Corina Russwurm

Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 microM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

Dual Acylation of PDE2A Splice Variant 3: TARGETING TO SYNAPTIC MEMBRANES*

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.

Activation of PDE10 and PDE11 Phosphodiesterases

Background: The cyclic nucleotide phosphodiesterases PDE10 and PDE11 contain putatively regulatory GAF domains with unknown function. Results: Synthetic GAF domain ligands can activate both PDEs. Conclusion: PDE10 is activated by cAMP, whereas the physiological ligand of the PDE11 GAF domains remains unknown. Significance: This is the first demonstration of a functional role of the PDE10 and PDE11 GAF domains. 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.

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

Background: In striatum, cortical glutamatergic and midbrain dopaminergic inputs are integrated via cAMP. Results: PDE10A, the major cAMP-hydrolyzing enzyme in striatum, is targeted into a signaling complex containing the scaffolding proteins AKAP150, PSD95, and the NMDA receptor and released upon phosphorylation. Conclusion: Targeting of PDE10 is under control of cAMP/PKA activity. Significance: Phosphorylation-dependent release of PDE10 gives rise to a feed forward mechanism. 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 a 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.

NO/cGMP: The Past, the Present, and the Future

The NO/cGMP signalling cascade participates in the regulation of physiological parameters such as smooth muscle relaxation, inhibition of platelet aggregation, and neuronal transmission. cGMP is formed in response to nitric oxide (NO) by NO-sensitive guanylyl cyclases that exist in two isoforms (NO-GC1 and NO-GC2). Much has been learned about the regulation of NO-GC; however the precise role of cGMP in complex physiological and especially in pathophysiological settings and its alteration by biological factors needs to be established. Despite reports on a variety of cGMP-independent NO effects, KO mice with a complete lack of NO-GC provide evidence that the vasorelaxing and platelet-inhibiting effects of NO are solely mediated by NO-GC. Isoform-specific KOs demonstrate that low cGMP increases are sufficient to induce smooth muscle relaxation and that either NO-GC isoform is sufficient in most instances outside the central nervous system. In the neuronal system, however, the NO-GC isoforms obviously serve distinct functions as both isoforms are required for long-term potentiation and NO-GC1 was shown to enhance glutamate release in excitatory neurons in the hippocampal CA1 region by gating HCN channels. Future studies have to clarify the role of NO-GC2, to show whether HCN channels are general targets of cGMP in the nervous system and whether the NO/cGMP signalling cascade participates in synaptic transmission in other brain regions.

Activation of PDE10 and PDE11

Phosphodiesterases (PDEs) are critically involved in the determination of intracellular cyclic nucleotide levels. PDEs are heterodimers containing a conserved C-terminal catalytic domain and an N-terminal regulatory domain. Within their regulatory domain, 5 PDE families contain GAF domains, which are potential cyclic nucleotide binding domains. cGMP is known to activate PDEs 2 and 5. Also PDE10 and 11 contain GAF domains in their N termini. However, the functional role of these domains remains a matter of debate. In chimeric constructs of PDE GAF domains and cyanobacterial adenylyl cyclase catalytic domains, cAMP and cGMP activated constructs containing the PDE10 and PDE11 GAF domains, respectively. On the other hand, binding of cAMP and cGMP was claimed not to activate the PDE10 and PDE11 holoenzymes. Here we used synthetic ligands identified by fluorescence resonance energy transfer (FRET)-based analysis of isolated GAF domains to demonstrate that PDE10 and 11 holoenzymes are activated by their GAF domains. Furthermore, we show that PDE10 is activated by cAMP and that PDE11 albeit sensitive to synthetic GAF ligands is not activated by the physiological nucleotides cGMP and cAMP.

In striatum phosphodiesterase 10A is part of a synaptic signalling complex

Background A number of neurological disorders (i.e. Schizophrenia and Parkinson’s disease) that result from dysfunction of striatal signal transduction pathways underline the importance of the striatum for motor function and procedural learning. Most striatal neurons are medium spiny neurons (MSN) that receive input via dopaminergic and glutamatergic terminals and project to the basal ganglia. The MSN express dopamine receptors that are either positively (D1) or negatively (D2) coupled to adenylyl cyclase therefore dopamine directly influences intracellular levels of cAMP. Participants in cAMP mediated signalling pathways are often organized in multiprotein complexes around A-kinase anchoring proteins (AKAPs). The proximity of the cyclases and phosphodiesterases (PDEs) as well as protein kinase A ensures fine tuning of cAMP levels in a given compartment and allows for selective regulation of target proteins such as NMDA or AMPA receptors by phosphorylation.

Comparative analysis of PDE10A isoforms

Phosphodiesterases (PDE) are considered to be key players in many signal transduction pathways. They degrade the second messengers cGMP and cAMP, thereby controlling their intracellular levels. So far, eleven PDE families, typically having several isoforms and splice variants are known. The PDE10 family is encoded by one gene that gives rise to at least two different isoforms in humans: the soluble PDE10A1 and the membrane associated PDE10A2. Distribution studies in human and mouse tissues showed highest expression in medium spiny neurons of the striatum and peripherally in testis. The localization of PDE10A in medium spiny neurons has led to much attention on PDE10 as a potential therapeutic target for novel antipsychotics. In neurons, membrane targeting of PDE10A2 is regulated at least in part by PKA-dependent phosphorylation of Thr16. On the other hand, little attention has been paid to PDE10A1 and the regulation of both isoforms. Here, we compared PDE10A isoforms with regard to their tissue distribution, subcellular localization and biochemical properties.

GAF domain-induced activation of phosphodiesterases 2 and 5.

The second messenger cGMP is involved in several physiological functions such as smooth muscle relaxation and inhibition of platelet aggregation. Besides cGMP synthesis, hydrolysis of cGMP by cyclic nucleotide phosphodiesterases (PDEs) determines the shape of cGMP signals. Eleven PDE families are known differing in regulation and cAMP/cGMP specificity. PDEs are homodimers; each monomer being composed of different N-terminal regulatory domains and a C-terminal catalytic domain highly conserved between the PDE families. Five PDEs contain a tandem of so called GAF domains in their N termini that have been shown to bind cGMP or cAMP. At least in PDEs 2, 5 and 6, binding of cGMP to the GAF domains leads to stimulation of the enzymes. Here, we focussed on the GAF-mediated stimulation of PDEs 2 and 5.

The neuronal PDE2A3 splice variant is targeted to membranes via dual acylation

Background The cyclic nucleotide second messengers cAMP and cGMP play key roles in mammalian signal transduction. Their intracellular levels are tightly controlled by the rate of synthesis and degradation. Hydrolysis of the cyclic nucleotides is dependent on the activity of phosphodiesterases (PDE) of which to date 11 families have been identified. The cGMP-stimulated cGMP/cAMP phosphodiesterase 2 is encoded by one gene, which has been shown to be spliced into three variants that have been cloned from different species. Besides the ubiquitous soluble PDE2A1, two membrane associated forms, PDE2A2 and PDE2A3, have been described. The PDE2A splice variants only differ in short stretches of amino acids at their very N terminus. This region has previously been suggested to determine the subcellular localization of the enzyme, but data about the mechanism underlying membrane attachment of PDE2A3 are lacking.