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

Abstract

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.

Development of [18F]LU14 for PET Imaging of Cannabinoid Receptor Type 2 in the Brain

“…Two types of cannabinoid receptors, namely cannabinoid receptors type 1 (CB1R) [ 5 ] and cannabinoid receptors type 2 (CB2R) [ 6 ], were identified so far and are being thoroughly investigated [ 7 , 8 ]. Both CB1R and CB2R transduce signals from extracellular to intracellular space by inhibiting the activity of adenylyl cyclase over G i/o proteins, thereby suppressing the subsequent cyclic adenosine monophosphate (cAMP) pathway [ 9 , 10 ]. While the CB1R is associated with the central nervous system and is abundantly expressed in the brain (highest density in the hippocampus, cerebellum and striatum), the CB2R is found in the spleen, tonsils and the thymus gland and modulates immune cell functions.…”

β-Caryophyllene inhibits monoacylglycerol lipase activity and increases 2-AG levels: a new mechanism of endocannabinoid-mediated analgesia?

“…Following the findings of Gertsch et al [8] BCP has been considered a putative CB2 agonist. However, although there is a lot of evidence from Gertsch et al and others [8,[24][25][26][27], it remains controversial, whether BCP binds directly to CB2 and acts as an agonist or if the interaction is rather weak [9][10][11]. In this study we showed that β-caryophyllene inhibits monoacylglycerol lipase at pharmacologically relevant concentrations and consequently increased the levels of the Cannabinoid receptor 2 (CB2) agonist 2-arachidonoylglycerol.…”

“…It has been proposed that BCP is a putative CB2 receptor agonist [8,[24][25][26][27]. However, recently it was disputed whether BCP binds directly to CB2 and acts as an agonist or if the interaction is rather weak [9][10][11].…”

Impact of the Cannabinoid System in Alzheimer's Disease

“…As a G protein-coupled receptor, signal transduction initiated by the CB2R is mediated by Gi/o [ 113 ], increasing intracellular calcium levels by activating the phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) signaling pathways [ 114 ]. CB2Rs inhibit cAMP, thereby reducing intracellular cAMP levels [ 115 ]. In addition, CB2R activation can also be combined with other cellular pathways, including PKA, ERK1/2, and P38 [ 116 , 117 ].…”