Abstract
Sensors based on fluorescence resonance energy transfer (FRET) are powerful tools to monitor signaling events in living mammalian cells. Here we describe development and use of new sensors for cyclic GMP (cGMP) based on cGMP binding domains from cGMP-dependent protein kinase I (GKI) and from phosphodiesterases (PDEs). The temporal and spatial resolution attained with the new sensors is superior to that of existing techniques, and permits direct recording and imaging of rapid cGMP-signaling events.
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Acknowledgements
We thank J. Beavo, S. Cawley and W. Dostmann for plasmids, U. Walter and M. Bünemann for advice and discussion, and Bayer HealthCare AG for providing vardenafil. This work was supported by grants from the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG; Leibniz award to M.J.L., Grant WA366 for S.G.) and the Fonds der Chemischen Industrie (German Chemical Industry Fund, FCI; grants to M.J.L.).
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Supplementary information
Supplementary Fig. 1
In vitro characterization of the sensors based on single GAF-domains of PDE2 and PDE5. (PDF 167 kb)
Supplementary Fig. 2
Concentration-response curves of cGES-DE2, cGES-DE5 and Cygnet 2.1 for SNP measured in intact cells. (PDF 95 kb)
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Nikolaev, V., Gambaryan, S. & Lohse, M. Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods 3, 23–25 (2006). https://doi.org/10.1038/nmeth816
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DOI: https://doi.org/10.1038/nmeth816
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