title: Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity.
abstract: C-terminally truncated and mutated forms of GLUT1 have been constructed to determine the minimum structure at the C-terminus required for glucose transport activity and ligand binding at the outer and inner binding sites. Four truncated mutants have been constructed (CTD24 to CTD27) in which 24 to 27 amino acids are deleted. In addition, point substitutions of R468-->L, F467-->L and G466-->E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wild-type clone. Wild-type levels of 2-deoxy-D-glucose transport activity were retained only in the clone transfected with the construct in which 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 clones showed markedly reduced transport activity. From a kinetic comparison of the CTD24 and CTD26 clones it was found that the reduced transport was mainly associated with a reduced Vmax. value for 2-deoxy-D-glucose uptake but with a slight lowering of the Km. These data establish that the 24 amino acids at the C-terminus of GLUT1 are not required for the transport catalysis. However, the point mutations of F467L and G466E (26 and 27 residues from the C-terminus) did not significantly perturb the kinetics of 2-deoxy-D-glucose transport. The substitution of R468L produced a slight, but significant, lowering of the Km. The ability of the truncated GLUt1s to bind the exofacial ligand, 2-N-4-(1-zai-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yl-oxy) -2-propylamine (ATB-BMPA), and the endofacial ligand, cytochalasin B, were assessed by photolabelling procedures. The ability to bind ATB-BMPA was retained only in the CTD24 truncated mutant and was reduced to levels comparable with those of the non-transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids are not individually essential.
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D016466:CHO Cells / Q000378:metabolism; D002384:Catalysis; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D003847:Deoxyglucose / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D006027:Glycosides; D006801:Humans; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011437:Propylamines; D011817:Rabbits; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection
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; C495677:SLC2A1 protein, human; C065412:2-N-(4-(1-azitrifluoroethyl)benzoyl)-1,3-bis-(mannos-4-yloxy)-2-propylamine; D003847:Deoxyglucose; D005947:Glucose