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. 2000 Aug 15;350(Pt 1):99–107.

The cytosolic C-terminus of the glucose transporter GLUT4 contains an acidic cluster endosomal targeting motif distal to the dileucine signal.

A M Shewan 1, B J Marsh 1, D R Melvin 1, S Martin 1, G W Gould 1, D E James 1
PMCID: PMC1221230  PMID: 10926832

Abstract

The insulin-responsive glucose transporter GLUT4 is targeted to a post-endocytic compartment in adipocytes, from where it moves to the cell surface in response to insulin. Previous studies have identified two cytosolic targeting motifs that regulate the intracellular sequestration of this protein: FQQI(5-8) in the N-terminus and LL(489,490) (one-letter amino acid notation) in the C-terminus. In the present study we show that a GLUT4 chimaera in which the C-terminal 12 amino acids in GLUT4 have been replaced with the same region from human GLUT3 is constitutively targeted to the plasma membrane when expressed in 3T3-L1 adipocytes. To further dissect this domain it was divided into three regions, each of which was mutated en bloc to alanine residues. Analysis of these constructs revealed that the targeting information is contained within the residues TELEYLGP(498-505). Using the transferrin-horseradish peroxidase endosomal ablation technique in 3T3-L1 adipocytes, we show that mutants in which this C-terminal domain has been disrupted are more sensitive to chemical ablation than wild-type GLUT4. These data indicate that GLUT4 contains a targeting signal in its C-terminus, distal to the dileucine motif, that regulates its sorting into a post-endosomal compartment. Similar membrane-distal, acidic-cluster-based motifs are found in the cytosolic tails of the insulin-responsive aminopeptidase IRAP (insulin-regulated aminopeptidase) and the proprotein convertase PC6B, indicating that this type of motif may play an important role in the endosomal sequestration of a number of different proteins.

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Selected References

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

  1. Bremnes T., Lauvrak V., Lindqvist B., Bakke O. A region from the medium chain adaptor subunit (mu) recognizes leucine- and tyrosine-based sorting signals. J Biol Chem. 1998 Apr 10;273(15):8638–8645. doi: 10.1074/jbc.273.15.8638. [DOI] [PubMed] [Google Scholar]
  2. Corvera S., Chawla A., Chakrabarti R., Joly M., Buxton J., Czech M. P. A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal. J Cell Biol. 1994 Aug;126(4):979–989. doi: 10.1083/jcb.126.4.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Garippa R. J., Johnson A., Park J., Petrush R. L., McGraw T. E. The carboxyl terminus of GLUT4 contains a serine-leucine-leucine sequence that functions as a potent internalization motif in Chinese hamster ovary cells. J Biol Chem. 1996 Aug 23;271(34):20660–20668. doi: 10.1074/jbc.271.34.20660. [DOI] [PubMed] [Google Scholar]
  4. Garippa R. J., Judge T. W., James D. E., McGraw T. E. The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain. J Cell Biol. 1994 Mar;124(5):705–715. doi: 10.1083/jcb.124.5.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Geisler C., Dietrich J., Nielsen B. L., Kastrup J., Lauritsen J. P., Odum N., Christensen M. D. Leucine-based receptor sorting motifs are dependent on the spacing relative to the plasma membrane. J Biol Chem. 1998 Aug 14;273(33):21316–21323. doi: 10.1074/jbc.273.33.21316. [DOI] [PubMed] [Google Scholar]
  6. Glickman J. N., Conibear E., Pearse B. M. Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor. EMBO J. 1989 Apr;8(4):1041–1047. doi: 10.1002/j.1460-2075.1989.tb03471.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Haney P. M., Levy M. A., Strube M. S., Mueckler M. Insulin-sensitive targeting of the GLUT4 glucose transporter in L6 myoblasts is conferred by its COOH-terminal cytoplasmic tail. J Cell Biol. 1995 May;129(3):641–658. doi: 10.1083/jcb.129.3.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harris D. S., Slot J. W., Geuze H. J., James D. E. Polarized distribution of glucose transporter isoforms in Caco-2 cells. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7556–7560. doi: 10.1073/pnas.89.16.7556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hirst J., Robinson M. S. Clathrin and adaptors. Biochim Biophys Acta. 1998 Aug 14;1404(1-2):173–193. doi: 10.1016/s0167-4889(98)00056-1. [DOI] [PubMed] [Google Scholar]
  10. Höning S., Sandoval I. V., von Figura K. A di-leucine-based motif in the cytoplasmic tail of LIMP-II and tyrosinase mediates selective binding of AP-3. EMBO J. 1998 Aug 10;17(5):1304–1314. doi: 10.1093/emboj/17.5.1304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Johnson K. F., Kornfeld S. The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi. J Cell Biol. 1992 Oct;119(2):249–257. doi: 10.1083/jcb.119.2.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kao A. W., Ceresa B. P., Santeler S. R., Pessin J. E. Expression of a dominant interfering dynamin mutant in 3T3L1 adipocytes inhibits GLUT4 endocytosis without affecting insulin signaling. J Biol Chem. 1998 Sep 25;273(39):25450–25457. doi: 10.1074/jbc.273.39.25450. [DOI] [PubMed] [Google Scholar]
  14. Le Borgne R., Schmidt A., Mauxion F., Griffiths G., Hoflack B. Binding of AP-1 Golgi adaptors to membranes requires phosphorylated cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor. J Biol Chem. 1993 Oct 25;268(30):22552–22556. [PubMed] [Google Scholar]
  15. Livingstone C., James D. E., Rice J. E., Hanpeter D., Gould G. W. Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes. Biochem J. 1996 Apr 15;315(Pt 2):487–495. doi: 10.1042/bj3150487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Marks M. S., Woodruff L., Ohno H., Bonifacino J. S. Protein targeting by tyrosine- and di-leucine-based signals: evidence for distinct saturable components. J Cell Biol. 1996 Oct;135(2):341–354. doi: 10.1083/jcb.135.2.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marsh B. J., Alm R. A., McIntosh S. R., James D. E. Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes. J Cell Biol. 1995 Sep;130(5):1081–1091. doi: 10.1083/jcb.130.5.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marsh B. J., Martin S., Melvin D. R., Martin L. B., Alm R. A., Gould G. W., James D. E. Mutational analysis of the carboxy-terminal phosphorylation site of GLUT-4 in 3T3-L1 adipocytes. Am J Physiol. 1998 Sep;275(3 Pt 1):E412–E422. doi: 10.1152/ajpendo.1998.275.3.E412. [DOI] [PubMed] [Google Scholar]
  19. Marshall B. A., Murata H., Hresko R. C., Mueckler M. Domains that confer intracellular sequestration of the Glut4 glucose transporter in Xenopus oocytes. J Biol Chem. 1993 Dec 15;268(35):26193–26199. [PubMed] [Google Scholar]
  20. Martin L. B., Shewan A., Millar C. A., Gould G. W., James D. E. Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes. J Biol Chem. 1998 Jan 16;273(3):1444–1452. doi: 10.1074/jbc.273.3.1444. [DOI] [PubMed] [Google Scholar]
  21. Martin S., Tellam J., Livingstone C., Slot J. W., Gould G. W., James D. E. The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells. J Cell Biol. 1996 Aug;134(3):625–635. doi: 10.1083/jcb.134.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Matter K., Yamamoto E. M., Mellman I. Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells. J Cell Biol. 1994 Aug;126(4):991–1004. doi: 10.1083/jcb.126.4.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mellman I. Endocytosis and molecular sorting. Annu Rev Cell Dev Biol. 1996;12:575–625. doi: 10.1146/annurev.cellbio.12.1.575. [DOI] [PubMed] [Google Scholar]
  24. Melvin D. R., Marsh B. J., Walmsley A. R., James D. E., Gould G. W. Analysis of amino and carboxy terminal GLUT-4 targeting motifs in 3T3-L1 adipocytes using an endosomal ablation technique. Biochemistry. 1999 Feb 2;38(5):1456–1462. doi: 10.1021/bi980988y. [DOI] [PubMed] [Google Scholar]
  25. Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. doi: 10.1093/nar/18.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pear W. S., Nolan G. P., Scott M. L., Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392–8396. doi: 10.1073/pnas.90.18.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Quon M. J., Guerre-Millo M., Zarnowski M. J., Butte A. J., Em M., Cushman S. W., Taylor S. I. Tyrosine kinase-deficient mutant human insulin receptors (Met1153-->Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5587–5591. doi: 10.1073/pnas.91.12.5587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rapoport I., Chen Y. C., Cupers P., Shoelson S. E., Kirchhausen T. Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site. EMBO J. 1998 Apr 15;17(8):2148–2155. doi: 10.1093/emboj/17.8.2148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rodionov D. G., Bakke O. Medium chains of adaptor complexes AP-1 and AP-2 recognize leucine-based sorting signals from the invariant chain. J Biol Chem. 1998 Mar 13;273(11):6005–6008. doi: 10.1074/jbc.273.11.6005. [DOI] [PubMed] [Google Scholar]
  33. Ross S. A., Scott H. M., Morris N. J., Leung W. Y., Mao F., Lienhard G. E., Keller S. R. Characterization of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes. J Biol Chem. 1996 Feb 9;271(6):3328–3332. doi: 10.1074/jbc.271.6.3328. [DOI] [PubMed] [Google Scholar]
  34. Schäfer W., Stroh A., Berghöfer S., Seiler J., Vey M., Kruse M. L., Kern H. F., Klenk H. D., Garten W. Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin. EMBO J. 1995 Jun 1;14(11):2424–2435. doi: 10.1002/j.1460-2075.1995.tb07240.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Simonsen A., Stang E., Bremnes B., Røe M., Prydz K., Bakke O. Sorting of MHC class II molecules and the associated invariant chain (Ii) in polarized MDCK cells. J Cell Sci. 1997 Mar;110(Pt 5):597–609. doi: 10.1242/jcs.110.5.597. [DOI] [PubMed] [Google Scholar]
  36. Slot J. W., Geuze H. J., Gigengack S., James D. E., Lienhard G. E. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7815–7819. doi: 10.1073/pnas.88.17.7815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Slot J. W., Moxley R., Geuze H. J., James D. E. No evidence for expression of the insulin-regulatable glucose transporter in endothelial cells. Nature. 1990 Jul 26;346(6282):369–371. doi: 10.1038/346369a0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Verhey K. J., Birnbaum M. J. A Leu-Leu sequence is essential for COOH-terminal targeting signal of GLUT4 glucose transporter in fibroblasts. J Biol Chem. 1994 Jan 28;269(4):2353–2356. [PubMed] [Google Scholar]
  41. 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]
  42. Verhey K. J., Yeh J. I., Birnbaum M. J. Distinct signals in the GLUT4 glucose transporter for internalization and for targeting to an insulin-responsive compartment. J Cell Biol. 1995 Sep;130(5):1071–1079. doi: 10.1083/jcb.130.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Voorhees P., Deignan E., van Donselaar E., Humphrey J., Marks M. S., Peters P. J., Bonifacino J. S. An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface. EMBO J. 1995 Oct 16;14(20):4961–4975. doi: 10.1002/j.1460-2075.1995.tb00179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Xiang Y., Molloy S. S., Thomas L., Thomas G. The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct trans-Golgi network/endosomal compartments. Mol Biol Cell. 2000 Apr;11(4):1257–1273. doi: 10.1091/mbc.11.4.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Zhang Y., Allison J. P. Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein. Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9273–9278. doi: 10.1073/pnas.94.17.9273. [DOI] [PMC free article] [PubMed] [Google Scholar]

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