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

Abstract

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.

A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

“…We have earlier suggested that GAF domains can act as sinks for cGMP, in the absence of an associated effector domain (66). The USPs that we have characterized, being devoid of an associated effector domain, perhaps represent the first example of a "bank" for cAMP, where the currency is released depending on the needs of the organism.…”

Functional Characterization of Core Components of the Bacillus subtilis Cyclic-Di-GMP Signaling Pathway

“…6E and F). We conclude that sensory inputs may well modulate the stimulatory activity of the GAF and PAS domains of DgcP and DgcW, respectively, in the cell (43)(44)(45)(46)(47)(48)(49)(50)(51)(52).…”

“…GAF domains have been shown to regulate enzymes, including histidine kinases and diguanylate cyclases, in response to cGMP, light, and oxygen levels (42)(43)(44)(45)(46). PAS domains similarly use cofactors to sense environmental cues including light or oxygen in the larger context of multidomain proteins (42,(47)(48)(49)(50)(51)(52).…”

Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases

“…have recently utilized BRET as a means of monitoring conformational changes in proteins that interact with cyclic nucleotides (31,41). BRET is based on energy transfer between luminescence donor and fluorescent acceptor proteins (e.g.…”

“…We used a fusion protein construct of KATms inserted between GFP 2 and luciferase ( Fig. 1A) (41). The addition of cAMP to lysates expressing the KATms BRET sensor led to a significant increase in BRET ratio (Fig.…”