title: Substitution of tyrosine 293 of GLUT1 locks the transporter into an outward facing conformation.
abstract: Tyrosines 292 and 293 in the mammalian glucose transporter GLUT1 have been substituted by either isoleucine or phenylalanine. Chinese hamster ovary clones that were transfected with Tyr-292-->Ile, Tyr-292-->Phe, Tyr-293-->Ile, and Tyr-293-->Phe constructs of GLUT1 were shown, by Western blotting and cell surface carbohydrate labeling, to have expression levels that were comparable with the wild type. The Vmax for 2-deoxy-D-glucose transport was markedly reduced only as a result of the Tyr-293-->Ile mutation. The ability of the Tyr-293-->Ile mutated GLUT1 to bind the exofacial ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) and the endofacial ligand cytochalasin B were assessed by photolabeling procedures. The ability to bind the bis-mannose compound was unimpaired, whereas the ability to bind cytochalasin B was totally abolished, and the level of labeling was lower than in the nontransfected clone. Affinities of the wild-type and Tyr-293-->Ile GLUT1 for D-glucose, the exofacial ligands (ATB-BMPA and 4,6-O-ethylidene-D-glucose), and the endofacial ligand (cytochalasin B) were assessed by the ability of these agents to displace the radioactive ATB-BMPA photolabel. These data indicated that the Tyr-293-->Ile substitution produced no change in the affinity for D-glucose, a relatively small enhancement in the affinity for exofacial ligands, but a large approximately 300-fold reduction in affinity for cytochalasin B, suggesting that the mutated GLUT1 is locked in an outward facing conformation. The observation that the Tyr-293-->Ile mutant transporter can bind nontransported C4 and C6 substituted hexose analogues but cannot catalyze transport is interpreted as indicating that Tyr-293 is involved in closing the exofacial site around C4 and C6 of D-glucose in the transport catalysis process.
authors.affiliation: Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Mori
authors.forename: M
authors.initials: M
authors.lastname: Hashiramoto
authors.forename: A E
authors.initials: AE
authors.lastname: Clark
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: A
authors.initials: A
authors.lastname: Muraoka
authors.forename: Y
authors.initials: Y
authors.lastname: Tamori
authors.forename: M
authors.initials: M
authors.lastname: Kasuga
authors.forename: G D
authors.initials: GD
authors.lastname: Holman
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003571:Cytochalasin B / Q000378:metabolism; D017931:DNA Primers; D003847:Deoxyglucose / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D051272:Glucose Transporter Type 1; D006027:Glycosides; D006801:Humans; D007532:Isoleucine; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D011437:Propylamines; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection; D014443:Tyrosine
chemical_list: D000345:Affinity Labels; D001386:Azides; D017931:DNA Primers; D004187:Disaccharides; D051272:Glucose Transporter Type 1; D006027:Glycosides; D009004:Monosaccharide Transport Proteins; D011437:Propylamines; D011994:Recombinant Proteins; C495677:SLC2A1 protein, human; D007532:Isoleucine; C065412:2-N-(4-(1-azitrifluoroethyl)benzoyl)-1,3-bis-(mannos-4-yloxy)-2-propylamine; D003571:Cytochalasin B; D014443:Tyrosine; D010649:Phenylalanine; D003847:Deoxyglucose