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Measuring Spatiotemporal Dynamics of Cyclic AMP Signaling in Real-Time Using FRET-Based Biosensors

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Receptor Signal Transduction Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 746))

Abstract

Cyclic AMP governs many fundamental signaling events in eukaryotic cells. Although cAMP signaling has been a major research focus for a long time, recent technological developments are revealing novel aspects of this paradigmatic pathway. In this chapter, we give an overview over current fluorescence resonance energy transfer (FRET)-based sensors for detection of cAMP dynamics, and their application in monitoring local, compartmentalized cAMP signals within living cells. A basic step-by-step protocol is given for conducting a FRET experiment in primary cells with a unimolecular cAMP sensor, which can easily be adapted to a user’s specific requirements.

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References

  1. Lee, D. C., Carmichael, D. F., Krebs, E. G. and McKnight, G. S. (1983) Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 80, 3608–3612.

    Article  PubMed  CAS  Google Scholar 

  2. Takio, K., Smith, S. B., Krebs, E. G., Walsh, K. A. and Titani, K. (1984) Amino acid sequence of the regulatory subunit of bovine type II adenosine cyclic 3′,5′-phosphate dependent protein kinase. Biochemistry 23, 4200–4206.

    Article  PubMed  CAS  Google Scholar 

  3. Taylor, S. S., Kim, C., Vigil, D., Haste, N. M., Yang, J., Wu, J. and Anand, G. S. (2005) Dynamics of signaling by PKA. Biochim. Biophys. Acta 15754, 25–37.

    Article  PubMed  CAS  Google Scholar 

  4. Wong, W. and Scott, J. D. (2004) AKAP signalling complexes: focal points in space and time. Nat. Rev. Mol. Cell. Biol. 5, 959–970.

    Article  PubMed  CAS  Google Scholar 

  5. Gronholm, M., Vossebein, L., Carlson, C. R., Kuja-Panula, J., Teesalu, T., Alfthan, K., Vaheri, A., Rauvala, H., Herberg, F. W., Tasken, K. and Carpen, O. (2003) Merlin links to the cAMP neuronal signaling pathway by anchoring the RIβ subunit of protein kinase A. J. Biol. Chem. 278, 41167–41172.

    Article  PubMed  Google Scholar 

  6. Miki, K. and Eddy, E. M. (1998) Identification of tethering domains for protein kinase A type Iα regulatory subunits on sperm fibrous sheath protein FSC1. J. Biol. Chem. 273, 34384–34390.

    Article  PubMed  CAS  Google Scholar 

  7. Huang, L. J., Durick, K., Weiner, J. A., Chun, J. and Taylor, S. S. (1997) D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain. Proc. Natl. Acad. Sci. USA 94, 11184–11189.

    Article  PubMed  CAS  Google Scholar 

  8. Huang, L. J., Durick, K., Weiner, J. A., Chun, J. and Taylor, S. S. (1997) Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits. J. Biol. Chem. 272, 8057–8064.

    Article  PubMed  CAS  Google Scholar 

  9. Di Benedetto, G., Zoccarato, A., Lissandron, V., Terrin, A., Li, X., Houslay, M. D., Baillie, G. S. and Zaccolo, M. (2008) Protein kinase A type I and type II define distinct intracellular signaling compartments. Circ. Res. 103, 836–844.

    Article  PubMed  Google Scholar 

  10. Wrighton, K. H. (2009) Sensing second messengers. Nat. Cell Biol. 11, S20–S21.

    Google Scholar 

  11. Steiner, A. L., Kipnis, D. M., Utiger, R. and Parker, C. (1969) Radioimmunoassay for the measurement of adenosine 3′,5′-cyclic phosphate. Proc. Natl. Acad. Sci. USA 64, 367–373.

    Article  PubMed  CAS  Google Scholar 

  12. Kariv, I. I., Stevens, M. E., Behrens, D. L. and Oldenburg, K. R. (1999) High throughput quantitation of cAMP production mediated by activation of seven transmembrane domain receptors. J. Biomol. Screen. 4, 27–32.

    Article  PubMed  CAS  Google Scholar 

  13. Prystay, L., Gagne, A., Kasila, P., Yeh, L. A. and Banks, P. (2001) Homogeneous cell-based fluorescence polarization assay for the direct detection of cAMP. J. Biomol. Screen. 6, 75–82.

    PubMed  CAS  Google Scholar 

  14. Gabriel, D., Vernier, M., Pfeifer, M. J., Dasen, B., Tenaillon, L. and Bouhelal, R. (2003) High throughput screening technologies for direct cyclic AMP measurement. Assay Drug Dev. Technol. 1, 291–303.

    Article  PubMed  CAS  Google Scholar 

  15. Kumar, M., Hsiao, K., Vidugiriene, J. and Goueli, S. A. (2007) A bioluminescent-based, HTS-compatible assay to monitor G-protein-coupled receptor modulation of cellular cyclic AMP. Assay Drug Dev. Technol. 5, 237–245.

    Article  PubMed  CAS  Google Scholar 

  16. Förster, T. (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Annalen der Physik 437, 55–75.

    Article  Google Scholar 

  17. Lakowicz, J. (2006) Energy transfer, in Principles of fluorescence spectroscopy pp 443–471, Springer, New York.

    Google Scholar 

  18. 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 

  19. Adams, S. R., Harootunian, A. T., Buechler, Y. J., Taylor, S. S. and Tsien, R. Y. (1991) Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349, 694–697.

    Article  PubMed  CAS  Google Scholar 

  20. Goaillard, J. M., Vincent, P. V. and Fischmeister, R. (2001) Simultaneous measurements of intracellular cAMP and L-type Ca2+ current in single frog ventricular myocytes. J. Physiol. 530, 79–91.

    Article  PubMed  CAS  Google Scholar 

  21. Zaccolo, M., De Giorgi, F., Cho, C. Y., Feng, L., Knapp, T., Negulescu, P. A., Taylor, S. S., Tsien, R. Y. and Pozzan, T. (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat. Cell Biol. 2, 25–29.

    Article  PubMed  CAS  Google Scholar 

  22. Lissandron, V., Terrin, A., Collini, M., D’Alfonso, L., Chirico, G., Pantano, S. and Zaccolo, M. (2005) Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction. J. Mol. Biol. 354, 546–555.

    Article  PubMed  CAS  Google Scholar 

  23. Zawadzki, K. M. and Taylor, S. S. (2004) cAMP-dependent protein kinase regulatory subunit type IIβ: active site mutations define an isoform-specific network for allosteric signaling by cAMP. J. Biol. Chem. 279, 7029–7036.

    Article  PubMed  CAS  Google Scholar 

  24. Mongillo, M., McSorley, T., Evellin, S., Sood, A., Lissandron, V., Terrin, A., Huston, E., Hannawacker, A., Lohse, M. J., Pozzan, T., Houslay, M. D. and Zaccolo, M. (2004) Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ. Res. 95, 67–75.

    Article  PubMed  CAS  Google Scholar 

  25. de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A. and Bos, J. L. (1998) Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396, 474–477.

    Article  PubMed  Google Scholar 

  26. Bos, J. L. (2003) Epac: a new cAMP target and new avenues in cAMP research. Nat. Rev. Mol. Cell. Biol. 4, 733–738.

    Article  PubMed  CAS  Google Scholar 

  27. Ponsioen, B., Zhao, J., Riedl, J., Zwartkruis, F. J., van der Krogt, G., Zaccolo, M., Moolenaar, W. H., Bos, J. L. and Jalink, K. (2004) Detecting cAMP-induced activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep. 5, 1–5.

    Article  Google Scholar 

  28. DiPilato, L. M., Cheng, X. and Zhang, J. (2004) Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signalling within discrete subcellular compartments. Proc. Natl. Acad. Sci. USA 101, 16513–16518.

    Article  PubMed  CAS  Google Scholar 

  29. De Arcangelis, V., Liu, R., Soto, D. and Xiang, Y. (2009) Differential association of phosphodiesterase 4D isoforms with β2-adrenoceptor in cardiac myocytes. J. Biol. Chem. 284, 33824–33832.

    Article  PubMed  Google Scholar 

  30. Terrin, A., Di Benedetto, G., Pertegato, V., Cheung, Y. F., Baillie, G., Lynch, M. J., Elvassore, N., Prinz, A., Herberg, F. W., Houslay, M. D. and Zaccolo, M. (2006) PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases. J. Cell Biol. 175, 441–451.

    Article  PubMed  CAS  Google Scholar 

  31. Nikolaev, V. O., Bunemann, M., Hein, L., Hannawacker, A. and Lohse, M. J. (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J. Biol. Chem. 279, 37215–37218.

    Article  PubMed  CAS  Google Scholar 

  32. Resh, M. D. (1999) Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451, 1–16.

    Article  PubMed  CAS  Google Scholar 

  33. Roder, I. V., Lissandron, V., Martin, J., Petersen, Y., Di Benedetto, G., Zaccolo, M. and Rudolf, R. (2009) PKA microdomain organisation and cAMP handling in healthy and dystrophic muscle in vivo. Cell. Signal. 21, 819–826.

    Article  PubMed  Google Scholar 

  34. Kenworthy, A. K. (2005) Photobleaching FRET microscopy, in Molecular Imaging: FRET Microscopy and Spectroscopy (Periasamy, A., and Day, R. N., Eds.), Oxford University Press, New York.

    Google Scholar 

  35. Periasamy, A., Elangovan, M., Elliott, E. and Brautigan, D. L. (2002) Fluorescence lifetime imaging (FLIM) of green fluorescent fusion proteins in living cells. Methods Mol. Biol. 183, 89–100.

    PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Institute of Neuroscience and Psychology, College of Medical Veterinary and Life Sciences, Glasgow University, Glasgow, UK

    Manuela Zaccolo

Authors
  1. Frank Gesellchen
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  2. Alessandra Stangherlin
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  3. Nicoletta Surdo
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  4. Anna Terrin
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  5. Anna Zoccarato
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  6. Manuela Zaccolo
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Corresponding author

Correspondence to Manuela Zaccolo .

Editor information

Editors and Affiliations

  1. , Dept. Cell Physiology & Pharmacology, University Leicester, University Road, Leicester, LE1 9HN, United Kingdom

    Gary B. Willars

  2. , Dept. Cell Physiology & Pharmacology, University Leicester, University Road, Leicester, LE1 9HN, United Kingdom

    R.A. John Challiss

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Gesellchen, F., Stangherlin, A., Surdo, N., Terrin, A., Zoccarato, A., Zaccolo, M. (2011). Measuring Spatiotemporal Dynamics of Cyclic AMP Signaling in Real-Time Using FRET-Based Biosensors. In: Willars, G., Challiss, R. (eds) Receptor Signal Transduction Protocols. Methods in Molecular Biology, vol 746. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-126-0_16

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  • DOI: https://doi.org/10.1007/978-1-61779-126-0_16

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-125-3

  • Online ISBN: 978-1-61779-126-0

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