G_s Coupling of CB1 Cannabinoid Receptors: Ligand Bias and the Influence of Receptor Number

CB1 is a widely expressed G protein-coupled receptor (GPCR) which couples to Gi/o proteins, leading to the inhibition of cAMP. However, by a mechanism which is not understood, CB1 can also couple to Gs, leading to stimulation of cAMP. We have previously shown this switch occurs through co-activation of CB1 and dopamine D2 receptors (another Gi linked GPCR) (1), and often encounter a Gs phenotype when transiently expressing CB1 in heterologous cell lines (resulting in very high receptor levels). This led us to speculate that CB1 couples to Gs when Gi is limited. In this study we examined the role of receptor number in signalling outcome, and characterised the ability of agonists to modify the pathways.

Methods: HEK cells were transfected with haemagglutinin (HA)-tagged human CB1 with (pplss-HEK-hCB1) and without (HEK-hCB1) a preprolactin signal sequence (pplss) to generate a wide range of expression levels. Agonists (CP55,940, WIN55,212-2, BAY 59-3074 (BAY), Δ9-tetrahydrocannabinol (THC), anandamide (AEA) and 2-arachidonyl glycerol (2AG)) were tested for the ability to stimulate or inhibit cAMP in cells stably expressing either hCB1 or pplss-hCB1 in the presence or absence of pertussis toxin (PTX). cAMP was measured by BRET biosensor over 20 minutes (2). To test the impact of different receptor expression levels, pharmacological receptor knockdown was achieved with the irreversible antagonist AM6544. Gi was transiently transfected into cells. pERK activation was determined by Perkin Elmer Alphascreen assay (2).

Figure 1 cAMP signalling in HEK-hCB1 (A) and pplss-hCB1 (B) in response to agonists. Graph shown is representative data from one experiment carried out in duplicate. Table shows agonist EC50 (mean±SEM) for five independent experiments.

Results: Cannabinoid agonists induce inhibition of cAMP in HEK-hCB1 (Fig 1A). In contrast, pplss-hCB1 couples to stimulation of cAMP with a wide range of efficacies (Fig 1B). For THC and BAY, stimulation of cAMP was enhanced by PTX treatment. To determine if this altered signalling is due to high receptor expression, cells were pre-incubated with increasing concentrations of the irreversible antagonist AM6544. As AM6544 concentration increased, receptor activation by CB1 agonists CP55,940 and WIN55,212-2 progressed from stimulating cAMP to inhibiting cAMP, while THC activation transitioned from no response to inhibition of cAMP. Furthermore, increasing the Gi protein expression level in cells by transient transfection also reversed the signalling phenotype from stimulatory to inhibitory. High expression of CB1 also resulted in more prolonged pERK activation.

Conclusions. High CB1 receptor number has been correlated with poor outcome in many cancer studies (3), and our own work has shown CB1 mediated Gs activation to be detrimental in Huntington’s disease (4). This study helps to explain the mechanism by which this signalling switch occurs and informs the design of potential therapeutic biased agonists for these opposing pathways.

(1) Glass M, Felder CC (1997). J Neurosci 17:5327-5333.

(2) Cawston et al (2013). Br J Pharmacol 170:893-907.

(3) Velasco et al (2016). Prog Neuropsychopharmacol Biol Psychiatry 64:259-66.

(4) Scotter et al (2010). Br J Pharmacol 160:747-61