FUNCTIONAL CLONING OF PROTEINS USED IN GLUT4 TRAFFICKING

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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.