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).