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Fluorescence Resonance Energy Transfer to Study Receptor Dimerization in Living Cells

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Receptor Signal Transduction Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 259))

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Abstract

The versatility, sensitivity, and feasibility of fluorescence methods are very attractive to study protein-protein interaction at low levels of protein expression. However, one of the most severe limits in protein chemistry has been the difficulty of introducing site-specific fluorescent labels. The development of genetically encoded fluorescent probes, that is, green fluorescent protein (GFP) and its variants therefore opened up a broad field of novel applications. To characterize protein-protein interactions and determine detailed spatio-temporal dynamics of partners that are molecularly well characterized, fluorescence energy transfer methods are excellent nondestructive tools in living cells. Cellular responses to external factors are extensively based on direct molecular interaction and especially G-protein-coupled receptors (GPCRs) have been shown to interact with an unexpected level of complexity. Classical models of signal transduction describe GPCRs as monomeric proteins, while recent studies using fluorescence resonance energy transfer (FRET) and other methods show that GPCRs can also function as homo- or heterodimers. Theoretical background information on FRET technology and its diverse applications are summarized here. A detailed description of a spectroscopic method for FRET studies in the field of GPCR interaction is presented to facilitate and propagate studies to increase our understanding of protein-protein interactions involving GPCRs.

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References

  1. van Roessel, P. and Brand, A. H. (2002) Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins. Nat. Cell Biol. 4, E15–E20.

    Article  PubMed  Google Scholar 

  2. Hovius, R., Vallotton, P., Wohland, T., and Vogel, H. (2000) Fluorescence techniques: shedding light on ligand-receptor interactions. Trends Pharmacol. Sci. 21, 266–273.

    Article  PubMed  CAS  Google Scholar 

  3. Wu, P. and Brand, L. (1994) Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1–13.

    Article  PubMed  CAS  Google Scholar 

  4. Selvin, P. R. (1995) Fluorescence resonance energy transfer. Methods Enzymol. 246, 300–334.

    Article  PubMed  CAS  Google Scholar 

  5. Day, R. N., Periasamy, A., and Schaufele, F. (2001) Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. Methods 25, 4–18.

    Article  PubMed  CAS  Google Scholar 

  6. Förster, T. (1946) Energy transfer and fluorescence (in German). Naturwissenschaften 6, 166–175.

    Article  Google Scholar 

  7. Förster, T. (1948) Intermolecular energy transfer and fluorescence (in German). Ann. Physik 2, 55–75.

    Article  Google Scholar 

  8. Patterson, G. H., Piston, D. W., and Barisas, B. G. (2000) Forster distances between green fluorescent protein pairs. Anal. Biochem. 284, 438–440.

    Article  PubMed  CAS  Google Scholar 

  9. Clegg, R. M. (1995) Fluorescence resonance energy transfer. Curr. Opin. Biotechnol. 6, 103–110.

    Article  PubMed  CAS  Google Scholar 

  10. Overton, M. C. and Blumer, K. J. (2002) Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast. Methods 27, 324–332.

    Article  PubMed  CAS  Google Scholar 

  11. Patel, R. C., Lange, D. C., and Patel, Y. C. (2002) Photobleaching fluorescence resonance energy transfer reveals ligand-induced oligomer formation of human somatostatin receptor subtypes. Methods 27, 340–348.

    Article  PubMed  CAS  Google Scholar 

  12. Harpur, A. G., Wouters, F. S., and Bastiaens, P. I. (2001) Imaging FRET between spectrally similar GFP molecules in single cells. Nat. Biotechnol. 19, 167–169.

    Article  PubMed  CAS  Google Scholar 

  13. Wouters, F. S. and Bastiaens, P. I. (1999) Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells. Curr. Biol. 9, 1127–1130.

    Article  PubMed  CAS  Google Scholar 

  14. Clegg, R. M. (1992) Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 211, 353–388.

    Article  PubMed  CAS  Google Scholar 

  15. Cornea, A., Janovick, J. A., Maya-Nunez, G., and Conn, P. M. (2001) Gonadotropin-releasing hormone receptor microaggregation. Rate monitored by fluorescence resonance energy transfer. J. Biol. Chem. 276, 2153–2158.

    Article  PubMed  CAS  Google Scholar 

  16. Gordon, G. W., Berry, G., Liang, X. H., Levine, B., and Herman, B. (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702–2713.

    Article  PubMed  CAS  Google Scholar 

  17. Sorkin, A., McClure, M., Huang, F., and Carter, R. (2000) Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy. Curr. Biol. 10, 1395–1398.

    Article  PubMed  CAS  Google Scholar 

  18. Tadrous, P. J. (2000) Methods for imaging the structure and function of living tissues and cells: 2. Fluorescence lifetime imaging. J. Pathol. 191, 229–234.

    Article  PubMed  CAS  Google Scholar 

  19. Emptage, N. J. (2001) Fluorescent imaging in living systems. Curr. Opin. Pharmacol. 1, 521–525.

    Article  PubMed  CAS  Google Scholar 

  20. Tadrous, P. J. (2000) Methods for imaging the structure and function of living tissues and cells: 3. Confocal microscopy and micro-radiology. J. Pathol. 191, 345–354.

    Article  PubMed  CAS  Google Scholar 

  21. Haraguchi, T., Shimi, T., Koujin, T., Hashiguchi, N., and Hiraoka, Y. (2002) Spectral imaging fluorescence microscopy. Genes Cells 7, 881–887.

    Article  PubMed  CAS  Google Scholar 

  22. Shimomura, O., Johnson, F. H., and Saiga, Y. (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59, 223–239.

    Article  CAS  Google Scholar 

  23. Shimomura, O., Johnson, F. H., and Saiga, Y. (1963) Further data on the bioluminescent protein, aequorin. J. Cell. Physiol. 62, 1–8.

    PubMed  CAS  Google Scholar 

  24. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233.

    Article  PubMed  CAS  Google Scholar 

  25. Tsien, R. Y. (1998) The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544.

    Article  PubMed  CAS  Google Scholar 

  26. Matz, M. V., Fradkov, A. F., Labas, Y. A., et al. (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973.

    Article  PubMed  CAS  Google Scholar 

  27. Wiehler, J., von Hummel, J., and Steipe, B. (2001) Mutants of Discosoma red fluorescent protein with a GFP-like chromophore. FEBS Lett. 487, 384–389.

    Article  PubMed  CAS  Google Scholar 

  28. Bevis, B. J. and Glick, B. S. (2002) Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20, 83–87.

    Article  PubMed  CAS  Google Scholar 

  29. Mizuno, H., Sawano, A., Eli, P., Hama, H., and Miyawaki, A. (2001) Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. Biochemistry 40, 2502–2510.

    Article  PubMed  CAS  Google Scholar 

  30. Campbell, R. E., Tour, O., Palmer, A. E., et al. (2002) A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  32. Devi, L. A. (2000) G-protein-coupled receptor dimers in the lime light. Trends Pharmacol. Sci. 21, 324–326.

    Article  PubMed  CAS  Google Scholar 

  33. Eidne, K. A., Kroeger, K. M., and Hanyaloglu, A. C. (2002) Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells. Trends Endocrinol. Metabol. 13, 415–421.

    Article  CAS  Google Scholar 

  34. Dean, M. K., Higgs, C., Smith, R. E., et al. (2001) Dimerization of G-protein-coupled receptors. J. Med. Chem. 44, 4595–4614.

    Article  PubMed  CAS  Google Scholar 

  35. Overton, M. C. and Blumer, K. J. (2000) G-protein-coupled receptors function as oligomers in vivo. Curr. Biol. 10, 341–344.

    Article  PubMed  CAS  Google Scholar 

  36. Devi, L. A. (2001) Heterodimerization of G-protein-coupled receptors: pharmacology, signaling and trafficking. Trends Pharmacol. Sci. 22, 532–537.

    Article  PubMed  CAS  Google Scholar 

  37. Maggio, R., Barbier, P., Colelli, A., Salvadori, F., Demontis, G., and Corsini, G. U. (1999) G protein-linked receptors: pharmacological evidence for the formation of heterodimers. J. Pharmacol. Exp. Ther. 291, 251–257.

    PubMed  CAS  Google Scholar 

  38. Jordan, B. A. and Devi, L. A. (1999) G-protein-coupled receptor heterodimerization modulates receptor function. Nature 399, 697–700.

    Article  PubMed  CAS  Google Scholar 

  39. Milligan, G. (2001) Oligomerisation of G-protein-coupled receptors. J. Cell. Sci. 114, 1265–1271.

    PubMed  CAS  Google Scholar 

  40. Edwards, S. W., Tan, C. M., and L. E., L. (2000) Localisation of G-protein-coupled receptors in health and disease. Trends Pharmacol. Sci. 21, 304–308.

    Article  PubMed  CAS  Google Scholar 

  41. Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M. (2000) Detection of β 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684–3689.

    Article  PubMed  CAS  Google Scholar 

  42. Hebert, T. E., Moffett, S., Morello, J. P., et al. (1996) A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J. Biol. Chem. 271, 16,384–16,392.

    Article  PubMed  CAS  Google Scholar 

  43. Rocheville, M., Lange, D. C., Kumar, U., Sasi, R., Patel, R. C., and Patel, Y. C. (2000) Subtypes of the somatostatin receptor assemble as functional homo-and heterodimers. J. Biol. Chem. 275, 7862–7869.

    Article  PubMed  CAS  Google Scholar 

  44. Cvejic, S. and Devi, L. A. (1997) Dimerization of the delta opioid receptor: implication for a role in receptor internalization. J. Biol. Chem. 272, 26,959–26,964.

    Article  PubMed  CAS  Google Scholar 

  45. Gomes, I., Jordan, B. A., Gupta, A., Trapaidze, N., Nagy, V., and Devi, L. A. (2000) Heterodimerization of μ and δ opioid receptors: a role in opiate synergy. J. Neurosci. 20, RC110.

    PubMed  CAS  Google Scholar 

  46. Jordan, B. A., Trapaidze, N., Gomes, I., Nivarthi, R., and Devi, L. A. (2001) Oligomerization of opioid receptors with β 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation. Proc. Natl. Acad. Sci. USA 98, 343–348.

    Article  PubMed  CAS  Google Scholar 

  47. Robbins, M. J., Ciruela, F., Rhodes, A., and McIlhinney, R. A. (1999) Characterization of the dimerization of metabotropic glutamate receptors using an N-terminal truncation of mGluR1α. J. Neurochem. 72, 2539–2547.

    Article  PubMed  CAS  Google Scholar 

  48. Cornea, A. and Michael Conn, P. (2002) Measurement of changes in fluorescence resonance energy transfer between gonadotropin-releasing hormone receptors in response to agonists. Methods 27, 333–339.

    Google Scholar 

  49. Dinger, M. C., Bader, J. E., Kobor, A. D., Kretzschmar, A. K., and Beck-Sickinger, A. G. (2003) Homodimerization of neuropeptide Y receptors investigated by fluorescence resonance energy transfer in living cells. J. Biol. Chem. 278, 10,362–10,572.

    Article  Google Scholar 

  50. Tatemoto, K., Carlquist, M., and Mutt, V. (1982) Neuropeptide Y—a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296, 659–660.

    Article  PubMed  CAS  Google Scholar 

  51. Ganten, D., Paul, M., and Lang, R. E. (1991) The role of neuropeptides in cardiovascular regulation. Cardiovasc. Drugs Ther. 5, 119–130.

    Article  PubMed  CAS  Google Scholar 

  52. Gerald, C., Walker, M. W., Criscione, L., et al. (1996) A receptor subtype involved in neuropeptide-Y-induced food intake. Nature 382, 168–171.

    Article  PubMed  CAS  Google Scholar 

  53. Flood, J. F. and Morley, J. E. (1989) Dissociation of the effects of neuropeptide Y on feeding and memory: evidence for pre-and postsynaptic mediation. Peptides 10, 963–966.

    Article  PubMed  CAS  Google Scholar 

  54. Vezzani, A., Civenni, G., Rizzi, M., Monno, A., Messali, S., and Samanin, R. (1994) Enhanced neuropeptide Y release in the hippocampus is associated with chronic seizure susceptibility in kainic acid treated rats. Brain Res. 660, 138–143.

    Article  PubMed  CAS  Google Scholar 

  55. Michel, M. C., Beck-Sickinger, A., Cox, H., et al. (1998) XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol. Rev. 50, 143–150.

    PubMed  CAS  Google Scholar 

  56. Cabrele, C. and Beck-Sickinger, A. G (2000) Molecular characterization of the ligand-receptor interaction of the neuropeptide Y family. J. Pept. Sci. 6, 97–122.

    Article  PubMed  CAS  Google Scholar 

  57. Daniels, A. J., Matthews, J. E., Slepetis, R. J., et al. (1995) High-affinity neuropeptide Y ceceptor antagonists. Proc. Natl. Acad. Sci. USA 92, 9067–9071.

    Article  PubMed  CAS  Google Scholar 

  58. Matthews, J. E., Jansen, M., Lyerly, D., et al. (1997) Pharmacological characterization and selectivity of the NPY antagonist GR231118 (1229U91) for different NPY receptors. Regul. Pept. 72, 113–119.

    Article  PubMed  CAS  Google Scholar 

  59. CLONTECH (2003) Features of Living Colors™ vectors. http://www.clontech.com/.

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Author information

Authors and Affiliations

  1. Institute of Biochemistry, University of Leipzig, Leipzig, Germany

    Jürgen E. Bader & Annette G. Beck-Sickinger

Authors
  1. Jürgen E. Bader
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  2. Annette G. Beck-Sickinger
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Editors and Affiliations

  1. Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK

    Gary B. Willars  & R. A. John Challiss  & 

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© 2004 Humana Press Inc.

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Bader, J.E., Beck-Sickinger, A.G. (2004). Fluorescence Resonance Energy Transfer to Study Receptor Dimerization in Living Cells. In: Willars, G.B., Challiss, R.A.J. (eds) Receptor Signal Transduction Protocols. Methods in Molecular Biology, vol 259. Humana Press. https://doi.org/10.1385/1-59259-754-8:335

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  • DOI: https://doi.org/10.1385/1-59259-754-8:335

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-329-9

  • Online ISBN: 978-1-59259-754-3

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