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

Abstract

Background and purpose: 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 approach: Using 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 results: The PDE2 ligands identified, 5,6-DM-cBIMP and 5,6-DCl-cBIMP, caused pronounced, up to 40-fold increases of the cAMP- and 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 1µM cGMP after approximately 10min.

Conclusions and implications: We 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.

Figure 4

Kinetics of PDE5 activation. Time courses of PDE activation were obtained by measuring GMP accumulation in cytosolic fractions of PDE5 overexpressing HEK cells at 0.03 µM (A, D, E), 1 µM (B, F) or 100 µM (C) cGMP as substrate either in the absence (A, B, C) or in the presence of 30 µM (D) or 300 µM (E, F) 8-Br-cGMP. The reactions were carried out as described in detail in Methods. Average PDE activity in the indicated time intervals was calculated as described in Methods. The width of a bar depicts the length of the respective interval, the height represents the average enzymatic velocity during the interval and the area of the bar is therefore equivalent to GMP formed during the interval. Data shown are mean ± SD of at least three independent experiments performed in duplicate.

Figure 1

Cyclic nucleotide binding properties of the GAF domains as fluorescence resonance energy transfer (FRET) constructs. Changes in the emission ratio of PDE5 (A) and PDE2 (B) GAF domain FRET constructs induced by cGMP and cAMP were recorded as described in Methods. Data shown are mean ± SD of three independent experiments performed in duplicate.

Figure 2

Stimulation of PDE2 by the GAF domain ligands 5,6-DM-cBIMP and 5,6-DCI-cBIMP. PDE activity was measured in cytosolic fractions of PDE2 overexpressing HEK cells in the presence of increasing concentrations of 5,6-DM-cBIMP (A, D), 5,6-DCI-cBIMP (B, E) or cGMP (C) at the indicated concentrations of cAMP (A–C) and cGMP (D–E) as substrate. The reactions were carried out in 5 min incubations as described in detail in Methods. Data shown are mean ± SD of at least three independent experiments performed in duplicate.

Figure 3

Stimulation of PDE5 by the GAF domain ligand 8-Br-cGMP. (A) PDE5 activity was measured in cytosolic fractions of PDE5 overexpressing HEK cells in the presence of increasing concentrations of 8-Br-cGMP at the indicated concentrations of cGMP as substrate. (B) Activity of a PDE5 construct containing only the catalytic domain was measured at 1 µM cGMP in the presence of increasing concentrations of 8-Br-cGMP or 8-Br-cAMP. The reactions were carried out for 5 min as described in detail in Methods. Data shown are mean ± SD of at least three independent experiments performed in duplicate.

Figure 5

PDE5 activation kinetics: GMP accumulation and statistical analysis. GMP accumulation was measured at the indicated time points in cytosolic fractions of PDE5 overexpressing HEK cells at 0.03 µM (A, D, E), 1 µM (B, F) or 100 µM (C) cGMP as substrate either in the absence (A, B, C) or in the presence of 30 µM (D) or 300 µM (E, F) 8-Br-cGMP. The reactions were carried out as described in detail in Methods. Data are normalized to the GMP value at 240 s to reduce scatter caused by different PDE5 expression levels. Data shown are mean ± SD of at least three independent experiments performed in duplicate. Values for half-life and 95% confidence intervals (in parentheses) were calculated by non-linear regression as described in Methods.

Figure 6

(A) cGMP degradation by PDE2 was measured at the indicated time points in cytosolic fractions of PDE2 overexpressing HEK cells at 1 µM cGMP as substrate. Data were normalized to the GMP value at 240 s. (B) Time course of PDE5 activation was determined in human platelet cytosolic fractions at a substrate concentration of 0.03 µM cGMP as described in Methods and the legend to Figure 4. (C) Statistical analysis of the GMP accumulation determined in platelet cytosolic fractions. Data were normalized to the GMP value at 240 s. Values for half-life and 95% confidence interval (in parentheses) were calculated by non-linear regression as described in Methods. Data shown are mean ± SD of at least three independent experiments performed in duplicate.

Figure 7

Phosphorylated PDE5 shows higher catalytic rates and a slower deactivation. PDE5 was phosphorylated as described in detail in the Methods. (A) Comparison of activity of phosphorylated versus non-phosphorylated PDE5 at the indicated concentrations of cGMP as substrate. The reactions were carried out as described in detail in Methods. P < 0.05 in paired t-test for phospho- versus non-phospho-PDE5 at all substrate concentrations. (B) Aliquots were analysed in Western blots with antibodies specifically detecting phosphorylated PDE5 and antibodies against PDE5 to check loading. The blot shown is representative of four performed with similar results (full lanes of the blots are depicted in Figure S4). cAK, cAMP-dependent protein kinase (C) PDE5 deactivation was monitored by measuring PDE activity of phosphorylated versus non-phosphorylated PDE5 in 10 s incubations at the indicated time points after a 1000-fold dilution to reduce the GAF ligand 8-Br-cGMP to concentrations below 0.3 µM. (D) Data from (C) normalized to the 1 min value. Data shown are mean ± SD of at least three independent experiments performed in duplicate.