Skip to main content
Book cover

Chemotaxis pp 179–198Cite as

Chemokine Receptor Dimerization and Chemotaxis

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 571))

Summary

A broad array of biological responses ranging from cell polarization, movement, immune and inflammatory responses, as well as prevention of HIV-1 infection, are triggered by the chemokines, a family of structurally related chemoattractant proteins that bind to specific seven-transmembrane receptors linked to G proteins. Although it was initially believed that chemokine receptors act as monomeric entities, it has now been shown that they function as oligomers. Chemokine receptor homo– and heterodimers are found on the cell membrane; binding to their ligands stabilizes specific receptor conformations and activates distinct signaling cascades. Thorough analysis of the conformations adopted by the receptors at the membrane is therefore a prerequisite for understanding the function of these inflammatory mediators.

For study of the chemokine receptor conformations at the cell surface, we focus here on conventional biochemical and genetic methods, as well as on new imaging techniques such as those based on resonance energy transfer; we also evaluate in vitro and in vivo methods to determine certain chemokine receptor functions.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Baggiolini, M. (1998) Chemokines and leukocyte traffic. Nature 392, 565–568.

    Article  PubMed  CAS  Google Scholar 

  2. Sallusto, F., Lanzavecchia, A., and Mackay, C. (1998) Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol. Today 19, 568–574.

    Article  PubMed  CAS  Google Scholar 

  3. Berger, E. A., Murphy, P. M., and Farber, J. M. (1999) Chemokines as HIV-1 coreceptors: roles in viral entry, tropism and disease. Annu. Rev. Immunol. 17, 657–700.

    Article  PubMed  CAS  Google Scholar 

  4. Belpario, J., Keane, M., Arenberg, D., Addison, C., Ehlert, J., Burdick, M., et al. (2000) CXC chemokines in angiogenesis. J. Leuk. Biol. 68, 1–8.

    Google Scholar 

  5. Zou, Y., Kottmann, A., Kuroda, M., Taniuchi, I., and Littman, D. (1998) Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393, 595–599.

    Article  PubMed  CAS  Google Scholar 

  6. Raz, E. (2003) Primordial germ-cell development: the zebrafish perspective. Nat. Rev. Genet. 4, 690–700.

    Article  PubMed  CAS  Google Scholar 

  7. Müller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M., et al. (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410, 50–56.

    Article  PubMed  Google Scholar 

  8. Wells, T. N. C., Power, C. A., Shaw, J. P., and Proudfoot, A. E. I. (2005) Chemokine blockers-therapeutics in the making? Trends Pharmacol. Sci. 27, 41–47.

    Article  PubMed  Google Scholar 

  9. Littman, D. R. (1998) chemokine receptors: keys to AIDS pathogenesis? Cell 93, 677–680.

    Article  PubMed  CAS  Google Scholar 

  10. El-Sawy, T., Fahmy, N. M., and Fairchild, R. L. (2002) Chemokines: directing leukocyte infiltration into allografts. Curr. Opin. Immunol. 14, 562–568.

    Article  PubMed  CAS  Google Scholar 

  11. Rossi, D., and Zlotnik, A. (2000) The biology of chemokines and their receptors. Annu. Rev. Immunol. 18, 217–242.

    Article  PubMed  CAS  Google Scholar 

  12. Murphy, P. M., Baggiolini, M., Charo, I. F., Hébert, C. A., Horuk, R., Matsushima, K., et al. (2000) International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 52, 145–176.

    PubMed  CAS  Google Scholar 

  13. Ganju, R. K., Brubaker, S. A., Meyer, J., Dutt, P., Yang, Y., Qin, S., et al. (1998) The alpha-chemokine, stromal cell-derived factor-1alpha, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J. Biol. Chem. 273, 23169–23175.

    Article  PubMed  CAS  Google Scholar 

  14. Knall, C., Worthen, G. S., and Johnson, G. L. (1997) Interleukin 8-stimulated phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Proc. Natl. Acad. Sci. USA 94, 3052–3057.

    Article  PubMed  CAS  Google Scholar 

  15. Myers, S. J., Wong, L. M., and Charo, I. F. (1995) Signal transduction and ligand specificity of the human monocyte chemoattractant protein-1 receptor in transfected embryonic kidney cells. J. Biol. Chem. 270, 5786–5792.

    Article  PubMed  CAS  Google Scholar 

  16. Arai, H., Tsou, C. L., and Charo, I. F. (1997) Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: evidence that directed migration is mediated by βγ dimers released by activation of Gαi-coupled receptors. Proc. Natl. Acad. Sci. USA 94, 14495–14499.

    Article  PubMed  CAS  Google Scholar 

  17. L’Heureux, G. P., Bourgoin, S., Jean, N., McColl, S. R., and Naccache, P. H. (1995) Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: interleukin-8, but not NAP-2 or GRO alpha, stimulates phospholipase D activity. Blood 85, 522–531.

    PubMed  Google Scholar 

  18. Mellado, M., Rodríguez-Frade, J. M., Mañes, S., and Martínez, A. C. (2001a) Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation. Ann. Rev. Immunol. 19, 397–421.

    Article  CAS  Google Scholar 

  19. Vila-Coro, A. J., Mellado, M., Martin de Ana, A., Lucas, P., del Real, G., Martinez, A. C., et al. (2000) HIV-1 infection through the CCR5 receptor is blocked by receptor dimerization. Proc. Natl. Acad. Sci. USA 97, 3388–3393.

    Article  PubMed  CAS  Google Scholar 

  20. Mellado, M., Rodriguez-Frade, J. M., Vila-Coro, A. J., Fernandez, S., Martin de Ana, A., Jones, D. R., et al. (2001) Chemokine receptor homo- or heterodimerization activates distinct signaling pathways. EMBO J. 20, 2497–2507.

    Article  PubMed  CAS  Google Scholar 

  21. Percherancier, Y., Berchiche, Y. A., Slight, I., Volkmer-Engert, R., Tamamura, H., Fujii, N., et al. (2005) Bioluminescence resonance energy transfer reveals ligand-induced conformational changes in CXCR4 homo- and heterodimers. J. Biol. Chem. 280, 9895–9903.

    Article  PubMed  CAS  Google Scholar 

  22. Wang, J., He, L., Combs, C. A., Roderiquez, G., and Norcross, M. A. (2006) Dimerization of CXCR4 in living malignant cells: control of cell migration by a synthetic peptide that reduces homologous CXCR4 interactions. Mol. Cancer Ther. 5, 2474–2483.

    Article  PubMed  CAS  Google Scholar 

  23. Hernanz-Falcon, P., Rodriguez-Frade, J. M., Serrano, A., Juan, D., del Sol, A., Soriano, S. F., et al. (2004) Identification of amino acid residues crucial for chemokine receptor dimerization. Nat. Immunol. 5, 216–223.

    Article  PubMed  CAS  Google Scholar 

  24. Wilson, S., Wilkinson, G., and Milligan, G. (2005) The CXCR1 and CXCR2 receptors form constitutive homo- and heterodimers selectively and with equal apparent affinities. J. Biol. Chem. 280, 28663–28674.

    Article  PubMed  CAS  Google Scholar 

  25. Tian, Y., New, D. C., Yung, L. Y., Allen, R. A., Slocombe, P. M., Twomey, B. M., et al. (2004) Differential chemokine activation of CC chemokine receptor 1-regulated pathways: ligand selective activation of Gα14-coupled pathways Eur. J. Immunol. 34, 785–795.

    Article  PubMed  CAS  Google Scholar 

  26. Rodriguez-Frade, J. M., Vila-Coro, A. J., de Ana, A. M., Albar, J. P., Martinez, A. C., and Mellado, M. (1999) The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2. Proc. Natl. Acad. Sci. USA 96, 3628–3633.

    Article  PubMed  CAS  Google Scholar 

  27. Pfleger, K. D., and Eidne, K. A. (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat. Methods 3, 165–174.

    Article  PubMed  CAS  Google Scholar 

  28. Cardullo, R. A. (2007) Theoretical principles and practical considerations for fluorescence resonance energy transfer microscopy. Methods Cell Biol. 81, 479–494.

    Article  PubMed  CAS  Google Scholar 

  29. Boute, N., Jockers, R., and Issad, T. (2002) The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol. Sci. 23, 351–354.

    Article  PubMed  CAS  Google Scholar 

  30. Coulon, V., Audet, M., Homburger, V., Bockaert, J., Fagni, L., Bouvier, M., et al. (2008) Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer. Biophys. J. 94, 1001–1009.

    Article  PubMed  CAS  Google Scholar 

  31. Pfleger, K. D., Seeber, R. M., and Eidne, K. A. (2006) Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Nat. Protoc. 1, 337–345.

    Article  PubMed  CAS  Google Scholar 

  32. McVey, M., Ramsay, D., Kellet, E., Rees, S., Wilson, S., Pope, A. J., et al. (2001) Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminiscence resonance energy transfer. J. Biol. Chem. 276, 14092–14099.

    PubMed  CAS  Google Scholar 

  33. Sekar, R. B., and Periasami, A. (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J. Cell Biol. 160, 629–623.

    Article  PubMed  CAS  Google Scholar 

  34. Pollok, B. A., and Heim, R. (1999) Using GFP in FRET-based applications. Trends Cell. Biol. 9, 57–60.

    Article  PubMed  CAS  Google Scholar 

  35. Periasami, A., Elangovan, M., Elliot, E., and Brautigan, D. L. (2002) Fluorescence lifetime imaging (FLIM) of green fluorescent fusion proteins in living cells. Methods Mol. Biol. 183, 189–200.

    Google Scholar 

  36. Kenworthy, A. K. (2001) Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, 289–96.

    Article  PubMed  CAS  Google Scholar 

  37. Springer, T. A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76, 301–314.

    Article  PubMed  CAS  Google Scholar 

  38. Terrillon, S., and Bouvier, M. (2004) Roles of G-protein-coupled receptor dimerization. EMBO Rep. 5, 30–34.

    Article  PubMed  CAS  Google Scholar 

  39. Rodríguez-Frade JM, del Real G, Serrano A, Hernanz-Falcón P, Soriano SF, Vila-Coro AJ, de Ana AM, Lucas P, Prieto I, Martínez AC, Mellado M. (2004) Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor. EMBO J. 23, 66–76.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank the members of the DIO chemokine group, who contributed to some of the work described in this review. We also thank C. Bastos and C. Mark for secretarial support and helpful editorial assistance, respectively. This work was partially funded by grants from the EU (LSHB-CT-2005-518167 and LSHG–CT-2003-503259), the Spanish Ministry of Science and Innovation (SAF2005-03388), and the Madrid Regional Government. The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (CSIC) and by Pfizer.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press

About this protocol

Cite this protocol

Rodríguez-Frade, J.M., Muñoz, L.M., Holgado, B.L., Mellado, M. (2009). Chemokine Receptor Dimerization and Chemotaxis. In: Jin, T., Hereld, D. (eds) Chemotaxis. Methods in Molecular Biology™, vol 571. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-198-1_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-198-1_12

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-197-4

  • Online ISBN: 978-1-60761-198-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics