Abstract
BACKGROUND AND PURPOSE:
CB1 cannabinoid receptor signalling is canonically mediated through inhibitory Gαi proteins, but signalling through other G proteins occurs under some circumstances; Gαs being the most characterised secondary pathway. Determinants of this signalling switch identified to date include Gαi blockade, CB1 /D2 dopamine receptor co-stimulation, CB1 agonist class, and cell background. The aim of this study was to examine the effects of receptor number and ligand dependence on CB1signalling.
EXPERIMENTAL APPROACH:
CB1 was expressed in HEK cells at different levels, and signalling characterisation was performed for cAMP by real-time BRET biosensor – CAMYEL – and for phospho-ERK by AlphaScreen. Characterisation of a novel irreversible antagonist of CB1 , AM6544, was performed by homogenate and whole cell radioligand binding assays.
KEY RESULTS:
In HEK cells expressing high levels of CB1 , agonist treatment resulted in cAMP stimulation; a response not previously known to be mediated by receptor number. Δ9 -THC and BAY59-3074 increased cAMP only in high-expressing cells that had been pre-treated with pertussis toxin, and agonists demonstrated more diverse signalling profiles in the stimulatory pathway than they elicited in the canonical inhibitory pathway. CB1 pharmacological knockdown and Gαi 1 supplementation were sufficient to restore canonical Gαisignalling to high expressing cells. Constitutive signalling in both lower- and higher-expressing cells was Gαi -mediated.
CONCLUSION AND IMPLICATIONS:
CB1 coupling to opposing G proteins is determined by both receptor and G protein number, and this underpins a mechanism for non-canonical signalling in a fashion consistent with Gαs signalling. CB1 is known to mediate opposite consequences in endpoints such as tumour viability receptor number-dependently, and our study may help to explain such effects at the level of G protein coupling.
This article is protected by copyright. All rights reserved.
- PMID: 28516479
- DOI: 10.1111/bph.13866
