Gα_s signalling of the CB₁ receptor and the influence of receptor number

Background and Purpose

CB₁ receptor signalling is canonically mediated through inhibitory Gα_i proteins, but occurs through other G proteins under some circumstances, Gα_s being the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gα_i blockade, CB₁/D₂ 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. Δ⁹‐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 Gα_i1 supplementation restored canonical Gα_i signalling to high‐expressing cells. Constitutive signalling in both low‐ and high‐expressing cells was Gα_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 Gα_s signalling. CB₁ 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.

DOI: 10.1111/bph.13866

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