Quantification and Comparison of Signals Generated by Different FRET-Based cAMP Reporters

  • Andreas KoschinskiEmail author
  • Manuela Zaccolo
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)


A variety of FRET-based biosensors are currently in use for real-time monitoring of dynamic changes of intracellular cAMP. Due to differences in sensor properties, unique features of the cell type under examination and diverse specifications of the imaging setups in different laboratories, data generated using these sensors may not be immediately comparable within the same study or across studies. To facilitate comparison, often FRET data are normalized and expressed as fractional change of the maximal FRET response at sensor saturation. However, this approach may lead to misinterpretation of the underlying cAMP change. In this chapter, we provide examples of the problems that may arise when using normalized FRET data and present a method based on the conversion of FRET ratio changes into actual cAMP concentrations that mitigates these issues.

Key words

Fluorescence resonance energy transfer FRET Biosensors cAMP Protein kinase A Real time imaging Intracellular signaling 



This work was supported by the British Heart Foundation (PG/10/75/28537 and RG/17/6/32944) and the BHF Centre of Research Excellence, Oxford (RE/13/1/30181).


  1. 1.
    Förster T (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann Phys 437:55–57. Scholar
  2. 2.
    Stryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47:819–846. Scholar
  3. 3.
    Adams SR, Harootunian AT, Buechler YJ et al (1991) Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349:694–697. Scholar
  4. 4.
    Zaccolo M, De Giorgi F, Cho CY et al (2000) A genetically encoded fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2:25–29. Scholar
  5. 5.
    DiPilato LM, Cheng X, Zhang J (2004) Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc Natl Acad Sci U S A 101(47):16513–16518. Scholar
  6. 6.
    Nikolaev VO, Bünemann M, Hein L et al (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218. Scholar
  7. 7.
    Klarenbeek J, Goedhart J, van Batenburg A et al (2015) Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity. PLoS One 10(4):e0122513. Scholar
  8. 8.
    Di Benedetto G, Zoccarato A, Lissandron V et al (2008) Protein kinase A type I and type II define distinct intracellular signaling compartments. Circ Res 103(8):836–844. Scholar
  9. 9.
    Sprenger JU, Perera RK, Steinbrecher JH et al (2015) In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease. Nat Commun 6:6965. Scholar
  10. 10.
    Surdo NC, Berrera M, Koschinski A et al (2017) FRET biosensor uncovers cAMP nano-domains at β-adrenergic targets that dictate precise tuning of cardiac contractility. Nat Commun 8:15031. Scholar
  11. 11.
    Nikolaev VO, Bünemann M, Schmitteckert E et al (2006) Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β1-adrenergic but locally confined β2-adrenergic receptor-mediated signaling. Circ Res 99:1084–1091. Scholar
  12. 12.
    Herget S, Lohse MJ, Nikolaev VO (2008) Real-time monitoring of phosphodiesterase inhibition in intact cells. Cell Signal 20:1423–1431. Scholar
  13. 13.
    Halls ML, Cooper DMF (2010) Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex. EMBO J 29:2772–2787. Scholar
  14. 14.
    Agarwal SR, Yang PC, Rice M et al (2014) Role of membrane microdomains in compartmentation of cAMP signaling. PLoS One 9(4):e95835. Scholar
  15. 15.
    Boerner S, Schwede F, Schlipp A et al (2011) FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells. Nat Protoc 6:427–438. Scholar
  16. 16.
    Koschinski A, Zaccolo M (2017) Activation of PKA in cell requires higher concentration of cAMP than in vitro: implications for compartmentalization of cAMP signalling. Sci Rep 7(1):14090. Scholar
  17. 17.
    Iancu RV, Ramamurthy G, Warrier S et al (2008) Cytoplasmic cAMP concentrations in intact cardiac myocytes. Am J Physiol Cell Physiol 295(2):C414–C422. Scholar
  18. 18.
    Wachten S, Masada N, Ayling LJ et al (2010) Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived. GH3B6 Cells 123(Pt 1):95–106. Scholar
  19. 19.
    Koschinski A, Zaccolo M (2015) A novel approach combining real-time imaging and the patch-clamp technique to calibrate FRET-based reporters for cAMP in their cellular microenvironment. Methods Mol Biol 1294:25–40. Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Physiology, Anatomy and GeneticsUniversity of OxfordOxfordUK

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