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. 1995 Sep 1;130(5):1081–1091. doi: 10.1083/jcb.130.5.1081

Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes

PMCID: PMC2120558  PMID: 7544796

Abstract

Insulin stimulates glucose transport in muscle and adipose tissue by triggering the movement of the glucose transporter GLUT-4 from an intracellular compartment to the cell surface. Fundamental to this process is the intracellular sequestration of GLUT-4 in nonstimulated cells. Two distinct targeting motifs in the amino and carboxy termini of GLUT-4 have been previously identified by expressing chimeras comprised of portions of GLUT-4 and GLUT-1, a transporter isoform that is constitutively targeted to the cell surface, in heterologous cells. These motifs-FQQI in the NH2 terminus and LL in the COOH terminus- resemble endocytic signals that have been described in other proteins. In the present study we have investigated the roles of these motifs in GLUT-4 targeting in insulin-sensitive cells. Epitope-tagged GLUT-4 constructs engineered to differentiate between endogenous and transfected GLUT-4 were stably expressed in 3T3-L1 adipocytes. Targeting was assessed in cells incubated in the presence or absence of insulin by subcellular fractionation. The targeting of epitope-tagged GLUT-4 was indistinguishable from endogenous GLUT-4. Mutation of the FQQI motif (F5 to A5) caused GLUT-4 to constitutively accumulate at the cell surface regardless of expression level. Mutation of the dileucine motif (L489L490 to A489A490) caused an increase in cell surface distribution only at higher levels of expression, but the overall cells surface distribution of this mutant was less than that of the amino- terminal mutants. Both NH2- and COOH-terminal mutants retained insulin- dependent movement from an intracellular to a cell surface locale, suggesting that neither of these motifs is involved in the insulin- dependent redistribution of GLUT-4. We conclude that the phenylalanine- based NH2-terminal and the dileucine-based COOH-terminal motifs play important and distinct roles in GLUT-4 targeting in 3T3-L1 adipocytes.

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Selected References

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  1. Alm R. A., Mattick J. S. Identification of a gene, pilV, required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa, whose product possesses a pre-pilin-like leader sequence. Mol Microbiol. 1995 May;16(3):485–496. doi: 10.1111/j.1365-2958.1995.tb02413.x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Balch W. E., Dunphy W. G., Braell W. A., Rothman J. E. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. doi: 10.1016/0092-8674(84)90019-9. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Benito M., Porras A., Nebreda A. R., Santos E. Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes. Science. 1991 Aug 2;253(5019):565–568. doi: 10.1126/science.1857988. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Blok J., Gibbs E. M., Lienhard G. E., Slot J. W., Geuze H. J. Insulin-induced translocation of glucose transporters from post-Golgi compartments to the plasma membrane of 3T3-L1 adipocytes. J Cell Biol. 1988 Jan;106(1):69–76. doi: 10.1083/jcb.106.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  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. 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]
  11. 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]
  12. 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]
  13. Fukumoto H., Kayano T., Buse J. B., Edwards Y., Pilch P. F., Bell G. I., Seino S. Cloning and characterization of the major insulin-responsive glucose transporter expressed in human skeletal muscle and other insulin-responsive tissues. J Biol Chem. 1989 May 15;264(14):7776–7779. [PubMed] [Google Scholar]
  14. 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]
  15. Gould G. W., Derechin V., James D. E., Tordjman K., Ahern S., Gibbs E. M., Lienhard G. E., Mueckler M. Insulin-stimulated translocation of the HepG2/erythrocyte-type glucose transporter expressed in 3T3-L1 adipocytes. J Biol Chem. 1989 Feb 5;264(4):2180–2184. [PubMed] [Google Scholar]
  16. Griffiths G., Parton R. G., Lucocq J., van Deurs B., Brown D., Slot J. W., Geuze H. J. The immunofluorescent era of membrane traffic. Trends Cell Biol. 1993 Jul;3(7):214–219. doi: 10.1016/0962-8924(93)90114-g. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. James D. E., Brown R., Navarro J., Pilch P. F. Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature. 1988 May 12;333(6169):183–185. doi: 10.1038/333183a0. [DOI] [PubMed] [Google Scholar]
  22. James D. E., Piper R. C. Insulin resistance, diabetes, and the insulin-regulated trafficking of GLUT-4. J Cell Biol. 1994 Sep;126(5):1123–1126. doi: 10.1083/jcb.126.5.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Kahn B. B. Facilitative glucose transporters: regulatory mechanisms and dysregulation in diabetes. J Clin Invest. 1992 May;89(5):1367–1374. doi: 10.1172/JCI115724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lawrence J. C., Jr, Hiken J. F., James D. E. Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact-cells and in vitro. J Biol Chem. 1990 Feb 5;265(4):2324–2332. [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. 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]
  32. 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]
  33. Reaves B., Banting G. Overexpression of TGN38/41 leads to mislocalisation of gamma-adaptin. FEBS Lett. 1994 Sep 12;351(3):448–456. doi: 10.1016/0014-5793(94)00813-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Robinson L. J., James D. E. Insulin-regulated sorting of glucose transporters in 3T3-L1 adipocytes. Am J Physiol. 1992 Aug;263(2 Pt 1):E383–E393. doi: 10.1152/ajpendo.1992.263.2.E383. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Simpson I. A., Yver D. R., Hissin P. J., Wardzala L. J., Karnieli E., Salans L. B., Cushman S. W. Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions. Biochim Biophys Acta. 1983 Dec 19;763(4):393–407. doi: 10.1016/0167-4889(83)90101-5. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. 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]
  40. Trowbridge I. S., Collawn J. F., Hopkins C. R. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol. 1993;9:129–161. doi: 10.1146/annurev.cb.09.110193.001021. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Wong S. H., Hong W. The SXYQRL sequence in the cytoplasmic domain of TGN38 plays a major role in trans-Golgi network localization. J Biol Chem. 1993 Oct 25;268(30):22853–22862. [PubMed] [Google Scholar]
  44. 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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