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 BRET² assays. BRET² ratios of the wild type GAFa fusion protein, but not GAFa_F163A, 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 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 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.