Chemotaxis pp 179-198 | Cite as

Chemokine Receptor Dimerization and Chemotaxis

  • José Miguel Rodríguez-Frade
  • Laura Martinez Muñoz
  • Borja L. Holgado
  • Mario Mellado
Part of the Methods in Molecular Biology™ book series (MIMB, volume 571)


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.

Key words:

Chemokine Chemokine receptor GPCR Dimerization BRET FRET Chemotaxis 



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.


  1. 1.
    Baggiolini, M. (1998) Chemokines and leukocyte traffic. Nature 392, 565–568.PubMedCrossRefGoogle Scholar
  2. 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.PubMedCrossRefGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 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. 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.PubMedCrossRefGoogle Scholar
  6. 6.
    Raz, E. (2003) Primordial germ-cell development: the zebrafish perspective. Nat. Rev. Genet. 4, 690–700.PubMedCrossRefGoogle Scholar
  7. 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.PubMedCrossRefGoogle Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 9.
    Littman, D. R. (1998) chemokine receptors: keys to AIDS pathogenesis? Cell 93, 677–680.PubMedCrossRefGoogle Scholar
  10. 10.
    El-Sawy, T., Fahmy, N. M., and Fairchild, R. L. (2002) Chemokines: directing leukocyte infiltration into allografts. Curr. Opin. Immunol. 14, 562–568.PubMedCrossRefGoogle Scholar
  11. 11.
    Rossi, D., and Zlotnik, A. (2000) The biology of chemokines and their receptors. Annu. Rev. Immunol. 18, 217–242.PubMedCrossRefGoogle Scholar
  12. 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.PubMedGoogle Scholar
  13. 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.PubMedCrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 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.PubMedCrossRefGoogle Scholar
  16. 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.PubMedCrossRefGoogle Scholar
  17. 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.PubMedGoogle Scholar
  18. 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.CrossRefGoogle Scholar
  19. 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.PubMedCrossRefGoogle Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. 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.PubMedCrossRefGoogle Scholar
  22. 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.PubMedCrossRefGoogle Scholar
  23. 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.PubMedCrossRefGoogle Scholar
  24. 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.PubMedCrossRefGoogle Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. 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.PubMedCrossRefGoogle Scholar
  27. 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.PubMedCrossRefGoogle Scholar
  28. 28.
    Cardullo, R. A. (2007) Theoretical principles and practical considerations for fluorescence resonance energy transfer microscopy. Methods Cell Biol. 81, 479–494.PubMedCrossRefGoogle Scholar
  29. 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.PubMedCrossRefGoogle Scholar
  30. 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.PubMedCrossRefGoogle Scholar
  31. 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.PubMedCrossRefGoogle Scholar
  32. 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.PubMedGoogle Scholar
  33. 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.PubMedCrossRefGoogle Scholar
  34. 34.
    Pollok, B. A., and Heim, R. (1999) Using GFP in FRET-based applications. Trends Cell. Biol. 9, 57–60.PubMedCrossRefGoogle Scholar
  35. 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. 36.
    Kenworthy, A. K. (2001) Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, 289–96.PubMedCrossRefGoogle Scholar
  37. 37.
    Springer, T. A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76, 301–314.PubMedCrossRefGoogle Scholar
  38. 38.
    Terrillon, S., and Bouvier, M. (2004) Roles of G-protein-coupled receptor dimerization. EMBO Rep. 5, 30–34.PubMedCrossRefGoogle Scholar
  39. 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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2009

Authors and Affiliations

  • José Miguel Rodríguez-Frade
    • 1
  • Laura Martinez Muñoz
    • 1
  • Borja L. Holgado
    • 1
  • Mario Mellado
    • 1
  1. 1.Department of Immunology and OncologyCentro Nacional de BiotecnologíaMadridSpain

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