Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach.
The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 microM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.
Russwurm
Michael
M
Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Federal Republic of Germany.
Mullershausen
Florian
F
Friebe
Andreas
A
Jäger
Ronald
R
Russwurm
Corina
C
Koesling
Doris
D
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism; D017033:Anabaena / Q000201:enzymology; D001665:Binding Sites; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D011863:Radioimmunoassay; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D015534:Trans-Activators; C067189:fhlA protein, E coli; D017869:Cyclic GMP-Dependent Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D000262:Adenylyl Cyclases; D006152:Cyclic GMP