The most recently identified cyclic nucleotide phosphodiesterases, PDE10 and PDE11, contain a tandem of so-called GAF domains in their N-terminal regulatory regions. In PDE2 and PDE5, the GAF domains mediate cGMP stimulation; however, their function in PDE10 and PDE11 remains controversial. Although the GAF domains of PDE10 mediate cAMP-induced stimulation of chimeric adenylyl cyclases, cAMP binding did not stimulate the PDE10 holoenzyme. Comparable data about cGMP and the PDE11 GAF domains exist. Here, we identified synthetic ligands for the GAF domains of PDE10 and PDE11 to reduce interference of the GAF ligand with the catalytic reaction of PDE. With these ligands, GAF-mediated stimulation of the PDE10 and PDE11 holoenzymes is demonstrated for the first time. Furthermore, PDE10 is shown to be activated by cAMP, which paradoxically results in potent competitive inhibition of cGMP turnover by cAMP. PDE11, albeit susceptible to GAF-dependent stimulation, is not activated by the native cyclic nucleotides cAMP and cGMP. In summary, PDE11 can be stimulated by GAF domain ligands, but its native ligand remains to be identified, and PDE10 is the only PDE activated by cAMP.

The cGMP-stimulated PDE2A hydrolyzes both cyclic nucleotides, cGMP and cAMP. Three splice variants have been cloned from several species. Whereas PDE2A1 is soluble, PDE2A2 and PDE2A3 are membrane-bound enzymes of rat and bovine origin, respectively. To date it is unclear whether one species expresses all three variants. The splice variants only differ in their N termini, which likely determine the subcellular localization. However, the mechanism for membrane attachment remains unknown. Here, we show that myristoylation underlies membrane targeting of PDE2A3. The myristoylated enzyme was bound to plasma membranes, whereas mutation of the myristoyl recipient Gly2 prevented incorporation of [3H]myristate and turned PDE2A3 completely soluble. Additionally, Cys5 and to a minor extent Cys11 are required for targeting of PDE2A3. Substitution of the putatively palmitoylated cysteines partially solubilized the enzyme and led to an accumulation in the endoplasmic reticulum/Golgi compartment, as shown by fluorescence microscopy in HEK 293 and PC12 cells. In vivo, PDE2A is expressed in many tissues. By using newly generated antibodies selectively detecting the splice variants PDE2A3 or PDE2A1, respectively, we demonstrate on the protein level PDE2A3 expression in mouse brain where it is entirely membrane-associated and a widespread expression of soluble PDE2A1 in mouse tissues. We show that PDE2A localizes to synaptosomal membranes and in primary cultures of hippocampal neurons partially overlaps with the presynaptic marker synaptophysin as demonstrated by immunofluorescence. In sum, these results demonstrate dual acylation as mechanism targeting neuronal PDE2A3 to synapses thereby ensuring local control of cyclic nucleotides.

Phosphodiesterase 10A (PDE10A) is a dual substrate PDE that can hydrolyze both cGMP and cAMP. In brain, PDE10A is almost exclusively expressed in the striatum. In several studies, PDE10A has been implicated in regulation of striatal output using either specific inhibitors or PDE10A knock out mice and has been suggested as promising target for novel antipsychotic drugs. In striatal medium spiny neurons, PDE10A is localized at the plasma membrane and in dendritic spines close to postsynaptic densities. In the present study, we identify PDE10A as the major cAMP PDE in mouse striatum and monitor PKA-dependent PDE10A phosphorylation. With recombinantly expressed PDE10A we demonstrate that phosphorylation does not alter PDE10A activity. In striatum, PDE10A was found to be associated with the A kinase anchoring protein AKAP150 suggesting the existence of a multiprotein signaling complex localizing PDE10A to a specific functional context at synaptic membranes. Furthermore, the cAMP effector PKA, the NMDA receptor subunits NR2A and B as well as PSD95 were tethered to the complex. In agreement, PDE10A was almost exclusively found in multiprotein complexes as indicated by migration in high molecular weight fractions in size exclusion chromatography. Finally, affinity of PDE10A to the signaling complexes formed around AKAP150 was reduced by PDE10A phosphorylation. The data indicate that phosphorylation of PDE10 has an impact on the interaction with other signaling proteins and adds an additional line of complexity to the role of PDE10 in regulation of synaptic transmission.

Objective—: In the vascular system, cyclic GMP (cGMP) in smooth muscle cells plays an important role for blood vessel relaxation. Intracellular concentrations of cGMP are thought to be determined by the action of cGMP-generating guanylyl cyclases (sensitive to nitric oxide or natriuretic peptides) and cGMP-degrading phosphodiesterases (PDE1, PDE3, and PDE5). Because functionally relevant cGMP elevations are not accessible to conventional methods, we applied real-time imaging with a fluorescence resonance energy transfer (FRET)-based cGMP indicator to follow nitric oxide– and natriuretic peptide–induced cGMP signals in living smooth muscle cells and analyzed the contribution of the miscellaneous cGMP-generating and cGMP-degrading enzymes. Approach and Results—: By comparison of cGMP signals in living smooth muscle cells and vascular relaxation of aortic strips in organ bath experiments, we show for the first time that FRET-based cGMP indicators permit the measurement of functionally relevant cGMP signals. PDE5 was the major cGMP phosphodiesterase responsible for reducing nitric oxide– and natriuretic peptide–induced cGMP signals. In contrast, PDE3—involved in the degradation of lower cGMP concentrations—displayed a preference for natriuretic peptide–stimulated cGMP. Unexpectedly, we found that cGMP is transported out of the cells by the ABC transporter multidrug resistance–associated protein 4 and this export turned out to be of similar importance for intracellular cGMP signals as degradation by PDE5. Functionally, inhibition of cGMP export enhanced vascular relaxation as much as inhibition of PDE5. Conclusions—: The findings indicate that cGMP export out of smooth muscle cells is a key player in the regulation of smooth muscle cGMP signals and blood vessel relaxation.

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 1 µM cGMP after approximately 10 min.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.