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Open AccessArticle

Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging

1
Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany
2
Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany
3
DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Martinistr. 52, D-20246 Hamburg, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(21), 7880; https://doi.org/10.3390/ijms21217880
Received: 22 September 2020 / Revised: 17 October 2020 / Accepted: 19 October 2020 / Published: 23 October 2020
G-protein coupled cannabinoid CB2 receptor signaling and function is primarily mediated by its inhibitory effect on adenylate cyclase. The visualization and monitoring of agonist dependent dynamic 3′,5′-cyclic adenosine monophosphate (cAMP) signaling at the single cell level is still missing for CB2 receptors. This paper presents an application of a live cell imaging while using a Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, for quantification of cAMP. We established HEK293 cells stably co-expressing human CB2 and Epac1-camps and quantified cAMP responses upon Forskolin pre-stimulation, followed by treatment with the CB2 ligands JWH-133, HU308, β-caryophyllene, or 2-arachidonoylglycerol. We could identify cells showing either an agonist dependent CB2-response as expected, cells displaying no response, and cells with constitutive receptor activity. In Epac1-CB2-HEK293 responder cells, the terpenoid β-caryophyllene significantly modified the cAMP response through CB2. For all of the tested ligands, a relatively high proportion of cells with constitutively active CB2 receptors was identified. Our method enabled the visualization of intracellular dynamic cAMP responses to the stimuli at single cell level, providing insights into the nature of heterologous CB2 expression systems that contributes to the understanding of Gαi-mediated G-Protein coupled receptor (GPCR) signaling in living cells and opens up possibilities for future investigations of endogenous CB2 responses. View Full-Text
Keywords: G-protein coupled receptor; cannabinoid; CB2; FRET; biosensor; cAMP; Epac1-camp; β-caryophyllene; live-cell imaging; signaling G-protein coupled receptor; cannabinoid; CB2; FRET; biosensor; cAMP; Epac1-camp; β-caryophyllene; live-cell imaging; signaling
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MDPI and ACS Style

Mensching, L.; Rading, S.; Nikolaev, V.; Karsak, M. Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging. Int. J. Mol. Sci. 2020, 21, 7880.

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