| Abstract No.: | C-B3029 |
| Country: | Canada |
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| Title: | PALMITOYLATION-DEPENDENT SUBCELLULAR TARGETING OF SPLICE VARIANTS OF CGMP-STIMULATED PHOSPHODIESTERASE IN NEURONS AND ITS ROLE IN NO-CGMP SIGNALING |
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| Authors/Affiliations: | 1 Pascale Fretier*; 1 Pam Arstikaitis; 1 David Lin; 1 Alaa El-Husseini; 1 Steven Vincent;
1 Psychiatry, Brain Research Centre, UBC, Vancouver, BC, Canada
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| Content: | Objectives: NO activates soluble guanylyl cyclase to increase intracellular cGMP, and the NO/cGMP signal transduction pathway is thought to play an important role in synaptic plasticity. NO acts via cGMP to regulate ion channels, protein kinases and phosphodiesterases. The cGMP-stimulated phosphodiesterase (PDE2A) is highly expressed in brain, and provides a unique mechanism for cGMP to directly regulate cAMP levels. The objectives of this study were to characterize PDE2A as a target for NO and cGMP signalling in the brain.
Materials and Methods: Immunohistochemistry was used to localize PDE2A in the brain. Molecular cloning identified 3 distinct splice variants of PDE2A and expression of GFP-fusion proteins was used to examine the subcellular localization of the isoforms in neurons. The role of PDE2A in regulating cyclic nucleotide levels in primary neuronal cultures was examined pharmacologically.
Results: We have found using immunohistochemistry that PDE2A is widely expressed in distinct populations of central neurons. In particular it is found in some NOS neurons (mesopontine tegmentum), as well as in neurons thought to be targets of NO signaling (Purkinje cells, striatonigral and striatopallidal neurons, habenulo-interpeduncular neurons). PDE2A is highly expressed in medium spiny striatal neurons, together with the α1β1 soluble guanylyl cyclase NO receptor. These neurons also display a well-characterized dopamine-stimulated adenylyl cyclase, and in primary cultures of striatal neurons we have found that activation of NO producing interneurons can stimulate PDE2A to attenuate dopamine-mediated cAMP signalling. Our RT-PCR analysis shows that PDE2 is expressed as three distinct splice variants in brain, which differ only at the N-terminal. One isoform (PDE2A3) contains a putative myristoylation site (G2) followed by two cysteine residues at positions 5 and 11 that could be palmitoylated. The other two splice variants lack these sites for lipid modification. Using [3H]-labelled palmitate, we found that PDE2A3 is the only palmitoylated splice variant. We next examined whether protein palmitoylation occurs on both cysteines and whether it depends on protein myristoylation. We find C5S or C11S mutants are still palmitoylated, but protein palmitoylation was abolished in the C5S,C11S double mutant and a G2A mutant. These results indicate that both cysteines are palmitoylated and this relies on prior protein myristoylation. We have found via expression of GFP-fusion proteins that one isoform (PDE2A3) is myristoylated and palmitoylated, and that these lipid modifications serve to target the enzyme to presynaptic boutons. In contrast, we have discovered that the non-palmitoylated isoform (PDE2A2) is targeted to neuronal mitochondria via a novel short helical N-terminal domain. This targeting domain is sufficient to target GFP to the mitochondria. Specific siRNA constructs have been developed which can knock-down the individual N-terminal splice variants of PDE2A in cells.
Conclusion: Expression of distinct N-terminal splice variants of PDE2A serves to target this cGMP-stimulated phosphodiesterase to distinct subcellular domains of neurons where NO can regulate cAMP signalling.
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