Roles of the N- and C-termini of GLUT4 in endocytosis.
In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane. To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells. Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4. However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4 in both the basal and insulin-stimulated states. Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis. Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins mu1, mu2, and mu3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles.
Al-Hasani
Hadi
H
Center for Molecular Medicine, Institute of Biochemistry, University of Cologne, Otto-Fischer-Str. 12-14, 50674 Cologne, Germany. hadi.al-hasani@uni-koeln.de
Kunamneni
Raghu K
RK
Dawson
Kevin
K
Hinck
Cynthia S
CS
Müller-Wieland
Dirk
D
Cushman
Samuel W
SW
D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D034281:Dynamins; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D051275:Glucose Transporter Type 4; D006388:Hemagglutinins / Q000378:metabolism; D007150:Immunohistochemistry; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009124:Muscle Proteins; D009154:Mutation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000077191:Wortmannin
D000730:Androstadienes; D051275:Glucose Transporter Type 4; D006388:Hemagglutinins; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D020558:GTP Phosphohydrolases; D034281:Dynamins; D000077191:Wortmannin