Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5

Abstract

By controlling intracellular cyclic nucleotide levels, phosphodiesterases (PDE) serve important functions within various signalling pathways. The PDE2 and PDE5 families are allosterically activated by their substrate cGMP via regulatory so-called GAF domains. Here, we set out to identify synthetic ligands for the GAF domains of PDE2 and PDE5. EXPERIMENTAL APPROACHUsing fluorophore-tagged, isolated GAF domains of PDE2 and PDE5, promising cGMP analogues were selected. Subsequently, the effects of these analogues on the enzymatic activity of PDE2 and PDE5 were analysed. KEY RESULTSThe PDE2 ligands identified, 5,6-DM-cBIMP and 5,6-DCl-cBIMP, caused pronounced, up to 40-fold increases of the cAMPand cGMP-hydrolysing activities of PDE2. The ligand for the GAF domains of PDE5, 8-Br-cGMP, elicited a 20-fold GAF-dependent activation and moreover revealed a time-dependent increase in PDE5 activity that occurred independently of a GAF ligand. Although GAF-dependent PDE5 activation was fast at high ligand concentrations, it was slow at physiologically relevant cGMP concentrations; PDE5 reached its final catalytic rates at 1 mM cGMP after approximately 10 min. CONCLUSIONS AND IMPLICATIONSWe conclude that the delayed activation of PDE5 is required to shape biphasic, spike-like cGMP signals. Phosphorylation of PDE5 further enhances activity and conserves PDE5 activation, thereby enabling PDE5 to act as a molecular memory balancing cGMP responses to nitric oxide or natriuretic peptide signals. AbbreviationsFRET, fluorescence resonance energy transfer; GAF, protein domain first identified in cGMP-specific and cGMP

Activation of PDE10 and PDE11 Phosphodiesterases

“…This is in stark contrast to the observations made for PDE10 (see above) and for PDE2 and PDE5 (31). Here, analogue-mediated stimulation is observed only at low concentrations of the physiological nucleotides; higher concentrations of the physiological nucleotides led to stimulation of the enzymes by themselves, thereby masking stimulation by the respective analogue.…”

“…and PDE11 (Ala 174 -Val 580 ) were amplified by PCR and subcloned into pcDNA3 between the open reading frames of CFP and YFP as described previously (29). HEK 293 cells were grown in 75-cm 2 flasks (30) and transfected as described (31). Two or 3 days post-transfection, cells were harvested and lysed by sonication (Branson) in 500 l of buffer (either 50 mM NaCl, 1 mM EDTA, 50 mM triethanolamine/HCl, pH 7.4 for expression of PDE holoenzymes or 25 mM triethanolamine/HCl, pH 7.4 for FRET constructs with both buffers containing 2 mM DTT and mammalian protease inhibitor cocktail (Sigma-Aldrich)).…”

Phosphodiesterase 2: anti-adrenergic friend or hypertrophic foe in heart disease?

“…Intriguingly, PDE2 is stimulated up to 40-fold by cGMP, establishing a potentially important cellular cross talk to the cardioprotective natriuretic peptide and NO systems ( Fig. 1) (Jager et al 2010). We have shown that PDE2 abundance, activity, and transcript levels are upregulated in human and animal models of HF (Mehel et al 2013).…”

Phosphodiesterase 10A Is Tethered to a Synaptic Signaling Complex in Striatum

“…Assay of PDE Activity-PDE activity was measured by the conversion of [ 32 P]cyclic nucleotide monophosphate into [ 32 P]nucleotide monophosphate as described previously (21). Reaction mixtures contained 0.1 l of the striatal or HEK cell homogenates.…”