Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes.

Abstract

Abstract. Insulin stimulates glucose transport in muscle and adipose tissue by triggering the movement of the glucose transporter GLUT-4 from an intracellular compartment to the cell surface. Fundamental to this process is the intracellular sequestration of GLUT-4 in nonstimulated ceils. Two 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-l, a transporter isoform that is constitutively targeted to the cell surface, in heterologous cells. These motifs-FQQI in the NH 2 terminus and LL in the COOH terminus-resemble endocytic signals that have been described in other proteins. In the present study we have investigated the roles of these motifs in GLUT-4 targeting in insulinsensitive cells. Epitope-tagged GLUT-4 constructs engineered to differentiate between endogenous and transfected GLUT-4 were stably expressed in 3T3-L1 adipocytes. Targeting was assessed in cells incubated in the presence or absence of insulin by subcellular fractionation. The targeting of epitope-tagged GLUT-4 was indistinguishable from endogenous GLUT-4. Mutation of the FQQI motif (F 5 to A 5) caused GLUT-4 to constitutively accumulate at the cell surface regardless of expression level. Mutation of the dileucine motif (t489L 490 to A489A 49°) caused an increase in cell surface distribution only at higher 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 from an intracellular to a cell surface locale, suggesting that neither of 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.

Trafficking of Glut4–Green Fluorescent Protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface Glut4 levels

“…FAG-GFP was observed to localize predominantly to the plasma membrane, in agreement with studies with epitope-tagged FAG stably expressed in these cells [32]. LAG-GFP expression was observed both intracellularly and, in part, at the plasma membrane (Figure 3), which was in good agreement with a range of studies examining the subcellular distribution of similar Glut4 mutants [32,33,40].…”

“…We also studied a mutant Glut4 species using this approach ( Figure 3) [31][32][33]. FAG-GFP was observed to localize predominantly to the plasma membrane, in agreement with studies with epitope-tagged FAG stably expressed in these cells [32].…”

“…Glut4 containing the point mutations F& A (FAG-GFP) and LL%)* ,%*! AA (LAG-GFP) were those described in [31][32][33], which were PCRamplified and fused in frame with GFP, as described for the wildtype protein above. PCR reactions were performed using either Vent2 or Taq polymerases and all products were completely sequenced on both strands to ensure fidelity of construction.…”

p115 Interacts with the GLUT4 Vesicle Protein, IRAP, and Plays a Critical Role in Insulin-stimulated GLUT4 Translocation

“…IRAP has a single transmembrane domain with the Nterminus projecting into the cytosol (Keller et al, 1995), whereas GLUT4 has potential sorting and targeting motifs in three cytosolic domains corresponding to N-and C-termini as well as the central loop that connects helices 6 and 7 (Fukumoto et al, 1989). The N-terminus of IRAP has dileucine motifs and an acidic cluster domain similar to that found in the C-terminus of GLUT4, which is thought to be important region for its trafficking (Verhey et al, 1993(Verhey et al, , 1995Corvera et al, 1994;Verhey and Birnbaum, 1994;Haney et al, 1995;Marsh et al, 1995). In this study, we used the N-terminal cytoplasmic domain of IRAP, residues 1-109, conjugated to a chitin-binding protein in order to find cytosolic proteins involved in GSV trafficking, and we identified p115 as one such protein.…”

Recycling of the Insulin-sensitive Glucose Transporter GLUT4

“…The 12 transmembrane domain protein displays, in its amino and carboxy-cytoplasmic tails, Phe 5 -Gln 6 -Gln 7 -Ile 8 -based (13) and Leu 489 -Leu 490 -based motifs (14) that when inactivated provoke its cellular redistribution. Both motifs have been involved in GLUT4 endocytosis, intracellular retention, and targeting in transfected 3T3-L1fibroblasts, COS-7 and Chinese hamster ovary cells (15)(16)(17)(18)(19)(20), myoblasts (21), and adipocytes (18,(22)(23)(24). Whether the dileucine motif mediates the trafficking of GLUT4 in adipocytes is, however, the subject of much debate (18,22,23).…”