DIRECT DEMONSTRATION OF INSULIN-INDUCED GLUT4 TRANSLOCATION TO THE SURFACE OF INTACT-CELLS BY INSERTION OF A C-MYC EPITOPE INTO AN EXOFACIAL GLUT4 DOMAIN

Stimulation of glucose transport is the main physiological effect of i nsulin in target tissues. This effect is linked to translocation of th e GLUT4 glucose transporter from an intracellular pool to the cell sur face. To elucidate the molecular mechanisms involved in this effect, w e developed a simple direct sensitive method to detect GLUT4 immunolog ically on the cell surface. cDNA containing GLUT4 inserted by a c-myc epitope in the first ectodomain (GLUT4myc) was constructed without dis rupting the functions of GLUT4 and was expressed in 3T3-L1 and Chinese hamster ovary fibroblast cells. In response to insulin, the GLUT4myc expressed in 3T3-L1 adipocytes was translocated to the cell surface fr om the intracellular pool, as shown by assays of exofacial antibody bi nding against the myc epitope and of the uptake of 2-deoxyglucose. Ins ulin, guanosine 5'-O-(3-thiotriphosphate), guanylyl imidodiphosphate, NaF, and phorbol 12-myristate 13-acetate also induced the translocatio n of GLUT4myc in Chinese hamster ovary cells coexpressing the human in sulin receptor.