title: Roles of the N- and C-termini of GLUT4 in endocytosis.
abstract: 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.
authors.affiliation: Center for Molecular Medicine, Institute of Biochemistry, University of Cologne, Otto-Fischer-Str. 12-14, 50674 Cologne, Germany. hadi.al-hasani@uni-koeln.de
authors.forename: Hadi
authors.initials: H
authors.lastname: Al-Hasani
authors.forename: Raghu K
authors.initials: RK
authors.lastname: Kunamneni
authors.forename: Kevin
authors.initials: K
authors.lastname: Dawson
authors.forename: Cynthia S
authors.initials: CS
authors.lastname: Hinck
authors.forename: Dirk
authors.initials: D
authors.lastname: Müller-Wieland
authors.forename: Samuel W
authors.initials: SW
authors.lastname: Cushman
mesh_terms: 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
chemical_list: 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