BACKGROUND AND PURPOSE: CB1 receptor signalling is canonically mediated through inhibitory Gαi proteins, but occurs through other G proteins under some circumstances, Gαs being the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gαi 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 Gαi 1 supplementation restored canonical Gαi signalling to high-expressing cells. Constitutive signalling in both low- and high-expressing cells was Gαi -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 Gαs 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.
BACKGROUND AND PURPOSE:CB1 receptor signalling is canonically mediated through inhibitory Gαi proteins, but occurs through other G proteins under some circumstances, Gαs being the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gαi 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 Gαi 1 supplementation restored canonical Gαi signalling to high-expressing cells. Constitutive signalling in both low- and high-expressing cells was Gαi -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 Gαs 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.
Authors: Christopher S Kearn; Katherine Blake-Palmer; Emma Daniel; Ken Mackie; Michelle Glass Journal: Mol Pharmacol Date: 2005-02-14 Impact factor: 4.436
Authors: K L Laugwitz; A Allgeier; S Offermanns; K Spicher; J Van Sande; J E Dumont; G Schultz Journal: Proc Natl Acad Sci U S A Date: 1996-01-09 Impact factor: 11.205
Authors: Erin E Cawston; William J Redmond; Courtney M Breen; Natasha L Grimsey; Mark Connor; Michelle Glass Journal: Br J Pharmacol Date: 2013-10 Impact factor: 8.739
Authors: Eric Sparkes; Elizabeth A Cairns; Richard C Kevin; Felcia Lai; Katharina Elisabeth Grafinger; Shuli Chen; Marie H Deventer; Ross Ellison; Rochelle Boyd; Lewis J Martin; Iain S McGregor; Roy R Gerona; David E Hibbs; Volker Auwärter; Michelle Glass; Christophe Stove; Samuel D Banister Journal: RSC Med Chem Date: 2021-10-25
Authors: Thomas F Gamage; Charlotte E Farquhar; Timothy W Lefever; Julie A Marusich; Richard C Kevin; Iain S McGregor; Jenny L Wiley; Brian F Thomas Journal: J Pharmacol Exp Ther Date: 2018-03-16 Impact factor: 4.030
Authors: Thomas F Gamage; Daniel G Barrus; Richard C Kevin; David B Finlay; Timothy W Lefever; Purvi R Patel; Megan A Grabenauer; Michelle Glass; Iain S McGregor; Jenny L Wiley; Brian F Thomas Journal: Pharmacol Biochem Behav Date: 2020-04-02 Impact factor: 3.533