IDENTIFICATION OF THE CARBOXY-TERMINUS AS IMPORTANT FOR THE ISOFORM-SPECIFIC SUBCELLULAR TARGETING OF GLUCOSE-TRANSPORTER PROTEINS

Differential trafficking of glucose transporters contributes significantly to the establishment of a cell's capacity for hormone-regulatable hexose uptake. In the true insulin-sensitive peripheral target tissues, muscle and adipose, the transporter isoform GLUT1 residues on the cell surface and interior of the cell whereas the highly homologous isoform GLUT4 displays virtually exclusive intracellular sequestration, allowing the latter to redistribute to the cell surface in response to hormone. These patterns are equally pronounced in cells into which the transporters have been introduced by DNA-mediated gene transfer, suggesting that signals for isoform-specific sorting are recognized in diverse cell types. To determine the primary sequences responsible for the characteristic distributions, chimeric transporters were constructed in which reciprocal domains were exchanged between GLUT1 and GLUT4. In addition, a non-disruptive, species-specific epitope ''tag'' was introduced into a neutral region of the transporter to allow analysis of reciprocal chimeras using a single antibody. These recombinant transporters were stably expressed in HIH 3T3 and PC12 cells by retrovirus-mediated gene transfer, and were localized by indirect immunofluorescence and laser scanning confocal microscopy, as well as by staining of plasma membrane sheets prepared from these cells. The results indicate that the carboxy-terminal 30 amino acids are primarily responsible for the differential targeting of the glucose transporter isoforms GLUT1 and GLUT4, though there is a lesser additional contribution by the amino-terminal 183 amino acids.