FUNCTIONAL CLONING OF PROTEINS USED IN GLUT4 TRAFFICKING

描述：（改编自申请人的摘要）GLUT贩运4 往返于细胞表面的葡萄糖转运蛋白对于 葡萄糖稳态，因为这种蛋白质存在于质膜 决定了脂肪和肌肉对葡萄糖的利用率。胰岛素 刺激GLUT 4从细胞内向质膜的再分布， 这种反应在胰岛素抵抗状态下受损，例如 2型糖尿病。PI希望了解GLUT 4 在缺乏胰岛素的情况下被隔离在细胞内， 胰岛素引起其再分布。他开发了一种新的分析方法， 定量和快速测量GLUT 4相对比例的变化 存在于3 T3-L1和中国仓鼠卵巢细胞的表面。该测定 依赖于检测外部化的myc表位标签 细胞表面的蛋白质量，以及在细胞表面与GFP框内融合的GFP。 蛋白质的羧基末端，作为蛋白质的量的内部对照， GLUT 4在每个细胞中表达。通过使用流式细胞术来测量 在两个波长的荧光强度，PI已经获得了相对 测量单个细胞表面的GLUT 4表达。的 因此，该测定能够功能性克隆编码蛋白质的cDNA， 规范GLUT 4贩运。将使用两种方法;第一种涉及 富集方法，其中感染表达转运蛋白的CHO细胞 用编码脂肪细胞cDNA文库的逆转录病毒， 使每个细胞表达单个cDNA。流式细胞术 将用于富集细胞，这些细胞将表现出增强或降低的 在胰岛素存在和不存在下表面GLUT 4的表达。后 分选细胞的几轮富集和扩增，PCR将用于 扩增并鉴定赋予改变的GLUT 4分布的cDNA。的 第二种方法涉及同胞选择策略，以鉴定改变 GLUT 4靶向。将1000个cDNA转染到表达以下的CHO细胞中： 转运蛋白和流式细胞术将用于鉴定 其靶向（表面与内部标签的比率）因此改变 生理操纵克隆池的进一步细分将导致 在鉴定含有改变GLUT 4的cDNA的单细胞系中， 将通过PCR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.