Abstract
Phosphodiesterases (PDEs) are hydrolytic enzymes, which convert cyclic
AMP (cAMP) and cyclic GMP (cGMP) into their corresponding monophosphates.
PDE-dependent hydrolysis shape gradients of these second messengers
in cells, which may form the basis of their compartmentation and
play a key role in a vast number of physiological and pathological
processes. Here, we present a novel approach for real-time monitoring
of local cAMP and cGMP levels associated with particular PDEs. We
used HEK 293 cells expressing genetic constructs encoding a PDE of
interest (PDE3A, PDE4A1 or PDE5A) fused to cAMP and cGMP sensors,
which allow to directly visualize changes in cyclic nucleotide concentrations
in the vicinity of PDE molecules by fluorescence resonance energy
transfer (FRET). FRET was detected by imaging of single cells on
96-well plates and demonstrated specific effects of PDE inhibitors
on local cyclic nucleotide levels. In addition, this approach reported
physiological regulation of PDE3A activity, its activation by PKA-dependent
phosphorylation and inhibition by cGMP. In conclusion, our assay
provides a unique and highly sensitive method to analyze PDE activity
in living cells. It allows to sense cAMP gradients around particular
PDE molecules and to study the pharmacological effects of selective
inhibitors on localized cAMP signalling.
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