进运输) 询问改变的Glut1寡聚体大小是否会影响这种合作行为。 碳水化合物文库也将被筛选出潜在的“动力激活剂”。 交通工具。 具体目标2和3针对的是Glut1结构。具体目标2建议 色氨酸诱变鉴定介导Glut1色氨酸残基(S) Glut1-色氨酸荧光猝灭当配体与糖特异结合 摄取或糖出口站点。特定目标3映射Glut1糖的摄取和输出 利用特定位点的光配体、多肽图谱和多肽构建结构域 测序。已确定的位置将接受丙氨酸扫描突变 重组的Glut1将被重新映射并进行活性测试，以确认 或驳斥功能丧失的问题。

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.