Interactions of the cannabinoid CB1 and dopamine D2 receptors

Abstract

The effects of cannabinoids in the nervous system are predominantly mediated by cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR). GPCRs are known to form homodimeric and heterodimeric structures, which affect the signalling and regulation of each constituent receptor. CB1 has been shown to have functional interactions with the dopamine D2 receptor (D2). This thesis explores the structure, regulation and function of the CB1-D2 heterodimer. A bioluminescence resonance energy transfer (BRET) assay was utilised to detect constitutive CB1-D2 heterodimer, which was not detectibly altered by receptor agonists. BRET was also utilised to test a proposed heterodimer interface containing four key residues on transmembrane helix 1 of each receptor, however mutation of these residues did not significantly disrupt detection of heterodimer. Previous studies on GPCR heterodimers have suggested that interactions may occur throughout the protein synthesis and ligand-mediated trafficking pathways. Immunocytochemistry-based receptor trafficking and expression assays were used to determine whether CB1 and D2 interact in their regulation. Subtle differences were found in CB1 agonist-driven internalisation in the presence of a D2 agonist. Co-expression of CB1 and a flag-tagged D2 resulted in changes to flag-D2 processing, perhaps by the addition of a post-translational modification, although it is not clear if this is solely a modification of the flag-tag. When activated concurrently, CB1 and D2 have been shown to “switch” signalling phenotype from Gαi-like to Gαs-like activity, resulting in accumulation of cAMP. When this signalling interaction was first observed by Glass et al. (1997), it was hypothesised that this may be a result of the receptors competing for a limited pool of Gαi proteins. If this were the case, this mechanism would also be in effect when CB1 was expressed alone. In order to test this, a mixed population of cells was created and sorted by flow cytometry on the basis of CB1 surface expression. cAMP assays performed on these cells showed that cells with low to moderate expression of CB1 inhibit cAMP production, while cells with high CB1 expression increase cAMP accumulation. In conclusion, while it is likely that CB1 and D2 form a constitutive heterodimer, this does not affect ligand-mediated receptor trafficking. CB1 expression does, however, change the synthesis and processing of flag-tagged D2 in a manner that has yet to be determined. Since CB1 expression alone is sufficient to change the predominant cAMP phenotype to Gαs, presumably by competition of G proteins, this work suggests that CB1-D2 heterodimerisation may function simply to increase the local competition for G proteins, rather than the dimer itself mediating the functional signalling switch.