Abstract
BackgroundWhile intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).Methods/Principal FindingsHere we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.ConclusionsThis newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.
Highlights
Cyclic adenosine 39, 59-monophosphate has long been regarded as a ‘‘simple’’ freely diffusible second messenger, wellknown for its ability to modulate multiple cellular functions such as motility, secretion, growth, metabolism, and synaptic plasticity[1,2]
CAMP signals are initiated by the binding of a specific extracellular ligand to a G-protein-coupled receptor (GPCR) that is linked to a heterotrimeric G-protein containing a Gas subunit
We cloned the protein kinase A (PKA)-RIb C-terminus (AA 133–380), purposely omitting the PKA catalytic inhibitory domain located at Nterminus (AA 90–100)
Summary
Cyclic adenosine 39, 59-monophosphate (cAMP) has long been regarded as a ‘‘simple’’ freely diffusible second messenger, wellknown for its ability to modulate multiple cellular functions such as motility, secretion, growth, metabolism, and synaptic plasticity[1,2]. CAMP signals are initiated by the binding of a specific extracellular ligand to a G-protein-coupled receptor (GPCR) that is linked to a heterotrimeric G-protein containing a Gas subunit. The binding of a single ligand to a hormone receptor can set into motion a complex ramifying cascade of signal transduction events that form unpredictable, nonlinear signaling networks[3]. The betagamma subunits of heterotrimeric G-proteins, which dissociate following receptor activation, have their own set of biological activities (e.g. modulation of plasma membrane ion channels). While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP)
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