FUNCTIONAL CLONING OF PROTEINS USED IN GLUT4 TRAFFICKING

描述：(改编自申请者的摘要)贩运GLUT4 进出细胞表面的葡萄糖转运蛋白对 葡萄糖动态平衡，因为这种蛋白存在于质膜 决定脂肪和肌肉对葡萄糖的利用率。胰岛素 刺激GLUT4从细胞内重新分布到质膜 这种反应在胰岛素抵抗状态下受损，如 2糖尿病。PI希望了解GLUT4通过什么机制 在没有胰岛素的情况下被隔离在细胞内，是通过什么机制 胰岛素会导致胰岛素的重新分配。他开发了一种新的分析方法来 定量而快速地测量GLUT4相对比例的变化 存在于3T3-L1和中国仓鼠卵巢细胞表面。这个化验 依赖于检测到外化的myc表位标签，该标签表明 细胞表面的蛋白质数量，以及框内融合的GFP 蛋白质的羧基末端，作为蛋白质的量的内部控制 各细胞均有GLUT4的表达。通过使用流式细胞术来测量 在两个波长的荧光强度上，等电点都得到了相对的 测量单个细胞表面GLUT4的表达。这个 因此，化验方法能够对编码蛋白质的cDNA进行功能性克隆 规范GLUT4贩运。将使用两种方法；第一种方法涉及 表达转运蛋白的CHO细胞被感染的浓缩方法 带有编码脂肪细胞cDNA文库的逆转录病毒；这将被感染 转化成CHO细胞，使每个细胞表达一个单独的cDNA.流式细胞术 将用于丰富将显示增强或减少的细胞 胰岛素存在和不存在时表面GLUT4的表达之后 对已分选的细胞进行几轮的浓缩和扩增，将PCR用于 扩增并鉴定导致GLUT4分布改变的基因。这个 第二种方法涉及同胞选择策略以识别改变的cDNA GLUT4靶向。CDNA库将被导入CHO细胞，表达 转运体和流式细胞术将用于识别克隆池 哪个目标(表面与内部标签的比率)因此而改变 生理操控。克隆池的进一步细分将导致 在鉴定含有改变GLUT4的cDNA的单个细胞系中 贩运，这将通过聚合酶链式反应进行测序。

DESCRIPTION: (Adapted from the applicant's abstract) Trafficking of the GLUT4 glucose transporter to and from the cell surface is of central importance for glucose homeostasis, since the presence of this protein in the plasma membrane determines the rate of glucose utilization by fat and muscle. Insulin stimulates redistribution of GLUT4 to the plasma membrane from an intracellular compartment; this response is impaired in insulin-resistant states such as type 2 diabetes mellitus. The PI wishes to understand the mechanism by which GLUT4 is sequestered intracellularly in the absence of insulin and by which mechanism insulin causes its redistribution. He has developed a novel assay to quantitatively and rapidly measure changes in the relative proportion of GLUT4 present at the surface of 3T3-L1 and Chinese hamster ovary cells. This assay relies upon the detection of an externalized myc epitope tag which indicates the amount of proteins at the surface of the cell, and an GFP fused in-frame at the carboxyl terminus of the protein, as an internal control of the amount of GLUT4 expressed in each cell. By using flow cytometry to measure a ratio of fluorescence intensities at the two wavelengths, the PI has obtained a relative measure of the expression of GLUT4 at the surface of individual cells. The assay thus enables the functional cloning of cDNAs encoding proteins that regulate GLUT4 trafficking. Two approaches will be used; the first involves an enrichment approach in which CHO cells expressing the transporter are infected with retroviruses encoding an adipocyte cDNA library; this will be infected into CHO cells such that each cell expresses an individual cDNA. Flow cytometry will be used to enrich for cells that will exhibit enhanced or decreased expression of surface GLUT4 in the presence and absence of insulin. After several rounds of enrichment and expansion of sorted cells, PCR will be used to amplify and identify the cDNA which confers the altered GLUT4 distribution. The second approach involves a sib selection strategy to identify cDNAs that alter GLUT4 targeting. Pools of cDNAs will be transfected into CHO cells expressing the transporter and flow cytometry will be used to identify pools of clones in which targeting (ratio of surface to internal tag) is altered as a consequence of physiological manipulation. Further subdivision of clonal pools will result in the identification of single cell lines containing cDNAs which alter GLUT4 trafficking, which will be sequenced by PCR.