A study of functional selectivity at the cannabinoid type 1 receptor

The cannabinoid CB1 receptor is a G protein-coupled receptor (GPCR) which is important in the regulation of neuronal function, predominately via coupling to heterotrimeric Gi/o proteins. The receptor has also been shown to interact with a variety of other intracellular signalling mediators, including other G proteins, several members of the mitogen activated kinase (MAP) superfamily and β-arrestins. The CB1 receptor is recognised by an array of structurally distinct endogenous and exogenous ligands and a growing body of evidence indicates that ligands acting at GPCRs are able to differently activate specific signalling pathways, a phenomenon known as functional selectivity or biased agonism. This is important in future drug development as it may be possible to produce drugs which selectively activate signalling pathways linked to therapeutic benefits, while minimising activation of those associated with unwanted side effects. The main aim of this thesis, therefore, was to investigate ligand-selective functional selectivity at the cannabinoid CB1 receptor both endogenously and exogenously expressed in a variety of cell lines. Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293) cells stably transfected with the human recombinant CB1 receptor and untransfected murine Neuro 2a (N2a) cells, were exposed to a number of cannabinoid receptor agonists, including the endogenous agonist anandamide, the phytocannabinoid Δ9-THC, and several synthetic ligands. Ligand affinity was determined using competition radioligand binding assays. Regulation of several CB1 receptor-coupled intracellular signalling mediators was investigated; G protein activation was measured using the [35S]-GTPγS binding assay; phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK) and p38 MAP kinases was measured using the immunocytochemical In-cell Western technique. Modulation of cAMP accumulation and β-arrestin recruitment were measured using DiscoveRx detection assay kits. Using concentration-response data, agonist bias was analysed using equimolar comparison plots, and by comparison of intrinsic relative activities for all agonists, calculated relative to the high potency cannabinoid receptor agonist HU-210 for the transfected cells or WIN 55,212-2 for the Neuro 2a cells. The PPARα agonist fenofibrate was identified as a previously unrecognized cannabinoid receptor agonist, exhibiting modest selectivity for the CB2 receptor subtype (~25-fold). In addition to its functioning as an orthosteric agonist, fenofibrate, at high concentrations, appeared to act as a negative allosteric modulator at the CB1 receptor expressed in CHO cells, identified by radioligand binding assays, and non-competitive inhibition of the orthosteric agonist CP 55,940 in the [35S]-GTPγS binding assay. All three cell lines showed CB1- and Gi/o protein-dependent activation of ERK and inhibition of forskolin-stimulated cAMP accumulation: however, the magnitude of these responses differed between the three cell lines, with the responses in the Neuro 2a cells being markedly smaller than in the recombinant cell lines. The synthetic agonists WIN 55,212-2 and ACEA both exhibited bias towards ERK activation, in comparison to inhibition of cAMP accumulation, in both the CHO and HEK cell lines. HU-210, Δ9-THC, methanandamide and fenofibrate all exhibited bias towards ERK activation in the Neuro 2a cells, but only the HU-210 bias was quantified, as the other three agonists did not couple to cAMP formation or inhibition in this cell line. In addition, time-course experiments revealed further novel patterns of agonist bias in ERK signalling, with CP 55,940 alone producing a second phase sustained ERK response in CHO cells, while higher concentrations of WIN 55-212-2 produced a CB1-receptor-dependent reversal of ERK phosphorylation in HEK cells at 20 min, but not 5 min, agonist stimulation. Additional cell-dependent responses were observed with HEK cells alone, exhibiting Gi/o protein-independent ERK activation and increase in cAMP levels. Despite reports in the literature to the contrary, none of the cell lines tested showed receptor-mediated activation of JNK or p38 MAP kinase. In conclusion, this thesis has demonstrated functional selectivity at the cannabinoid type 1 receptor in a number of cell lines, expressing both native and transfected recombinant receptors. These findings contribute to our increasing understand of the complexity of GPCR signalling, and potentially allow for the development of more targeted drugs able to selectively engage with beneficial signal transduction pathways.