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. 1994 Mar 1;124(5):705–715. doi: 10.1083/jcb.124.5.705

The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

PMCID: PMC2119955  PMID: 8120093

Abstract

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.

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Selected References

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  1. Asano T., Takata K., Katagiri H., Tsukuda K., Lin J. L., Ishihara H., Inukai K., Hirano H., Yazaki Y., Oka Y. Domains responsible for the differential targeting of glucose transporter isoforms. J Biol Chem. 1992 Sep 25;267(27):19636–19641. [PubMed] [Google Scholar]
  2. Bell G. I., Burant C. F., Takeda J., Gould G. W. Structure and function of mammalian facilitative sugar transporters. J Biol Chem. 1993 Sep 15;268(26):19161–19164. [PubMed] [Google Scholar]
  3. Birnbaum M. J. Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell. 1989 Apr 21;57(2):305–315. doi: 10.1016/0092-8674(89)90968-9. [DOI] [PubMed] [Google Scholar]
  4. Breitfeld P. P., Casanova J. E., McKinnon W. C., Mostov K. E. Deletions in the cytoplasmic domain of the polymeric immunoglobulin receptor differentially affect endocytotic rate and postendocytotic traffic. J Biol Chem. 1990 Aug 15;265(23):13750–13757. [PubMed] [Google Scholar]
  5. Calderhead D. M., Kitagawa K., Tanner L. I., Holman G. D., Lienhard G. E. Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 1990 Aug 15;265(23):13801–13808. [PubMed] [Google Scholar]
  6. Collawn J. F., Kuhn L. A., Liu L. F., Tainer J. A., Trowbridge I. S. Transplanted LDL and mannose-6-phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor. EMBO J. 1991 Nov;10(11):3247–3253. doi: 10.1002/j.1460-2075.1991.tb04888.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collawn J. F., Stangel M., Kuhn L. A., Esekogwu V., Jing S. Q., Trowbridge I. S., Tainer J. A. Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell. 1990 Nov 30;63(5):1061–1072. doi: 10.1016/0092-8674(90)90509-d. [DOI] [PubMed] [Google Scholar]
  8. Corvera S., Graver D. F., Smith R. M. Insulin increases the cell surface concentration of alpha 2-macroglobulin receptors in 3T3-L1 adipocytes. Altered transit of the receptor among intracellular endocytic compartments. J Biol Chem. 1989 Jun 15;264(17):10133–10138. [PubMed] [Google Scholar]
  9. Cushman S. W., Wardzala L. J. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem. 1980 May 25;255(10):4758–4762. [PubMed] [Google Scholar]
  10. Czech M. P., Chawla A., Woon C. W., Buxton J., Armoni M., Tang W., Joly M., Corvera S. Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization. J Cell Biol. 1993 Oct;123(1):127–135. doi: 10.1083/jcb.123.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davis C. G., van Driel I. R., Russell D. W., Brown M. S., Goldstein J. L. The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis. J Biol Chem. 1987 Mar 25;262(9):4075–4082. [PubMed] [Google Scholar]
  12. Dunn K. W., McGraw T. E., Maxfield F. R. Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome. J Cell Biol. 1989 Dec;109(6 Pt 2):3303–3314. doi: 10.1083/jcb.109.6.3303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ezaki O., Bono N., Itakura H., Kasahara M. Co-localization of a glucose transporter and the insulin receptor in microsomes of insulin-treated rat adipocytes. Biochem Biophys Res Commun. 1989 Mar 31;159(3):1368–1374. doi: 10.1016/0006-291x(89)92261-4. [DOI] [PubMed] [Google Scholar]
  14. Haney P. M., Slot J. W., Piper R. C., James D. E., Mueckler M. Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type. J Cell Biol. 1991 Aug;114(4):689–699. doi: 10.1083/jcb.114.4.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holman G. D., Kozka I. J., Clark A. E., Flower C. J., Saltis J., Habberfield A. D., Simpson I. A., Cushman S. W. Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J Biol Chem. 1990 Oct 25;265(30):18172–18179. [PubMed] [Google Scholar]
  16. Hudson A. W., Ruiz M., Birnbaum M. J. Isoform-specific subcellular targeting of glucose transporters in mouse fibroblasts. J Cell Biol. 1992 Feb;116(3):785–797. doi: 10.1083/jcb.116.3.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jadot M., Canfield W. M., Gregory W., Kornfeld S. Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor. J Biol Chem. 1992 Jun 5;267(16):11069–11077. [PubMed] [Google Scholar]
  18. James D. E., Piper R. C., Slot J. W. Targeting of mammalian glucose transporters. J Cell Sci. 1993 Mar;104(Pt 3):607–612. doi: 10.1242/jcs.104.3.607. [DOI] [PubMed] [Google Scholar]
  19. James D. E., Strube M., Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature. 1989 Mar 2;338(6210):83–87. doi: 10.1038/338083a0. [DOI] [PubMed] [Google Scholar]
  20. Jhun B. H., Rampal A. L., Liu H., Lachaal M., Jung C. Y. Effects of insulin on steady state kinetics of GLUT4 subcellular distribution in rat adipocytes. Evidence of constitutive GLUT4 recycling. J Biol Chem. 1992 Sep 5;267(25):17710–17715. [PubMed] [Google Scholar]
  21. Jing S. Q., Spencer T., Miller K., Hopkins C., Trowbridge I. S. Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization. J Cell Biol. 1990 Feb;110(2):283–294. doi: 10.1083/jcb.110.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johnson L. S., Dunn K. W., Pytowski B., McGraw T. E. Endosome acidification and receptor trafficking: bafilomycin A1 slows receptor externalization by a mechanism involving the receptor's internalization motif. Mol Biol Cell. 1993 Dec;4(12):1251–1266. doi: 10.1091/mbc.4.12.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Klausner R. D., van Renswoude J., Kempf C., Rao K., Bateman J. L., Robbins A. R. Failure to release iron from transferrin in a Chinese hamster ovary cell mutant pleiotropically defective in endocytosis. J Cell Biol. 1984 Mar;98(3):1098–1101. doi: 10.1083/jcb.98.3.1098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kono T., Robinson F. W., Blevins T. L., Ezaki O. Evidence that translocation of the glucose transport activity is the major mechanism of insulin action on glucose transport in fat cells. J Biol Chem. 1982 Sep 25;257(18):10942–10947. [PubMed] [Google Scholar]
  25. Ktistakis N. T., Thomas D., Roth M. G. Characteristics of the tyrosine recognition signal for internalization of transmembrane surface glycoproteins. J Cell Biol. 1990 Oct;111(4):1393–1407. doi: 10.1083/jcb.111.4.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lobel P., Fujimoto K., Ye R. D., Griffiths G., Kornfeld S. Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis. Cell. 1989 Jun 2;57(5):787–796. doi: 10.1016/0092-8674(89)90793-9. [DOI] [PubMed] [Google Scholar]
  27. Mayor S., Presley J. F., Maxfield F. R. Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process. J Cell Biol. 1993 Jun;121(6):1257–1269. doi: 10.1083/jcb.121.6.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McClelland A., Kühn L. C., Ruddle F. H. The human transferrin receptor gene: genomic organization, and the complete primary structure of the receptor deduced from a cDNA sequence. Cell. 1984 Dec;39(2 Pt 1):267–274. doi: 10.1016/0092-8674(84)90004-7. [DOI] [PubMed] [Google Scholar]
  29. McGraw T. E., Dunn K. W., Maxfield F. R. Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation. J Cell Biol. 1988 Apr;106(4):1061–1066. doi: 10.1083/jcb.106.4.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McGraw T. E., Greenfield L., Maxfield F. R. Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor. J Cell Biol. 1987 Jul;105(1):207–214. doi: 10.1083/jcb.105.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McGraw T. E., Maxfield F. R. Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain. Cell Regul. 1990 Mar;1(4):369–377. doi: 10.1091/mbc.1.4.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McGraw T. E., Pytowski B., Arzt J., Ferrone C. Mutagenesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second-site mutation is capable of reverting an internalization-defective phenotype. J Cell Biol. 1991 Mar;112(5):853–861. doi: 10.1083/jcb.112.5.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Peters C., Braun M., Weber B., Wendland M., Schmidt B., Pohlmann R., Waheed A., von Figura K. Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes. EMBO J. 1990 Nov;9(11):3497–3506. doi: 10.1002/j.1460-2075.1990.tb07558.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Piper R. C., Hess L. J., James D. E. Differential sorting of two glucose transporters expressed in insulin-sensitive cells. Am J Physiol. 1991 Mar;260(3 Pt 1):C570–C580. doi: 10.1152/ajpcell.1991.260.3.C570. [DOI] [PubMed] [Google Scholar]
  35. Piper R. C., Tai C., Kulesza P., Pang S., Warnock D., Baenziger J., Slot J. W., Geuze H. J., Puri C., James D. E. GLUT-4 NH2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration. J Cell Biol. 1993 Jun;121(6):1221–1232. doi: 10.1083/jcb.121.6.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Piper R. C., Tai C., Slot J. W., Hahn C. S., Rice C. M., Huang H., James D. E. The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus. J Cell Biol. 1992 May;117(4):729–743. doi: 10.1083/jcb.117.4.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Robinson L. J., Pang S., Harris D. S., Heuser J., James D. E. Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and GTP gamma S and localization of GLUT4 to clathrin lattices. J Cell Biol. 1992 Jun;117(6):1181–1196. doi: 10.1083/jcb.117.6.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Satoh S., Nishimura H., Clark A. E., Kozka I. J., Vannucci S. J., Simpson I. A., Quon M. J., Cushman S. W., Holman G. D. Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action. J Biol Chem. 1993 Aug 25;268(24):17820–17829. [PubMed] [Google Scholar]
  39. Shibasaki Y., Asano T., Lin J. L., Tsukuda K., Katagiri H., Ishihara H., Yazaki Y., Oka Y. Two glucose transporter isoforms are sorted differentially and are expressed in distinct cellular compartments. Biochem J. 1992 Feb 1;281(Pt 3):829–834. doi: 10.1042/bj2810829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Slot J. W., Geuze H. J., Gigengack S., Lienhard G. E., James D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol. 1991 Apr;113(1):123–135. doi: 10.1083/jcb.113.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Suzuki K., Kono T. Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc Natl Acad Sci U S A. 1980 May;77(5):2542–2545. doi: 10.1073/pnas.77.5.2542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tanner L. I., Lienhard G. E. Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization. J Biol Chem. 1987 Jul 5;262(19):8975–8980. [PubMed] [Google Scholar]
  43. Tanner L. I., Lienhard G. E. Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters. J Cell Biol. 1989 Apr;108(4):1537–1545. doi: 10.1083/jcb.108.4.1537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Verhey K. J., Hausdorff S. F., Birnbaum M. J. Identification of the carboxy terminus as important for the isoform-specific subcellular targeting of glucose transporter proteins. J Cell Biol. 1993 Oct;123(1):137–147. doi: 10.1083/jcb.123.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ward D. M., Ajioka R., Kaplan J. Cohort movement of different ligands and receptors in the intracellular endocytic pathway of alveolar macrophages. J Biol Chem. 1989 May 15;264(14):8164–8170. [PubMed] [Google Scholar]
  46. Yamashiro D. J., Tycko B., Fluss S. R., Maxfield F. R. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell. 1984 Jul;37(3):789–800. doi: 10.1016/0092-8674(84)90414-8. [DOI] [PubMed] [Google Scholar]
  47. Yang J., Holman G. D. Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells. J Biol Chem. 1993 Mar 5;268(7):4600–4603. [PubMed] [Google Scholar]

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