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Role of cyclic GMP-dependent protein kinase type II in the brain Viswanathan, Vijay


PKG is one of the most important downstream effector of the NO/natriuretic peptides cGMP pathway. Of the two types of PKG, within the brain PKG I is almost exclusively expressed in the cerebellum. By contrast PKG II has a much wider expression in the brain, indicating that it is most likely to be the mediator of cGMP actions. However, subcellular distribution and the function of PKG II in the brain is still unknown. Western blot analysis using a PKG II specific antibody confirmed previous reports that PKG II is highly expressed in the cortex, striatum, hippocampus, hypothalamus and thalamus and absent in the cerebellum. In comparison, we found that PKG I is highly expressed in the cerebellum and hypothalamus and very weakly expressed in the hippocampus and cortex. Western blot analysis of subcellular fractions from adult brain shows that PKG II is present in both the pre and postsynaptic fractions and is enriched in the membrane fractions. PKG II is also enriched in the synaptosomal membrane fraction of embryonic rat brain subcellular fractions. Immunocytochemisty using the PKG II antibody indicates that PKG II is present in both the cell body and processes of cortical, hippocampal and thalamic neurons in culture. PKG II was found to co-localize with both the presynaptic marker, synaptophysin and the postsynaptic marker PSD-95, indicating that PKG II is present at the synapse. Using wild type (wt) and non-myristoylated mutant PKG II proteins it was demonstrated that N-myristoylation (N-myr) is important for targeting of PKG II to membranes and distal ends of filopodia like structures in COS-1 and HEK-293 cells. Full length wt PKG II and wt regulatory PKG JJ proteins, when overexpressed in hippocampal neurons targeted to the synapse, however, a G2A PKG II mutant, which does not undergo myristoylation had a much more diffuse distribution in the entire cell and did not target to the synapse. This indicates that N-myr is important for proper targeting of PKG II. Overexpression of the dominant negative regulatory domain of PKG II in hippocampal neurons caused a 2.5-fold increase in filopodia in young neurons and a 2-fold increase in spine like structures in older neurons compared to GFP overexpressing cells. Such an effect was not observed with the FLwt PKG II. The non-myristoylated forms of PKG II overexpressing cells did not show an increase in filopodia/spine like structures, indicating that proper targeting of PKG II through myristoylation is important for the regulatory domain to have a dominant negative effect. This indicates that PKG II is most likely to play a role in neuronal development and regulation of synaptogenesis.

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