Gα signalling of the CB receptor and the influence of receptor number.

Background And Purpose: CB receptor signalling is canonically mediated through inhibitory Gα proteins, but occurs through other G proteins under some circumstances, Gα being the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gα blockade, CB /D receptor co-stimulation, CB agonist class and cell background. Hence, we examined the effects of receptor number and different ligands on CB receptor signalling.

Experimental Approach: CB 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 CB receptor antagonist.

Key Results: In HEK cells expressing high levels of CB receptors, agonist treatment stimulated cAMP, a response not known to be mediated by receptor number. Δ -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 CB receptor knockdown and Gα 1 supplementation restored canonical Gα signalling to high-expressing cells. Constitutive signalling in both low- and high-expressing cells was Gα -mediated.

Conclusion And Implications: CB 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 Gα signalling. CB receptors mediate 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.