Ubiquitination of G-Protein-Coupled Receptors

  • Adriano Marchese
  • Jeffrey L. Benovic
Part of the Methods in Molecular Biology book series (MIMB, volume 259)

Abstract

In this chapter we describe methods for detecting the ubiquitination state of G-protein-coupled receptors (GPCRs). This involves coexpression of a GPCR with an epitope-tagged ubiquitin construct in a heterologous expression system. Modification by ubiquitin of the GPCR resulting from agonist activation is detected by immunoprecipation and subsequent immunoblotting for the epitope-tagged ubiquitin. We use here the chemokine receptor CXCR4 as the model receptor; however, this could be easily modified to detect the ubiquitination state of any GPCR.

Key Words

Agonist CXCR4 degradation G-protein-coupled receptor immunoblot immunoprecipitation lysosome sorting ubiquitin 

References

  1. 1.
    Claing, A., Laporte, S. A., Caron, M. G., and Lefkowitz, R. J. (2002) Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and β-arrestin proteins. Prog. Neurobiol. 66, 61–79.PubMedCrossRefGoogle Scholar
  2. 2.
    Marchese, A. and Benovic, J. L. (2001) Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting. J. Biol. Chem. 276, 45,509–45,512.PubMedCrossRefGoogle Scholar
  3. 3.
    Shenoy, S. K., McDonald, P. H., Kohout, T. A., and Lefkowitz, R. J. (2001) Regulation of receptor fate by ubiquitination of activated β2-adrenergic receptor and β-arrestin. Science 294, 1307–1313.PubMedCrossRefGoogle Scholar
  4. 4.
    Weissman, A. M. (2001) Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell Biol. 2, 169–178.PubMedCrossRefGoogle Scholar
  5. 5.
    Hicke, L. (2001) Protein regulation by monoubiquitin. Nat. Rev. Mol. Cell Biol. 2, 195–201.PubMedCrossRefGoogle Scholar
  6. 6.
    Bonifacino, J. S. and Weissman, A. M. (1998) Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu. Rev. Cell Dev. Biol. 14, 19–57.PubMedCrossRefGoogle Scholar
  7. 7.
    Hicke, L. (1997) Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins. FASEB J. 11, 1215–1226.PubMedGoogle Scholar
  8. 8.
    Katzmann, D. J., Babst, M., and Emr, S. D. (2001) Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106, 145–155.PubMedCrossRefGoogle Scholar
  9. 9.
    Reggiori, F. and Pelham, H. R. (2001) Sorting of proteins into multivesicular bodies: ubiquitin-dependent and-independent targeting. EMBO J. 20, 5176–5186.PubMedCrossRefGoogle Scholar
  10. 10.
    Urbanowski, J. L. and Piper, R. C. (2001) Ubiquitin sorts proteins into the intralumenal degradative compartment of the late-endosome/vacuole. Traffic 2, 622–630.PubMedCrossRefGoogle Scholar
  11. 11.
    Seto, E. S., Bellen, H. J., and Lloyd, T. E. (2002) When cell biology meets development: endocytic regulation of signaling pathways. Genes Dev. 16, 1314–1336.PubMedCrossRefGoogle Scholar
  12. 12.
    Piper, R. C. and Luzio, J. P. (2001) Late endosomes: sorting and partitioning in multivesicular bodies. Traffic 2, 612–621.PubMedCrossRefGoogle Scholar
  13. 13.
    Raiborg, C., Bache, K. G., Gillooly, D. J., Madshus, I. H., Stang, E., and Stenmark, H. (2002) Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat. Cell Biol. 4, 394–398.PubMedCrossRefGoogle Scholar
  14. 14.
    Mundell, S. J., Orsini, M. J., and Benovic, J. L. (2002) Characterization of arrestin expression and function. Methods Enzymol. 343, 600–611.PubMedCrossRefGoogle Scholar
  15. 15.
    Ellison, M. J. and Hochstrasser, M. (1991) Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function. J. Biol. Chem. 266, 21,150–21,157.PubMedGoogle Scholar
  16. 16.
    Courbard, J. R., Fiore, F., Adelaide, J., Borg, J. P., Birnbaum, D., and Ollendorff, V. (2002) Interaction between two ubiquitin-protein isopeptide ligases of different classes, CBLC and AIP4/ITCH. J. Biol. Chem. 277, 45,267–45,275.PubMedCrossRefGoogle Scholar
  17. 17.
    Wilkinson, K. D. (2000) Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Semin. Cell Dev. Biol. 11, 141–148.PubMedCrossRefGoogle Scholar
  18. 18.
    Hicke, L., Zanolari, B., and Riezman, H. (1998) Cytoplasmic tail phosphorylation of the α-factor receptor is required for its ubiquitination and internalization. J. Cell Biol. 141, 349–358.PubMedCrossRefGoogle Scholar
  19. 19.
    Amerik, A. Y., Nowak, J., Swaminathan, S., and Hochstrasser, M. (2000) The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways. Mol. Biol. Cell 11, 3365–3380.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Adriano Marchese
    • 1
  • Jeffrey L. Benovic
    • 1
  1. 1.Department of Microbiology and Immunology, Kimmel Cancer CenterThomas Jefferson UniversityPhiladelphia

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