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Abstract
Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by Gs- and Gi/o-proteins, is a central issue in biomedical research. The classical biochemical method to monitor increases in intracellular cAMP concentrations consists of a radioactive multicellular assay, which is well established, highly sensitive, and reproducible, but precludes continuous spatial and temporal assessment of cAMP levels in single living cells. For this purpose, Förster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor Gs-induced cAMP increases and it has remained elusive whether Epac sensors can reliably detect decreased intracellular cAMP levels as well. In this study, we systematically optimize experimental strategies employing FRET-based cAMP sensors to monitor Gi/o-mediated cAMP reductions. FRET experiments with adrenergic α2A or μ opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon Gi/o-coupled receptor activation in single living cells, and this effect can be reversed by selective receptor antagonists. Moreover, pre-treatment with forskolin or 3-isobutyl-1-methylxanthine (IBMX) to artificially increase basal cAMP levels was not required to monitor Gi/o-coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.
Highlights
G-protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that transduce extracellular signals into cellular responses by activating intracellular signal transduction pathways
Optimized Förster resonance energy transfer (FRET)-based cAMP sensors are effective in monitoring Gs-mediated cAMP level increases To investigate whether exchange proteins directly activated by cAMP (Epac)-based cAMP sensors can be employed to detect cAMP decreases, we used a set of different Epac sensors: four sensors which are well-established to monitor Gsprotein-mediated cAMP increases (YFP-Epac1-cyan fluorescent protein (CFP), YFPEpac2-CFP, H74, and H187) and two constructs with modified fluorochromes generated by us which are based on the original Epac1 and Epac2 constructs
The summary of FRET signal amplitudes shows that the FRET pair yellow fluorescent protein (YFP)/CFP was more efficacious than the FRET pair mVenus/mTurquoise2 (Fig. 1g)
Summary
G-protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that transduce extracellular signals into cellular responses by activating intracellular signal transduction pathways. The two best characterized families of effector enzymes regulated by GPCRs are phospholipases C (PLC) and adenylyl cyclases (ACs). Several members of the former enzyme family are activated by Gq/11-proteins. Intracellular cAMP levels can either be elevated as a consequence of Gsprotein-dependent activation of ACs or decreased following engagement of Gi/o-protein-coupled receptors resulting in inhibition of AC [12]. The second messenger cAMP is tightly regulated by Gs- and Gi/o-coupled receptors
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