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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. [ABSTRACT FROM AUTHOR]

Background and Purpose: N-arachidonyl dopamine (NADA) has been identified as a putative endocannabinoid, but there is little information about which signalling pathways it activates. The purpose of this study was to identify the signalling pathways activated by NADA in vitro.Experimental Approach: Human or rat cannabinoid CB1 receptors were expressed in AtT20, CHO or HEK 293 cells. NADA displacement of radiolabelled cannabinoids, and CB1 receptor mediated activation of K channels or ERK phosphorylation, release of intracellular calcium ([Ca]i ) and modulation of adenylyl cyclase were measured in addition to NADA effects on CB1 receptor trafficking.Key Results: At concentrations up to 30 μM, NADA failed to activate any signalling pathways via CB1 receptors, with the exception of mobilization of [Ca]i . The elevations of [Ca]i were insensitive to pertussis toxin, and reduced or abolished by blockers of Gq /11 -dependent processes including U73122, thapsigargin and a peptide antagonist of Gq /11 activation. Prolonged NADA incubation produced modest loss of cell surface CB1 receptors. The prototypical cannabinoid agonist CP55940 signalled as expected in all assays.Conclusions and Implications: NADA is an ineffective agonist at most canonical cannabinoid receptor signalling pathways, but did promote mobilization of [Ca]i via Gq -dependent processes and some CB1 receptor trafficking. This signalling profile is distinct from that of any known cannabinoid, and suggests that NADA may have a unique spectrum of effects in vivo. Our results also indicate that it may be possible to identify highly biased CB1 receptor ligands displaying a subset of the pharmacological or therapeutic effects usually attributed to CB1 ligands. [ABSTRACT FROM AUTHOR]

Cholecystokinin (CCK) is a physiologically important gastrointestinal and neuronal peptide hormone, with roles in stimulating gallbladder contraction, pancreatic secretion, gastrointestinal motility and satiety. CCK exerts its effects via interactions with two structurally related class I guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs), the CCK(1) receptor and the CCK(2) receptor. Here, we focus on the CCK(1) receptor, with particular relevance to the broad spectrum of signalling initiated by activation with the natural full agonist peptide ligand, CCK. Distinct ligand-binding pockets have been defined for the natural peptide ligand and for some non-peptidyl small molecule ligands. While many CCK(1) receptor ligands have been developed and have had their pharmacology well described, their clinical potential has not yet been fully explored. The case is built for the potential importance of developing more selective partial agonists and allosteric modulators of this receptor that could have important roles in the treatment of common clinical syndromes. [ABSTRACT FROM AUTHOR]