Investigating Ligand-Induced Anterograde Trafficking of Cannabinoid Receptor 2

Abstract

Cannabinoid Receptor 2 (CB₂) is expressed abundantly in the immune system, tends to be immunosuppressive upon activation, and is considered a promising therapeutic target for modulating inflammation. Cellular responsiveness to receptor ligands is critically dependent on the number of functional cell surface receptors, and investigating receptor trafficking processes and their underlying mechanisms are therefore pertinent in understanding receptor biology. CB₂ trafficking has been characterised insofar as receptor internalisation and post-endocytic trafficking, whereas anterograde trafficking, and trafficking in regard to the widely-observed intracellular pool remain to be investigated. The overarching aim of this thesis is to investigate the molecular mechanisms underlying these trafficking pathways. At the outset of this study, preliminary data indicated that sustained stimulation with CP 55,940, a full agonist at CB₂, concurrently internalises cell surface CB₂ and stimulates delivery of a distinct population of CB₂ to the cell surface. Additionally, a di-lysine (KK) motif residing in the C-terminal tail of CB₂ is required for basal delivery to the cell surface. Interestingly, sustained CP 55,940 stimulation induces cell surface expression of the mutated receptor. This led to a hypothesis that intracellular CB₂ may be “mobilised” to the cell surface by CB₂ agonists. In this thesis human CB₂ trafficking, with and without mutation of the KK motif, is studied in response to a range of ligands. Signalling and trafficking inhibitors are also utilised to clarify the mechanisms driving the hypothesised mobilisation. These studies indicate that agonist-induced anterograde trafficking is not downstream of canonical CB₂ signalling pathways and, contrary to the original hypothesis, requires de novo protein synthesis. Enhanced cell surface delivery of CB₂ is also induced by multiple CB₂ ligands, including inverse agonists, corroborating that this phenomenon is not reliant on receptor activation. A set of ligands with increased polarity in comparison with traditional lipophilic cannabinoids is tested under the hypothesis that CB₂ ligands might act as pharmacological chaperones, whereby modulation of propensity to traverse the plasma membrane and access the cytoplasm may correlate with propensity to facilitate CB₂ anterograde trafficking. Some of these compounds elicited different efficacies of CB₂ mobilisation in comparison with CP 55,940, and/or unique signalling profiles and biased agonism. Finally, extensive method development toward establishment of a bimolecular fluorescence protein complementation (BiFC) assay aiming to identify CB₂ protein-protein interactions involved in anterograde trafficking is undertaken. The process taken highlights important, and under-recognised, pitfalls associated with this technique. The findings in this thesis characterise a novel trafficking phenotype, whereby CB₂ ligands can unconventionally manipulate CB₂ cell surface expression. Comprehensive understanding of this process may give rise to novel therapeutic avenues for modulating CB₂ surface expression and resulting drug responses. The biased ligands identified may exhibit unique functional properties in vivo and serve as useful tools in the development of CB₂-targeted therapies.