Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1994 Sep 1;302(Pt 2):355–361. doi: 10.1042/bj3020355

Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter.

K Inukai 1, T Asano 1, H Katagiri 1, M Anai 1, M Funaki 1, H Ishihara 1, K Tsukuda 1, M Kikuchi 1, Y Yazaki 1, Y Oka 1
PMCID: PMC1137236  PMID: 8092986

Abstract

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

Full text

PDF
355

Images in this article

357Figure 1
on p.357
357Figure 2
on p.357

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Asano T., Shibasaki Y., Kasuga M., Kanazawa Y., Takaku F., Akanuma Y., Oka Y. Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development. Biochem Biophys Res Commun. 1988 Aug 15;154(3):1204–1211. doi: 10.1016/0006-291x(88)90268-9. [DOI] [PubMed] [Google Scholar]
  2. Asano T., Shibasaki Y., Ohno S., Taira H., Lin J. L., Kasuga M., Kanazawa Y., Akanuma Y., Takaku F., Oka Y. Rabbit brain glucose transporter responds to insulin when expressed in insulin-sensitive Chinese hamster ovary cells. J Biol Chem. 1989 Feb 25;264(6):3416–3420. [PubMed] [Google Scholar]
  3. Bell G. I., Kayano T., Buse J. B., Burant C. F., Takeda J., Lin D., Fukumoto H., Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care. 1990 Mar;13(3):198–208. doi: 10.2337/diacare.13.3.198. [DOI] [PubMed] [Google Scholar]
  4. Birnbaum M. J., Haspel H. C., Rosen O. M. Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5784–5788. doi: 10.1073/pnas.83.16.5784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bruns R. F., Lawson-Wendling K., Pugsley T. A. A rapid filtration assay for soluble receptors using polyethylenimine-treated filters. Anal Biochem. 1983 Jul 1;132(1):74–81. doi: 10.1016/0003-2697(83)90427-x. [DOI] [PubMed] [Google Scholar]
  6. Cairns M. T., Alvarez J., Panico M., Gibbs A. F., Morris H. R., Chapman D., Baldwin S. A. Investigation of the structure and function of the human erythrocyte glucose transporter by proteolytic dissection. Biochim Biophys Acta. 1987 Dec 11;905(2):295–310. doi: 10.1016/0005-2736(87)90458-5. [DOI] [PubMed] [Google Scholar]
  7. Calderhead D. M., Lienhard G. E. Labeling of glucose transporters at the cell surface in 3T3-L1 adipocytes. Evidence for both translocation and a second mechanism in the insulin stimulation of transport. J Biol Chem. 1988 Sep 5;263(25):12171–12174. [PubMed] [Google Scholar]
  8. Carter-Su C., Pessin J. E., Mora R., Gitomer W., Czech M. P. Photoaffinity labeling of the human erythrocyte D-glucose transporter. J Biol Chem. 1982 May 25;257(10):5419–5425. [PubMed] [Google Scholar]
  9. Charron M. J., Brosius F. C., 3rd, Alper S. L., Lodish H. F. A glucose transport protein expressed predominately in insulin-responsive tissues. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2535–2539. doi: 10.1073/pnas.86.8.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Devés R., Krupka R. M. Cytochalasin B and the kinetics of inhibition of biological transport: a case of asymmetric binding to the glucose carrier. Biochim Biophys Acta. 1978 Jul 4;510(2):339–348. doi: 10.1016/0005-2736(78)90034-2. [DOI] [PubMed] [Google Scholar]
  11. Deziel M., Pegg W., Mack E., Rothstein A., Klip A. Labelling of the human erythrocyte glucose transporter with 3H-labelled cytochalasin B occurs via protein photoactivation. Biochim Biophys Acta. 1984 May 30;772(3):403–406. doi: 10.1016/0005-2736(84)90157-3. [DOI] [PubMed] [Google Scholar]
  12. Fukumoto H., Seino S., Imura H., Seino Y., Eddy R. L., Fukushima Y., Byers M. G., Shows T. B., Bell G. I. Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter-like protein. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5434–5438. doi: 10.1073/pnas.85.15.5434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garcia J. C., Strube M., Leingang K., Keller K., Mueckler M. M. Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes. J Biol Chem. 1992 Apr 15;267(11):7770–7776. [PubMed] [Google Scholar]
  14. Griffin J. F., Rampal A. L., Jung C. Y. Inhibition of glucose transport in human erythrocytes by cytochalasins: A model based on diffraction studies. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3759–3763. doi: 10.1073/pnas.79.12.3759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harrison S. A., Buxton J. M., Helgerson A. L., MacDonald R. G., Chlapowski F. J., Carruthers A., Czech M. P. Insulin action on activity and cell surface disposition of human HepG2 glucose transporters expressed in Chinese hamster ovary cells. J Biol Chem. 1990 Apr 5;265(10):5793–5801. [PubMed] [Google Scholar]
  16. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holman G. D., Rees W. D. Photolabelling of the hexose transporter at external and internal sites: fragmentation patterns and evidence for a conformational change. Biochim Biophys Acta. 1987 Mar 12;897(3):395–405. doi: 10.1016/0005-2736(87)90437-8. [DOI] [PubMed] [Google Scholar]
  18. Katagiri H., Asano T., Ishihara H., Lin J. L., Inukai K., Shanahan M. F., Tsukuda K., Kikuchi M., Yazaki Y., Oka Y. Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin. Biochem J. 1993 May 1;291(Pt 3):861–867. doi: 10.1042/bj2910861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Katagiri H., Asano T., Shibasaki Y., Lin J. L., Tsukuda K., Ishihara H., Akanuma Y., Takaku F., Oka Y. Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter. J Biol Chem. 1991 Apr 25;266(12):7769–7773. [PubMed] [Google Scholar]
  20. Kayano T., Burant C. F., Fukumoto H., Gould G. W., Fan Y. S., Eddy R. L., Byers M. G., Shows T. B., Seino S., Bell G. I. Human facilitative glucose transporters. Isolation, functional characterization, and gene localization of cDNAs encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle, and adipose tissue and an unusual glucose transporter pseudogene-like sequence (GLUT6). J Biol Chem. 1990 Aug 5;265(22):13276–13282. [PubMed] [Google Scholar]
  21. Kayano T., Fukumoto H., Eddy R. L., Fan Y. S., Byers M. G., Shows T. B., Bell G. I. Evidence for a family of human glucose transporter-like proteins. Sequence and gene localization of a protein expressed in fetal skeletal muscle and other tissues. J Biol Chem. 1988 Oct 25;263(30):15245–15248. [PubMed] [Google Scholar]
  22. Keller K., Strube M., Mueckler M. Functional expression of the human HepG2 and rat adipocyte glucose transporters in Xenopus oocytes. Comparison of kinetic parameters. J Biol Chem. 1989 Nov 15;264(32):18884–18889. [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Mueckler M., Caruso C., Baldwin S. A., Panico M., Blench I., Morris H. R., Allard W. J., Lienhard G. E., Lodish H. F. Sequence and structure of a human glucose transporter. Science. 1985 Sep 6;229(4717):941–945. doi: 10.1126/science.3839598. [DOI] [PubMed] [Google Scholar]
  25. Oka Y., Asano T., Shibasaki Y., Kasuga M., Kanazawa Y., Takaku F. Studies with antipeptide antibody suggest the presence of at least two types of glucose transporter in rat brain and adipocyte. J Biol Chem. 1988 Sep 15;263(26):13432–13439. [PubMed] [Google Scholar]
  26. Oka Y., Asano T., Shibasaki Y., Lin J. L., Tsukuda K., Katagiri H., Akanuma Y., Takaku F. C-terminal truncated glucose transporter is locked into an inward-facing form without transport activity. Nature. 1990 Jun 7;345(6275):550–553. doi: 10.1038/345550a0. [DOI] [PubMed] [Google Scholar]
  27. Pessin J. E., Tillotson L. G., Yamada K., Gitomer W., Carter-Su C., Mora R., Isselbacher K. J., Czech M. P. Identification of the stereospecific hexose transporter from starved and fed chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2286–2290. doi: 10.1073/pnas.79.7.2286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shanahan M. F. Characterization of cytochalasin B photoincorporation into human erythrocyte D-glucose transporter and F-actin. Biochemistry. 1983 May 24;22(11):2750–2756. doi: 10.1021/bi00280a024. [DOI] [PubMed] [Google Scholar]
  29. Shanahan M. F., Olson S. A., Weber M. J., Lienhard G. E., Gorga J. C. Photolabeling of glucose-sensitive cytochalasin B binding proteins in erythrocyte, fibroblast and adipocyte membranes. Biochem Biophys Res Commun. 1982 Jul 16;107(1):38–43. doi: 10.1016/0006-291x(82)91666-7. [DOI] [PubMed] [Google Scholar]
  30. Thorens B., Sarkar H. K., Kaback H. R., Lodish H. F. Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and beta-pancreatic islet cells. Cell. 1988 Oct 21;55(2):281–290. doi: 10.1016/0092-8674(88)90051-7. [DOI] [PubMed] [Google Scholar]
  31. Wheeler T. J., Hinkle P. C. Kinetic properties of the reconstituted glucose transporter from human erythrocytes. J Biol Chem. 1981 Sep 10;256(17):8907–8914. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES