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Microdomains of GPI-anchored proteins in living cells revealed by crosslinking

Nature volume 394, pages 802–805 (1998)Cite this article

Abstract

There is some discussion as to whether glycosyl-phosphatidylinositol(GPI)-anchored proteins occur in microdomains in the cell membrane1,2. These putative microdomains have been implicated in processes such as sorting in polarized cells3,4,5 and signal transduction6,7,8. Complexes enriched in GPI-anchored proteins, cholesterol and glycosphingolipids have been isolated from cell membranes by using non-ionic detergents: these complexes were thought to represent a clustered arrangement of GPI-anchored proteins9,10. However, results obtained when clustering of GPI-anchored proteins induced by antibodies or by detergents was prevented support the idea of a dispersed surface distribution of GPI-anchored proteins at steady state11,12,13. Here we use chemical crosslinking to show that membrane microdomains of a GPI-anchored protein exist at the surface in living cells. This clustering is specific for the GPI-anchored form, as two transmembrane forms bearing the same ectodomain do not form oligomers. Depletion of membrane cholesterol causes the clustering of GPI-anchored proteins to break up, whereas treatment of cells with detergent substantially increases the size of the complexes. We find that in living cells these GPI-anchored proteins reside in microdomains consisting of at least 15 molecules, which are much smaller than those seen after detergent extraction.

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Figure 1: Detection of oligomers upon crosslinking of GPI-anchored, transmembrane and secretory forms of GH.
Figure 2: Clustering of GPI-anchored proteins depends on cholesterol.
Figure 3: Detergent treatment leads to increased size of crosslinked oligomers.

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References

  1. Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    Article  ADS  CAS  Google Scholar 

  2. Weimbs, T., Low, S. H., Chapin, S. J. & Mostov, K. E. Apical targeting in polarized epithelial cells: there's more afloat than rafts. Trends Cell Biol. 7, 393–399 (1997).

    Article  CAS  Google Scholar 

  3. Lisanti, M. P., Le Bivic, A., Sargiacomo, M. & Rodriguez Boulan, E. Steady-state distribution and biogenesis of endogenous Madin–Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery. J. Cell Biol. 109, 2117–2127 (1989).

    Article  CAS  Google Scholar 

  4. Brown, D. A. & Rose, J. K. Dorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533–544 (1992).

    Article  CAS  Google Scholar 

  5. Kurzchalia, T. V.et al. VIP21, a 21-kD membrane protein is an integral component of trans -Golgi-network-derived transport vesicles. J. Cell Biol. 118, 1003–1014 (1992).

    Article  CAS  Google Scholar 

  6. Stefanova, I., Horejsi, V., Ansotegui, I. J., Knapp, W. & Stockinger, H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science 254, 1016–1019 (1991).

    Article  ADS  CAS  Google Scholar 

  7. Brown, D. The tyrosine kinase connection: how GPI-anchored proteins activate T cells. Curr. Opin. Immunol. 5, 349–354 (1993).

    Article  CAS  Google Scholar 

  8. Rodgers, W., Crise, B. & Rose, J. K. Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction. Mol. Cell Biol. 14, 5384–5391 (1994).

    Article  CAS  Google Scholar 

  9. Fiedler, K., Kobayashi, T., Kurzchalia, T. V. & Simons, K. Glycosphingolipid-enriched, detergent-insoluble complexes in protein sorting in epithelial cells. Biochemistry 32, 6365–6373 (1993).

    Article  CAS  Google Scholar 

  10. Sargiacomo, M., Sudol, M., Tang, Z. & Lisanti, M. P. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J. Cell Biol. 122, 789–807 (1993).

    Article  CAS  Google Scholar 

  11. Hannan, L. A., Lisanti, M. P., Rodriguez Boulan, E. & Edidin, M. Correctly sorted molecules of a GPI-anchored protein are clustered and immobile when they arrive at the apical surface of MDCK cells. J. Cell Biol. 120, 353–358 (1993).

    Article  CAS  Google Scholar 

  12. Mayor, S., Rothberg, K. G. & Maxfield, F. R. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 264, 1948–1951 (1994).

    Article  ADS  CAS  Google Scholar 

  13. Mayor, S. & Maxfield, F. R. Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment. Mol. Biol. Cell 6, 929–944 (1995).

    Article  CAS  Google Scholar 

  14. Lisanti, M. P., Caras, I. W., Davitz, M. A. & Rodriguez Boulan, E. Aglycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J. Cell Biol. 109, 2145–2156 (1989).

    Article  CAS  Google Scholar 

  15. Kurzchalia, T., Hartmann, E. & Dupree, P. Guilt by insolubility—Does a protein's detergent insolubility reflect caveolar location? Trends Cell Biol. 5, 187–189 (1995).

    CAS  PubMed  Google Scholar 

  16. Rothberg, K. G., Ying, Y. S., Kamen, B. A. & Anderson, R. G. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J. Cell Biol. 111, 2931–2938 (1990).

    Article  CAS  Google Scholar 

  17. Cerneus, D. P., Ueffing, E., Posthuma, G., Strous, G. J. & van der Ende, A. Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol. J. Biol. Chem. 268, 3150–3155 (1993).

    CAS  PubMed  Google Scholar 

  18. Hanada, K., Nishijima, M., Akamatsu, Y. & Pagano, R. E. Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes. J. Biol. Chem. 270, 6254–6260 (1995).

    Article  CAS  Google Scholar 

  19. Chang, W. J., Rothberg, K. G., Kamen, B. A. & Anderson, R. G. Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate. J. Cell Biol. 118, 63–69 (1992).

    Article  CAS  Google Scholar 

  20. Smart, E. J., Mineo, C. & Anderson, R. G. Clustered folate receptors deliver 5-methyltetrahydrofolate to cytoplasm of MA104 cells. J. Cell Biol. 134, 1169–1177 (1996).

    Article  CAS  Google Scholar 

  21. Stulnig, T. M.et al. Signal transduction via glycosyl phosphatidylinositol-anchored proteins in T cells is inhibited by lowering cellular cholesterol. J. Biol. Chem. 272, 19242–19247 (1997).

    Article  CAS  Google Scholar 

  22. Klein, U., Gimpl, G. & Fahrenholz, F. Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry 34, 13784–13793 (1995).

    Article  CAS  Google Scholar 

  23. Fiedler, K., Parton, R. G., Kellner, R., Etzold, T. & Simons, K. VIP36, a novel compoennt of glycolipid rafts and exocytic carrier vesicles in epithelial cells. EMBO J. 13, 1729–1740 (1994).

    Article  CAS  Google Scholar 

  24. Scheiffele, P., Peranen, J. & Simons, K. N -glycans as apical sorting signals in epithelial cells. Nature 378, 96–98 (1995).

    Article  ADS  CAS  Google Scholar 

  25. Fujimoto, T. GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking. J. Histochem. Cytochem. 44, 929–941 (1996).

    Article  CAS  Google Scholar 

  26. Varma, R. & Mayor, S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394, 798–801 (1998).

    Article  ADS  CAS  Google Scholar 

  27. Harder, T. & Simons, K. Caveolae, DIGs, and the dynamics of sphingolipid–cholesterol microdomains. Curr. Opin. Cell Biol. 9, 534–542 (1997).

    Article  CAS  Google Scholar 

  28. Schnitzer, J. E., McIntosh, D. P., Dvorak, A. M., Liu, J. & Oh, P. Separation of caveolae from associated microdomains of GPI-anchored proteins. Science 269, 1435–1439 (1995).

    Article  ADS  CAS  Google Scholar 

  29. Wu, M., Fan, J., Gunning, W. & Ratnam, M. Clustering of GPI-anchored folate receptor independent of both cross-linking and association with caveolin. J. Membr. Biol. 159, 137–147 (1997).

    Article  CAS  Google Scholar 

  30. Huang, C.et al. Organization of G proteins and adenylyl cyclase at the plasma membrane. Mol. Biol. Cell 8, 2365–2378 (1997).

    Article  CAS  Google Scholar 

  31. Matter, K., Hunziker, W. & Mellman, I. Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell 71, 741–753 (1992).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank G. Lewin, M. S. Bhojani and M. Wiedmann for critically reading the manuscript; W. Hunziker for plasmids pCBFL5-50 and pCBFL5-503YA; and A. Henske for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft and Human Frontier Science Program.

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  1. Department of Cell Biology, Max Delbrück Centre for Molecular Medicine, Robert-Rössle-Strasse 10, 13125, Berlin-Buch, Germany

    Tim Friedrichson & Teymuras V. Kurzchalia

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Correspondence to Teymuras V. Kurzchalia.

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Friedrichson, T., Kurzchalia, T. Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature 394, 802–805 (1998). https://doi.org/10.1038/29570

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