title: Mutational analysis of the carboxy-terminal phosphorylation site of GLUT-4 in 3T3-L1 adipocytes.
abstract: The carboxy terminus of GLUT-4 contains a functional internalization motif (Leu-489Leu-490) that helps maintain its intracellular distribution in basal adipocytes. This motif is flanked by the major phosphorylation site in this protein (Ser-488), which may play a role in regulating GLUT-4 trafficking in adipocytes. In the present study, the targeting of GLUT-4 in which Ser-488 has been mutated to alanine (SAG) has been examined in stably transfected 3T3-L1 adipocytes. The trafficking of SAG was not significantly different from that of GLUT-4 in several respects. First, in the absence of insulin, the distribution of SAG was similar to GLUT-4 in that it was largely excluded from the cell surface and was enriched in small intracellular vesicles. Second, SAG exhibited insulin-dependent movement to the plasma membrane (4- to 5-fold) comparable to GLUT-4 (4- to 5-fold). Finally, okadaic acid, which has previously been shown to stimulate both GLUT-4 translocation and its phosphorylation at Ser-488, also stimulated the movement of SAG to the cell surface similarly to GLUT-4. Using immunoelectron microscopy, we have shown that GLUT-4 is localized to intracellular vesicles containing the Golgi-derived gamma-adaptin subunit of AP-1 and that this localization is enhanced when Ser-488 is mutated to alanine. We conclude that the carboxy-terminal phosphorylation site in GLUT-4 (Ser-488) may play a role in intracellular sorting at the trans-Golgi network but does not play a major role in the regulated movement of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes.
authors.affiliation: Boulder Laboratory for Three-Dimensional Fine Structure, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA.
authors.forename: B J
authors.initials: BJ
authors.lastname: Marsh
authors.forename: S
authors.initials: S
authors.lastname: Martin
authors.forename: D R
authors.initials: DR
authors.lastname: Melvin
authors.forename: L B
authors.initials: LB
authors.lastname: Martin
authors.forename: R A
authors.initials: RA
authors.lastname: Alm
authors.forename: G W
authors.initials: GW
authors.lastname: Gould
authors.forename: D E
authors.initials: DE
authors.lastname: James
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D051274:Glucose Transporter Type 3; D051275:Glucose Transporter Type 4; D006801:Humans; D007930:Leucine; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D009124:Muscle Proteins; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D014162:Transfection
chemical_list: D051274:Glucose Transporter Type 3; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C495687:SLC2A3 protein, human; C495692:SLC2A4 protein, human; C495689:Slc2a3 protein, mouse; C495693:Slc2a4 protein, mouse; D012694:Serine; D007930:Leucine; D000409:Alanine