Abstract
Two arginine residues (RR333/334) in the conserved GRR motif located in the endofacial loop between helix 8 and 9 of the glucose transporter GLUT4 were substituted for leucine and alanine, respectively. Reconstituted glucose transport activity of the construct (GLUT4-RR333/4LA) expressed in COS-7 or LM(TK-) cells was less than 10% of that of the wild-type GLUT4. In contrast, binding of the inhibitory ligand cytochalasin B and glucose-inhibitable photolabeling with IAPS-forskolin were not significantly affected. Exchange of a histidine residue (H337Q) previously believed to be involved in the binding of inhibitory ligands failed to affect any of the investigated parameters. These data suggest that positive charges in the GRR motif at the cytoplasmic surface of the transporter participate in the conformational changes of the carrier protein during the process of facilitated diffusion.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Appleman JR, Lienhard GE (1989) Kinetics of the purified glucose transporter. Direct measurement of the rates of interconversion of transporter conformers. Biochemistry 28:8221–8227
Bell G, Kayano T, Buse J, Burant C, Takeda J, Lin D, Fukumoto H, Seino S (1990)Molecular biology of mammalian glucose transporters. Diabetes Care 13:198–206
Birnbaum MJ (1989) Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell 57: 305–315
Cushman SW, Wardzala LJ (1980) Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. J Biol Chem 255:4758–4762
Garcia JC, Strube M, Leingang K, Keller K, Mueckler MM (1992) Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG (GLUT1) glucose transporter inhibit transport activity and targeting to the plasma membrane in xenopus oocytes. J Biol Chem 267:7770–7776
Hashiramoto M, Kadowaki T, Clark AE, Muraoka A, Momomura K, Sakura H, Tobe K, Akanuma Y, Yazaki Y, Holman GD et al. (1992) Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding. J Biol Chem 267:17502–17507
Hellwig B, Joost HG (1991) Differentiation of erythrocyte- (GLUT1), liver- (GLUT2), and adipocyte-type (GLUT4) glucose transporters by binding of the inhibitory ligands cytochalasin B, forskolin, dipyridamole, and isobutylmethylxanthine. Mol Pharmacol 40: 383–389
Hellwig B, Brown FM, Schürmann A, Shanahan MF, Joost HG (1992) Localization of the binding domains of the inhibitory ligand forskolin in the glucose transporter GLUT4 by photolabeling, proteolytic cleavage and a site-specific antiserum. Biochim Biophys Acta 1111:178–184
Holman GD, Rees WD (1987) Photolabelling of the hexose transporter at external and internal sites: fragmentation patterns and evidence for a conformational change. Biochim Biophys Acta 897: 395–405
Ishihara H, Asano T, Katagiri H, Lin JL, Tsukuda K, Shibasaki Y, Oka Y (1991) The glucose transport activity of GLUTI is markedly decreased by substitution of a single amino acid with a different charge at residue 415. Biochem Biophys Res Commun 176: 922–930
Joost HG, Steinfelder HJ (1987) Forskolin inhibits insulin-stimulated glucose transport in rat adipose cells by a direct interaction with the glucose transporter. Mol Pharmacol 31:179–283
Joost HG, Wandel S, Schürmann A (1994) Structure-function relationship of glucose transporters catalyzing facilitated diffusion. Exp Clin Endocrinol 102:434–438
Katagiri H, Asano T, Shibasaki Y, Lin JL, Tsukuda K, Ishihara H, Akanuma Y, Takaku F, Oka Y (1991) Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labelling but markedly decreases the intrinsic activity of GLUTI glucose transporter. J Biol Chem 266:7769–7773
Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154: 367–382
Marger MD, Saier MH (1993) A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci 18:13–20
Mori H, Hashiramoto M, Clark AE, Yang J, Muraoka A, Tamori Y, Kasuga M, Holman GD (1994) Substitution of tyrosine 293 of GLUTI locks the transporter in an outward facing conformation. J Biol Chem 269:11578–11583
Robinson FW, Blevins TL, Suzuki K, Kono T (1982) An improved method of reconstitution of adipocyte glucose transport activity. Anal Biochem 122:10–19
Rosenthal H (1967) A graphic method for the determination and presentation of binding parameters in a complex system. Anal Biochem 20:525–532
Sauer N, Tanner W (1993) Molecular biology of sugar transporters in plants. Botanica Acta 106:277–286
Schürmann A, Rosenthal W Hinsch KD, Joost HG (1989) Differential sensitivity of guanine nucleotides of basal and insulin-stimulated glucose transporter activity reconstituted from adipocyte membrane fractions. FEBS Lett 255:259–264
Schürmann A, Monden I, Joost HG, Keller K (1992) Subcellular distribution and activity of glucose transporter isoforms (GLUTI and GLUT4) transiently expressed in COS-7 cells. Biochim Biophys Acta 1131:245–252
Schürmann A, Keller K, Monden I, Brown FM, Wandel S, Shanahan MF, Joost HG (1993) Glucose transport activity and photolabelling with 3-[125]iodo-4-azido-phenethylarnido-7–0-succinyldeacetyl (IAPS)-forskolin of two mutants at tryptophan-388 and -412 of the glucose transporter GLUTI: dissociation of the binding domains of forskolin and glucose. Biochem J 290:497–501
von Heijne G (1989) Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature 341:456–458
Wadzinsky B, Shanahan M, Ruoho AE (1987) Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label. J Biol Chem 262:17683–17689
Wandel S, Schürmann A, Becker W, Summers, SA, Shanahan ME Joost, HG (1994) Substitution of conserved tyrosine residues in helix 4 (Y143) and 7 (Y293) affects the activity, but not IAPS-forskolin binding, of the glucose transporter GLUT4. FEBS Lett 348:114–118
Weiland M, Schürmann A, Schmidt WE, Joost HG (1990) Development of the hormone-sensitive glucose transport activity in differentiating 3T3-Ll murine fibroblasts. Role of the two glucose transporter species and their subcellular localization. Biochem J 270:331–336
Wellner M, Monden I, Keller, K (1992) The differential role of Cys-421 and Cys-429 of the Glutl glucose transporter in transport inhibition by p-chloromercuribenzenesulfonic acid (pCMBS) or cytochalasin B (CB). FEBS Lett 309:293–296
Wellner M, Monden I, Mueckler MM, Keller K (1995) Functional consequences of proline mutations in the putative transmembrane segments 6 and 10 of the glucose transporter GLUTI. Eur J Biochem 227:454–458
Yamaguchi A, Someya Y, Sawai T (1992) Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. J Biol Chem 267:19155–19162
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wandel, S., Schürmann, A., Becker, W. et al. Mutation of two conserved arginine residues in the glucose transporter GLUT4 supresses transport activity, but not glucose-inhibitable binding of inhibitory ligands. Naunyn-Schmiedeberg's Arch Pharmacol 353, 36–41 (1995). https://doi.org/10.1007/BF00168913
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00168913