TARGETING OF GLUT1-GLUT5 CHIMERIC PROTEINS IN THE POLARIZED CELL-LINECACO-2

Caco-2, a human differentiated intestinal epithelial cell line, is a p romising model for investigating the mechanism of polarized targeting of apical and basolateral membrane proteins. We stably transfected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells With an expression vector. Immunohistochemical study revealed that the GLUT5 protein exp ressed was localized at apical membranes and that the GLUT1 expressed was present primarily in the basolateral membranes of cells grown on p ermeable support. Next, to investigate the domain responsible for dete rmining apical vs. basolateral sorting in glucose transporters, we pre pared several GLUT1-GLUT5 chimeric cDNAs and transfected them into Cac o-2 cells. A GLUT1 [N terminus similar to sixth transmembrane domain ( TM6)]-GLUT5 [intracellular loop (IL)similar to C terminus] chimera was observed exclusively at the apical membrane, while GLUT1 (N terminus similar to IL)-GLUT5 (TM7 similar to C terminus) and GLUT1 (N terminus similar to TM12)-GLUT5 (C-terminal domain) chimeras were observed mai nly at the basolateral membrane, a localization similar to that of GLU T1. Moreover, using a recombinant adenovirus expression system, we exp ressed a GLUT5 (N terminus similar to TM6)-GLUT1 (IL)-GLUT5(TM7 simila r to C-terminus)chimera, which was observed at the basolateral membran e. Based on these results, the C-terminal domain does not determine is oform-specific targeting of GLUT1 and GLUT5. Rather, it is the intrace llular loop in glucose transporters that appears to play a pivotal rol e in apical-basolateral sorting signals in Caco-2 cells.