PDE5 is converted to an activated state upon cGMP binding to the GAF A domain

Abstract

cGMP-specific, cGMP-binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N-terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time- and concentration-dependent reversible activation in response to cGMP, with the highest activation (9- to 11-fold) observed at low substrate concentrations (0.1 micro M cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non-activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non-activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain-containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.

Fig. 1. Time-dependent activation and reactivation of recombinant mouse PDE5 during pre-incubation with 50 µM cGMP on ice. (A and B) Lysate containing mouse recombinant PDE5 expressed in HEK 293 cells was pre-incubated with 50 µM cGMP on ice without PDE inhibitors (filled triangles, A and B) or with 0.2 µM sildenafil (filled circles, B). At different time points during pre-incubation, aliquots were taken and, after appropriate dilutions, PDE5 activity was assayed with 0.1 µM cGMP for 5 min at 30°C. An additional portion of 50 µM cGMP was added to the pre-incubation mixture at 60 min after the start of pre-incubation as indicated by an arrow (open triangles, A). PDE5 activity was expressed as pmol/min/µg of protein. (C) [³H]cGMP (total 100 000 c.p.m.) was added to the pre-incubation mixture containing 50 µM cGMP without (filled squares) or with 0.2 µM sildenafil (open squares). The percentage 50 µM cGMP hydrolysis at different time points was measured using the PDE5 activity assay.

Fig. 2. Recombinant PDE5 is activated after pre-incubation with cGMP only at low substrate concentration, and is not a result of phosphorylation. (A) After pre-incubation of PDE5 with 50 µM cGMP on ice, an aliquot was diluted and assayed with either 0.1, 1.0 or 10 µM cGMP for 5 min at 30°C. PDE5 activity for each substrate concentration was expressed as fold activation of PDE5 (after pre-incubation with cGMP on ice) (black bars) relative to the control samples (without cGMP pre-incubation) (white bars), which were defined as 1. The data represent three or more different experiments with the mean fold activation ± SD. (B) Samples were prepared in SDS sample buffer before and after pre-incubation with 50 µM cGMP on ice and analyzed by western blot analysis with phospho-PDE5 and total PDE5 antibodies. As a control for phospho-PDE5, the same amount of recombinant PDE5 was phosphorylated in the presence of ATP, PKG and cGMP for 30 min at 30°C.

Fig. 3. PDE5 has only one phosphorylation site: phospho-site mutant PDE5 cannot be phosphorylated by either PKG or PKA in vitro. (A) Recombinant PDE5 (control) and phospho-site mutant PDE5 (mutant) were incubated with either PKG or the catalytic subunit of PKA in the phosphorylation buffer with [³²P]ATP for 60 min at 30°C. After phosphorylation, PDE5 was immunoprecipitated, and the immunoprecipitates were analyzed by SDS–PAGE and then subjected to autoradiography to reveal ³²P incorporation. For western blot analysis, samples were prepared directly after the phosphorylation step. The immunoblots were probed with phospho-PDE5 and total PDE5 antibodies. (B) Phospho-site mutant PDE5 expressed in HEK 293 cells was pre-incubated with 50 µM cGMP on ice and, after appropriate dilutions, assayed with either 0.1, 1.0 or 10 µM cGMP for 5 min at 30°C. Data are expressed as fold activation of PDE5 activity after pre-incubation with cGMP (hatched bars) relative to the control samples (white bars) and defined as in Figure 2A.

Fig. 4. Pre-treatment of PDE5 with mAb P3B2 blocks cGMP binding to the GAF A domain of PDE5. (A) cGMP saturation binding curves of recombinant PDE5 were measured without (filled squares) or with pre-incubation with mAbs P3B2 (filled circles) or P4D8 (filled triangles) for 30 min on ice. The binding assays were performed with 0.01–20 µM cGMP in the presence of 0.1 mM IBMX for 30 min on ice. The specific binding (c.p.m.) is calculated by subtraction of non-specific binding from total binding. (B and C) The epitope for mAb P3B2 lies in the GAF A region of PDE5. Recombinant mouse GAF(A + B), GAF A and GAF B were resolved by 12% SDS–PAGE and then either processed for western blot analysis (B) with mAbs P3B2 and P4D8, or analyzed by silver staining (C).

Fig. 5. PDE5 has a low intrinsic catalytic activity: blocking cGMP binding by mAb P3B2 results in a significant reduction of PDE5 activity. PDE5 activity was measured at 0.1 µM (A) or 10 µM cGMP (B). Samples were analyzed without any treatments (filled squares), after pre-incubation with 50 µM cGMP on ice (filled triangles) or after pre-incubation with mAb P3B2 for 30 min on ice (filled circles). Treatment of PDE5 with mAb P3B2 for 30 min on ice preceded pre-incubation with 50 µM cGMP (open circles). PDE5 activity was expressed as pmol/µg of protein.

Fig. 6. Time-dependent activation of PDE5 during an activity assay. PDE5 activity was measured at 0.1 µM (A) or 1.0 µM (B) cGMP at 30°C without any pre-incubation (filled squares) or after pre-incubation with mAb P3B2 for 20 min on ice (filled circles). PDE5 activity was expressed as pmol/µg of protein.

Fig. 7. PDE5 in an activated state is more sensitive to inhibition by sildenafil and is not affected by further phosphorylation. (A) Non- activated (without any pre-incubations) (filled squares) and activated (after pre-incubation with cGMP on ice) (filled triangles) samples of PDE5 were assayed for 5 min at 0.1 µM in the presence of different concentrations of sildenafil (0.01–250 µM). PDE5 activities, assayed without sildenafil, were expressed as 100%. (B) Activated PDE5 (after pre-incubation with 50 µM cGMP on ice for 15 min and subsequent dilution) was assayed with 0.1 µM cGMP before and after phosphorylation by the catalytic subunit of PKA at 30°C for 20 min. PDE5 activity was expressed as the percentage of the maximum PDE5 activation measured immediately after PDE5 pre-incubation with cGMP on ice and defined as 100%. Corresponding samples were prepared in SDS sample buffer and analyzed by western blot analysis with phospho-PDE5 antibody.

Fig. 8. PDE5 catalytic activity increases after 1–2 weeks of storage on ice accompanied by loss of its ability to be stimulated by cGMP. (A) PDE5 activity was measured at 0.1 µM after 2 weeks of storage on ice (filled squares). Samples were prepared under the same conditions as in Figure 5 and pre-incubated with cGMP (filled triangles), mAb P3B2 (filled circles) or both (open circles). PDE5 activity was expressed as pmol/µg of protein. (B and C) Samples of immunoprecipitates with mAb/P3B2 (B) and the total lysates (C) freshly expressed (1) or stored for 2 weeks on ice (2) were prepared in SDS sample buffer and analyzed by western blotting with total PDE5 antibody.

Fig. 9. PDE5 is activated directly upon cGMP binding to its GAF A domain. Without cGMP bound, PDE5 is in a non-activated state. When bound to cGMP, PDE5 is converted into an activated state. After storage, PDE5 converts to an activated state and loses its ability to be stimulated by cGMP. Although PDE5 is likely to exist as a dimer in all conformational states, in this cartoon, a monomeric structure of PDE5 is shown for reasons of simplification.