Factors influencing the specificity and bias of CB1 cannabinoid receptor signalling

Abstract

CB1 cannabinoid receptor signalling is canonically mediated by inhibitory Gαi proteins, but signalling through other G proteins occurs under some circumstances. The most characterised alternative pathway gives rise to stimulatory cAMP signalling responses and is therefore thought to be Gαsmediated. Determinants of this signalling switch identified to date include Gαi blockade, CB1/D2 dopamine receptor co-stimulation, CB1 agonist class, and cell background. The ability of different G protein-coupled receptor ligands to preferentially activate different signalling pathways is also a current theme in molecular pharmacology (so-called functional selectivity or biased agonism). The phenomena of the CB1 cAMP signalling switch and biased agonism together form the basis of the current thesis. The first major finding of this thesis was that CB1 coupling to stimulatory signalling pathways is determined by both receptor and G protein expression level. This underpins a mechanism for noncanonical signalling consistent with Gαs activity. A panel of six diverse CB1 agonists were compared for their abilities to induce the signalling switch. Relative to the CB1 agonists 2-AG, WIN55,212-2, CP55,940 and AEA, the partial agonists Δ9-THC and BAY59-3074 demonstrated a reduced ability to induce cAMP increases and produced this response only in highly CB1-expressing cells that had been pre-treated with pertussis toxin. Agonists generally demonstrated more diverse signalling profiles in the stimulatory pathway than they elicited in the canonical inhibitory pathway, with a wide range of agonist efficacies observed. The activity of same panel of agonists was also characterised in three simultaneous pathway endpoints: receptor trafficking, canonical cAMP inhibition, and pERK activation. A formal functional selectivity study was undertaken, utilising current methods for quantification of bias (Operational analysis). Surprisingly, pronounced agonist bias was not detected for any pathway comparison, leading to a closer examination and empirically-based critique of the Operational analysis method. Concerns currently include the observations that Operational analysis fails to detect agonist-specific differences in pathway efficacies, and that the current method cannot adequately explicate kinetic features of agonist responses that alter agonist bias conclusions. Following the discovery of a novel mechanism for the switch in CB1-mediated cAMP signalling, the question is raised as to whether the stimulatory (Gαs-like) response is either induced (reflecting a change in receptor coupling preference) or unmasked (existing concurrently with the inhibitory signal, but becoming manifest) as a consequence of appropriate biochemical and biophysical conditions. Current research tools such as pertussis and cholera toxins cannot address this conundrum, so another study was conceived to probe the concept of utilising specific G protein pathway inhibitors (blocking peptides) for this purpose. It is hoped that prefabricated peptides may ultimately be able to be delivered exogenously in in vitro assays to facilitate gains in mechanistic insight (such as elucidating the specificity of G protein-receptor interactions). The outcome of the current project indicates that more work is required before this will be possible, however. CB1 is known to mediate opposite consequences in endpoints such as tumour viability in a manner that appears to be receptor number-dependent, and aspects of this thesis may help to explain such effects at the level of G protein coupling. Themes including biased agonism give hope that greater finesse in targeting desired responses may one day be possible, especially as CB1 is intimately involved in a plethora of physiological and pathological processes.