MOLECULAR REGULATION OF GLUT-4 TARGETING IN 3T3-L1 ADIPOCYTES

Insulin stimulates glucose transport in muscle and adipose tissue by t riggering the movement of the glucose transporter GLUT-4 from an intra cellular compartment to the cell surface. Fundamental to this process is the intracellular sequestration of GLUT-4 in nonstimulated cells. T wo distinct targeting motifs in the amino and carboxy termini of GLUT- 4 have been previously identified by expressing chimeras comprised of portions of GLUT-4 and GLUT-1, a transporter isoform that is constitut ively targeted to the eel surface, in heterologous cells, These motifs -FQQI in the NH2 terminus and LL in the COOH terminus-resemble endocyt ic signals that have been described in other proteins, In the present study we have investigated the roles of these motifs in GLUT-4 targeti ng in insulin-sensitive cells, Epitope-tagged GLUT-4 constructs engine ered to differentiate between endogenous and transfected GLUT-LC were stably expressed in 3T3-L1 adipocytes. Targeting was assessed in cells incubated in the presence or absence of insulin by subcellular fracti onation. The targeting of epitope-tagged GLUT-4 was indistinguishable from endogenous GLUT-4. Mutation of the FQQI motif (F-5 to A(5)) cause d GLUT-4 to constitutively accumulate at the cell surface regardless o f expression level, Mutation of the dileucine motif (L(489)L(490) to A (489)A(490)) caused an increase in cell surface distribution only at h igher levels of expression, but the overall cell surface distribution of this mutant was less than that of the amino-terminal mutants. Both NH2- and COOH-terminal mutants retained insulin-dependent movement fro m an intracellular to a cell surface locale, suggesting that neither o f these motifs is involved in the insulin-dependent redistribution of GLUT-4. We conclude that the phenylalanine-based NH2-terminal and the dileucine-based COOH-terminal motifs play important and distinct roles in GLUT-4 targeting in 3T3-L1 adipocytes.