Abstract:
Accessory proteins have been found to play a critical role in the function of G protein-coupled receptors. The expression and signalling phenotype of the calcitonin receptor-like receptor (CLR) is dependent on the interaction with the specific isoform of the receptor activity modifying protein (RAMP). The melanocortin receptor 2 (MC2) requires the melanocortin 2 receptor accessory proteins (MRAPs) for surface receptor expression. There are a number of inconsistencies in the literature about subcellular distribution and signalling of cannabinoid receptors (CBRs). This has led to the hypotheses that CBRs may also interact with accessory proteins.
The aim of this thesis was to investigate any potential interactions between CB1, CB2, GPR18, or GPR55 with RAMP1, RAMP2, RAMP3, MRAPα or MRAP2.
Immunocytochemistry of transiently transfected cells was used to detect any changes in the surface and total expression of the receptors and accessory proteins when co-expressed together. cAMP, pERK, β-arrestin signaling assays were utilised to detect any changes in ligand-induced CBR signaling with accessory protein co-expression. CB1, CB2, and GPR18 showed no distinct robust interactions with any of the accessory proteins, and there were no apparent changes in any signaling of the receptors with co-expression of any accessory protein. However, GPR55 co-expressed with either of the MRAPs resulted in a significant decrease in surface and total receptor expression. Concordantly, GPR55 was found to reduce cell surface expression of MRAPα.
The findings in this thesis indicate that the accessory proteins studied here generally speaking do not explain the atypical subcellular distribution of CBRs or literature discrepancies in CBR signalling, with the exception of GPR55 showing evidence of interactions with MRAPs. Although more investigation and characterisation is needed, the GPR55-MRAP findings are very interesting and raise important questions for future study regarding whether GPR55 can modulate the function of MC2 and vice versa via MRAP interactions in tissues that express these elements.
