Molecular mechanisms controlling μ-opioid receptor activation

The μ-opioid receptor (MOP) is a G protein-coupled receptor (GPCR) responsible for mediating the analgesic effects of opioids such as morphine. Despite the numerous adverse effects associated with opioids, the MOP is the main therapeutic target for the treatment of severe and chronic pain. Due to the current ‘opioid epidemic crisis’ within western societies, extensive research and drug discovery efforts are aiming at the generation of new opioid drugs with improved therapeutic windows. In this context, bias agonism towards the G protein pathway and away from the β-arrestin2 pathway is currently being exploited for the development of safer and more effective MOP-targeted analgesics. In chapter 2, we characterised a range of opioid ligands, including the most recent G protein-biased MOP agonists (TRV130, PZM21 and SR-17018) using Bioluminescence Resonance Energy Transfer (BRET) tools. In this study, a Venus-tagged nanobody (Nb33- Venus) was developed as a BRET biosensor to quantitatively measure MOP activation. Similarly, we also assessed the recruitment of a modified mini G protein (Venus-mGsi) to measure coupling to G protein. Additionally, we measured different assay endpoints representative of G protein or β-arrestin2 pathways. We found no significant bias for any of the so-called G protein biased agonists. Instead, we found that these ligands displayed lower efficacy than other MOP agonists in pathways proximal to receptor activation. Moreover, we found a strong correlation of efficacies between the G protein- and β-arrestin2- dependent pathways. Thus, we propose that the efficacy of the new generation opioid drugs, widely recognised to be biased ligands, is in fact due to weak partial agonist activity and additional pharmacological properties must be taken into consideration. This is critical information, as some of these new drugs are currently being approved by the FDA. The work presented in appendix 1 demonstrated that differences in spatiotemporal signalling caused by MOP agonists was due to the redistribution of the MOP at the plasma membrane. Thus, in chapter 3 we extended these observations by studying the dynamics and reorganisation of the MOP at the plasma membrane after agonist stimulation using advanced imaging techniques such as Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). Our data revealed that DAMGO, a stable analogue of an endogenous opioid peptide, caused very distinct plasma membrane reorganisation of the MOP compared to the prototypical analgesic, morphine. Interestingly, this study also revealed a novel regulatory role of G protein-coupled receptor kinases (GRKs) in mediating plasma membrane reorganisation of the MOP in an agonist-specific manner and independently of G protein activation and internalisation, providing insights into another molecular event differentially regulated by distinct opioids. GRKs and β-arrestins are key GPCR regulators in promoting receptor desensitisation and internalisation. Extensive research has shown that different opioid agonists mediate distinct regulatory processes through differential phosphorylation of serine and threonine (S/T) residues. In chapter 4 and 5, we studied the role of specific S/T sites located in the Cterminal tail (Ct) or in the intracellular loops (ICLs) of the MOP. We described that multisite phosphorylation of the 375STANT379 motif within the Ct controls the dynamics of GRK and β- arrestin interactions with the MOP and contributes to receptor desensitisation. Our results suggest that other residues outside this motif also participate in the initial and transient recruitment of GRKs and β-arrestins. We described that S/T residues within the ICLs also contribute to G protein coupling and β-arrestin2 recruitment. Moreover, we showed that β- arrestin2 recruitment is partially dependent on G protein activation and that β-arrestin2 can also be translocated to the plasma membrane in an agonist-independent manner by PKC activation. Together, this reveals multiple mechanisms of β-arrestin2 interaction with the MOP. Elucidating these complex receptor-effector interactions represents an essential step toward a mechanistic understanding of MOP function so that effective strategies can be applied for the development of improved opioid drugs