REGULATION OF GLUCOSE TRANSPORTER TARGETING AND ACTIVITY

并不是所有的分子事件都被理解，这提供了适当的 葡萄糖跨细胞质膜转运的调节 或破坏II型糖尿病患者的调节。 GLUT1 和GLUT 4是钠非依赖性的同源家族的两个成员， 葡萄糖转运蛋白，其特征在于低和高反应 运输，分别，主要是通过他们的相对能力， 在胰岛素的作用下重新分布到质膜上。 差异 在葡萄糖转运的动力学参数中，基底膜靶向 和胰岛素刺激状态，以及对降解的敏感性， 区分这两种转运蛋白的重要特性。 有 5个主要的非同一性领域，可以赋予独特的结构和 因此，每一个都具有独特的功能特性， 传送器 它们包括：1）N-末端，2）C-末端，3）大 细胞外环，4）大细胞内环，和5）跨膜 域2/3。 本提案的主要目的是比较 通过互换这些转运蛋白中的每一个而形成的嵌合转运蛋白的性质 GLUT 1和GLUT 4之间的结构域。 如果转让的域名赋予一个 或更多的独特的分布，动力学，或稳定性， 天然转运蛋白，那么该属性应该从 天然供体转运蛋白到受体嵌合转运蛋白。 一 最近分离的转运蛋白cDNA，命名为GLUT 4 B，编码一种变体 其中跨膜结构域XII和C-末端结构域 GLUT 4被一个独特的33个氨基酸序列所取代。 性能 GLUT 4 B的数量未知，因此本提案的另一个目标是 是表征和比较GLUT 4 B与GLUT 4，以评估 GLUT 4 B的结构差异可能改变转运蛋白的功能。 到 为了实现这些目标，将表达天然和嵌合转运蛋白 体外转录转运蛋白在非洲爪蟾卵母细胞中的显微注射 信息，以及通过测量确定的运输动力学常数 3-O-甲基葡萄糖通量。 嵌合转运蛋白也将在大肠杆菌中表达。 胰岛素应答性3 T3-L1脂肪细胞， 通过脉冲追踪标记3 T3-L1比较转运蛋白降解速率 脂肪细胞与35 S-甲硫氨酸和免疫沉淀，每个标记 具有结构域特异性抗体的嵌合转运蛋白。 膜 天然和嵌合转运蛋白的分布将在 卵母细胞和3 T3-L1脂肪细胞 记录每种细胞类型的技术，并定量溶解的 具有域特异性抗体的转运蛋白。 胰岛素的作用 嵌合转运蛋白的膜分布将在 3 T3-L1脂肪细胞。 确定哪些域赋予一个或多个独特的 GLUT 1、GLUT 4或GLUT 4 B的功能，以及了解 嵌合转运蛋白中的互换结构域改变或破坏 转运蛋白功能将进一步确定重要的结构区域， 每个传送器的正常功能 这些研究有望 为今后的研究奠定基础， 与这些结构域相互作用的蛋白质， 在这些蛋白质或转运域可能解释了某些形式的 2型糖尿病的胰岛素抵抗

Not all of the molecular events are understood which provide proper regulation of glucose transport across the plasma membrane of cells in healthy individuals, or disrupt regulation in type II diabetics. GLUT1 and GLUT4 are two members of a homologous family of sodium independent glucose transporters which can be characterized as low and high response transporters, respectively, primarily by their relative ability to redistribute to the plasma membrane in response to insulin. Differences in kinetic parameters for glucose transport, membrane targeting in basal and insulin stimulated states, and susceptibility to degradation are important properties which distinguish these two transporters. There are 5 major domains of non-identity which could confer unique structural and hence the unique functional properties which characterize each transporter. They include the: 1)N-terminus, 2)C-terminus, 3) large extracellular loop, 4)large intracellular loop, and 5)transmembrane domains 2/3. The primary goal of this proposal is to compare the properties of chimeric transporters formed by interchanging each of these domains between GLUT1 and GLUT4. If the domain transferred confers one or more of the unique distribution, kinetic, or stability properties upon the native transporter, then that property should be transferred from the native donor transporter to the recipient chimeric transporter. A recently isolated transporter cDNA, designated GLUT4B, encodes a variant transporter in which transmembrane domain XII and the C-terminal domain of GLUT4 are replaced by a unique 33 amino acid sequence. The properties of GLUT4B are unknown and therefore an additional goal of this proposal is to characterize and compare GLUT4B to GLUT4 to assess how the structural differences of GLUT4B might alter transporter function. To accomplish these goals native and chimeric transporters will be expressed in Xenopus oocytes by microinjecting in vitro transcribed transporter message, and the kinetic constants for transport determined by measuring 3-O-methylglucose flux. Chimeric transporters will also be expressed in insulin responsive 3T3-L1 adipocytes via a retroviral vector, and the rates of transporter degradation compared by pulse chase labelling 3T3-L1 adipocytes with 35S-methionine and immunoprecipitating each labelled chimeric transporter with domain specific antibodies. The membrane distribution of native and chimeric transporters will be determined in both oocytes and 3T3-L1 adipocytes by using membrane fractionation techniques documented for each cell type and quantifying the solubilized transporters with domain specific antibodies. The effect of insulin on the membrane distribution of chimeric transporters will be measured in 3T3-L1 adipocytes. Identifying which domains confer one or more unique functions to GLUT1,GLUT4, or GLUT4B, as well as understanding which interchanged domains in chimeric transporters alter or destroy transporter function will further define structural regions important to the normal function of each transporter. These studies hopefully will provide groundwork for future studies designed to identify regulatory proteins interacting with these domains and evaluate whether mutations in these proteins or transporter domains might account for some forms of insulin resistance in type II diabetes.