3.4 Functional characterization of the PDE4DIP mutation
We first examined whether the A123T substitution alters the interaction between PDE4D and PDE4DIP, using immunofluorescence staining and specific antibody against PDE4D. Wildtype (WT) and mutant PDE4DIPwere expressed in C2C12 cells and the expression was verified by positive mCherry auto-fluorescent tag. Careful examination showed that upon isoproterenol stimulation, PDE4D and wildtype PDE4DIP show increased colocalization but the co-localization of PDE4D and PDE4DIPpA123T was dramatically reduced (Figure 4).
To assess how the altered interaction between PDE4DIPA123T and PDE4D affects cAMP levels, cells were transfected with a plasmid either containing thePDE4DIPA123T or wild type PDE4DIP . Since cAMP activation occurs at compartmental level within the cells, the effect of PDE4DIPA123T on intracellular cAMP signaling was explored using a fluorescence resonance energy transfer (FRET)-based reporter. Isoproterenol was used to stimulate the endogenous beta-2 adrenergic receptor (β2AR). The FRET sensor imaging showed increased cAMP levels in mutant compared to wildtype transfected cells in response to isoproterenol stimulation (Figure 5).
Examination of the PKA and the and G protein-coupled receptor serine/threonine kinases (GRKs) phosphorylation sites on the β2AR revealed an increase in PKA mediated phosphorylation but no significant difference in β2AR phosphorylation at the GRK residues in cells expressing PDE4DIPA123T compared to wild type PDE4DIP (figure 6). The examination of the PKA phosphorylation of Desmin by immunofluorescent microscopy revealed decreased phosphorylation at its serine 31 site and diminished colocalization of Desmin with PDE4DIP (Figure 7). The reduced phospho-Desmin in the mutants suggests that PDE4DIPA123T mutation causes loss of compartmentalization of both PDE4D and PKA, resulting in increased PKA phosphorylation of β2AR but reduced phosphorylation of Desmin. These findings are highly relevant since inactivation of sarcoplasmic PDE4D and altered Desmin phosphorylation have both been linked to cardiomyopathy and arrhythmias (Beca et al., 2011; Lehnart et al., 2005; Rainer et al., 2018).