REGULATION OF GLUCOSE TRANSPORTER TARGETING AND ACTIVITY

并不是所有的分子事件都被理解，它们提供了适当的 细胞内葡萄糖跨膜转运的调节 健康个体，或扰乱II型糖尿病患者的调节。Glut1 和GLUT4是钠非依赖性同源家族的两个成员 葡萄糖转运蛋白，可分为低反应和高反应 转运体，主要是根据它们的相对能力 对胰岛素的反应重新分布到质膜上。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 区别这两个传送器的重要性质。确实有 5个主要的非同一性领域，可以授予独特的结构和 因此，每个组件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)大的细胞内环，5)跨膜 域2/3。此提案的主要目标是比较 通过相互交换形成的嵌合转运体的性质 GLUT1和GLUT4之间的域。如果转移的域授予一个 或更多独特的分布、动力学或稳定性特性 本地传送器，则该属性应从 原生供体转运体到受体嵌合转运体。一个 最近分离到的转运蛋白cDNA，命名为GLUT4B，编码一个变体 其中跨膜结构域XII和C-末端结构域的转运体 GLUT4被一个独特的33个氨基酸序列取代。这些属性 GLUT4B是未知的，因此这项提案的另一个目标 对GLUT4B和GLUT4进行表征和比较，以评估 GLUT4B的结构差异可能会改变转运蛋白的功能。至 实现这些目标的原生和嵌合转运蛋白将被表达 体外微量注射转录转运蛋白在非洲爪哇卵母细胞中的应用 信息，以及通过测量确定的传输的动力学常数 3-O-甲基葡萄糖通量。嵌合转运蛋白也将在 胰岛素反应性3T3-L1脂肪细胞通过逆转录病毒载体，以及 脉冲追踪法标记3T3-L1转运蛋白降解速率的比较 用35S-蛋氨酸和免疫沉淀标记脂肪细胞 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#年确定。 卵母细胞和3T3-L1脂肪细胞的膜分离 为每种细胞类型记录的技术并量化溶解的 具有结构域特异性抗体的转运体。胰岛素对血管紧张素转换酶的影响 嵌合转运体的膜分布将在 3T3-L1脂肪细胞。标识哪些域授予一个或多个唯一 GLUT1、GLUT4或GLUT4B的函数，以及了解哪些 嵌合转运蛋白中的互换结构域改变或破坏 运输机功能将进一步定义对以下方面重要的结构区域 每个传送器的正常功能。这些研究希望能 为未来旨在确定监管机构的研究奠定基础 与这些结构域相互作用的蛋白质并评估突变 在这些蛋白质或转运体结构域中可能解释了某些形式的 2型糖尿病患者的胰岛素抵抗。

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.