title: Identification of amino acid residues within the C terminus of the Glut4 glucose transporter that are essential for insulin-stimulated redistribution to the plasma membrane.
abstract: The Glut4 glucose transporter undergoes complex insulin-regulated subcellular trafficking in adipocytes. Much effort has been expended in an attempt to identify targeting motifs within Glut4 that direct its subcellular trafficking, but an amino acid motif responsible for the targeting of the transporter to insulin-responsive intracellular compartments in the basal state or that is directly responsible for its insulin-stimulated redistribution to the plasma membrane has not yet been delineated. In this study we define amino acid residues within the C-terminal cytoplasmic tail of Glut4 that are essential for its insulin-stimulated translocation to the plasma membrane. The residues were identified based on sequence similarity (LXXLXPDEXD) between cytoplasmic domains of Glut4 and the insulin-responsive aminopeptidase (IRAP). Alteration of this putative targeting motif (IRM, insulin-responsive motif) resulted in the targeting of the bulk of the mutant Glut4 molecules to dispersed membrane vesicles that lacked detectable levels of wild-type Glut4 in either the basal or insulin-stimulated states and completely abolished the insulin-stimulated translocation of the mutant Glut4 to the plasma membrane in 3T3L1 adipocytes. The bulk of the dispersed membrane vesicles containing the IRM mutant did not contain detectable levels of any subcellular marker tested. A fraction of the total IRM mutant was also detected in a wild-type Glut4/Syntaxin 6-containing perinuclear compartment. Interestingly, mutation of the IRM sequence did not appreciably alter the subcellular trafficking of IRAP. We conclude that residues within the IRM are critical for the targeting of Glut4, but not of IRAP, to insulin-responsive intracellular membrane compartments in 3T3-L1 adipocytes.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Xiao Mei
authors.initials: XM
authors.lastname: Song
authors.forename: Richard C
authors.initials: RC
authors.lastname: Hresko
authors.forename: Mike
authors.initials: M
authors.lastname: Mueckler
mesh_terms: D041721:3T3-L1 Cells; D000256:Adenoviridae; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism*; D010122:Cystinyl Aminopeptidase / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000737:chemistry* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D000596:Amino Acids; D051275:Glucose Transporter Type 4; D007328:Insulin; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase