Abstract:
Cannabinoid Receptor 1 (CB1), an abundant G-protein coupled receptor (GPCR) in the central
nervous system, is currently of significant interest as a therapeutic target. The cellular control
of receptor trafficking is intimately linked with drug effects, however in comparison with other
GPCRs, the study of CB1 trafficking is in its infancy. Although the existing literature suggests
CB1 should be classified as a “dual-fate” receptor, some conflicting evidence exists as to the
conditions under which CB1 recycles or degrades. Of particular interest is the widely noted
intracellular pool which has been speculated to form part of a constitutive internalisation and
recycling pathway.
This study performs a detailed quantification of CB1 trafficking in four cell lines, one of which
expresses CB1 endogenously. A novel high-throughput immunocytochemistry-based approach
is applied to quantitatively measure receptor trafficking. An important advance on previous
studies is the use of a proteolytic method to directly quantitate intracellular receptors. Contrary
to previous reports, the data suggests that CB1 does not recycle following constitutive or
agonist-induced internalisation but instead exhibits a primarily degradative phenotype.
Evidence is obtained through antibody “live-feeding” protocols and the effects of protein
synthesis inhibitors, among other approaches. In addition, the data suggests that the
intracellular pool does not traffic to the cell surface and therefore does not contribute to CB1
signalling via classical paradigms. The effects of Rab GTPase dominantly-acting positive and
negative mutants on basal CB1 localisation corroborate these results.
The findings of this thesis have significant implications for the interpretation of CB1 biochemical
studies and call for a revision of the currently held theories of CB1 intracellular trafficking. The
study provides a foundation for further mechanistic studies and may impact the design and
application of cannabinoid therapeutics.
