POSSIBLE DOMAINS RESPONSIBLE FOR INTRACELLULAR TARGETING AND INSULIN-DEPENDENT TRANSLOCATION OF GLUCOSE-TRANSPORTER TYPE-4

Translocation of the type 4 glucose transporter (GLUT4) to the cell su rface from an intracellular pool is the major mechanism of insulin-sti mulated glucose uptake in insulin-target cells. We developed a highly sensitive and quantitative method to detect GLUT4 immunologically on t he surface of intact cells, using c-myc epitope-tagged GLUT4 (GLUT4myc ). We constructed c-myc epitope-tagged glucose transporter type 1 (GLU T1myc) and found that the GLUT1myc was also translocated to the cell s urface of Chinese hamster ovary cells, 3T3-L1 fibroblasts and NIH 3T3 cells, in response to insulin, but the degree of translocation was les s than that of GLUT4myc. Since GLUT1 and GLUT4 have different intracel lular distributions and different degrees of insulin-stimulated transl ocation, we examined the domains of GLUT4, using c-myc epitope-tagged chimeric glucose transporters between these two isoforms. The results indicated that, (1) all the cytoplasmic N-terminal region, middle intr acellular loop and cytoplasmic C-terminal region of GLUT4 have indepen dent intracellular targeting signals, (2) these sequences for intracel lular targeting of GLUT4 were not sufficient to determine GLUT4 transl ocation in response to insulin, and (3) the N-terminal half of GLUT4 d evoid both of cytoplasmic N-terminus and of middle intracellular loop seems to be necessary for insulin-stimulated GLUT4 translocation.