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 (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. 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 BRET (2) 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-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.
Biswas
Kabir Hassan
KH
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Sopory
Shailaja
S
Visweswariah
Sandhya S
SS
D015374:Biosensing Techniques; D002460:Cell Line; D000242:Cyclic AMP / Q000737:chemistry; D006152:Cyclic GMP / Q000737:chemistry* / Q000378:metabolism*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000737:chemistry* / Q000378:metabolism*; D004735:Energy Transfer; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology; D049449:Luminescence; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
D000242:Cyclic AMP; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D006152:Cyclic GMP