The GAF Domain of the cGMP-Binding, cGMP-Specific Phosphodiesterase (PDE5) Is a Sensor and a Sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFaF163A) unable to bind cGMP w...

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Bibliographic Details
Published in:Biochemistry (Easton) 2008-03, Vol.47 (11), p.3534-3543
Main Authors: Biswas, Kabir Hassan, Sopory, Shailaja, Visweswariah, Sandhya S.
Format: Article
Language:English
Subjects:
Biosensing Techniques
Cell Line
Cyclic AMP - chemistry
Cyclic GMP - chemistry
Cyclic GMP - metabolism
Cyclic Nucleotide Phosphodiesterases, Type 5 - chemistry
Cyclic Nucleotide Phosphodiesterases, Type 5 - metabolism
Energy Transfer
Humans
Intracellular Fluid - enzymology
Luminescence
Molecular Sequence Data
Protein Binding - genetics
Protein Structure, Tertiary - genetics
Signal Transduction - genetics
Signal Transduction - physiology
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Summary:We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFaF163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET2 assays. BRET2 ratios of the wild type GAFa fusion protein, but not GAFaF163A, increased in the presence of cGMP but not cAMP. Higher basal BRET2 ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFaF163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET2 technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET2 sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi702025w