Abstract
BACKGROUND AND PURPOSE CB1 receptor signalling is canonically mediated through inhibitory G alpha(i) proteins, but occurs through other G proteins under some circumstances, G alpha(s) being the most characterized secondary pathway. Determinants of this signalling switch identified to date include G alpha(i) blockade, CB1/D-2 receptor co-stimulation, CB1 agonist class and cell background. Hence, we examined the effects of receptor number and different ligands on CB1 receptor signalling.
EXPERIMENTAL APPROACH CB1 receptors were expressed in HEK cells at different levels, and signalling characterized for cAMP by real-time BRET biosensor -CAMYEL - and for phospho-ERK by AlphaScreen. Homogenate and whole cell radioligand binding assays were performed to characterize AM6544, a novel irreversible CB1 receptor antagonist.
KEY RESULTS In HEK cells expressing high levels of CB1 receptors, agonist treatment stimulated cAMP, a response not known to be mediated by receptor number. Delta(9)-THC and BAY59-3074 increased cAMP only in high-expressing cells pretreated with pertussis toxin, and agonists demonstrated more diverse signalling profiles in the stimulatory pathway than the canonical inhibitory pathway. Pharmacological CB1 receptor knockdown and G alpha(i)1 supplementation restored canonical Gai signalling to high-expressing cells. Constitutive signalling in both low- and high-expressing cells was G alpha(i)-mediated.
CONCLUSION AND IMPLICATIONS CB1 receptor coupling to opposing G proteins is determined by both receptor and G protein expression levels, which underpins a mechanism for non-canonical signalling in a fashion consistent with G alpha(s) signalling. CB1 receptorsmediate opposite consequences in endpoints such as tumour viability depending on expression levels; our results may help to explain such effects at the level of G protein coupling.