Regulation of the μ-opioid receptor signalling in naturally occurring variants and phosphorylation site mutants

Opioid drugs are highly effective for the treatment of moderate to severe nociceptive pain. They exert their analgesic and rewarding effects primarily by signalling through the μ-opioid receptor (MOPr). The focus of this project was to better understand MOPr signalling regulation by investigating natural variants of MOPr and MOPr phosphosite mutants. Isogenic, stably transfected mouse pituitary adenoma (AtT20) cell lines expressing eight naturally occurring human MOPr variants and four phosphomutants were created. Opioid-stimulated changes in membrane potential were measured using a membrane potential-sensitive dye, while receptor phosphorylation of Ser 377 residue was determined by Western Blot and whole-cell ELISA was used to obtain the receptor surface loss dynamics. The N-terminal MOPr variants, A6V and N40D, are the most common single-nucleotide polymorphisms found worldwide. Their signalling regulation was quite similar to the wild-type MOPr in each assay, where buprenorphine was the opiod with the most variance observed. In AtT20-hMOPr-L85I cells morphine mediated internalisation was not as substantial as previously reported, while the second intracellular loop (ICL2) polymorphism R181C dramatically impacted receptor ability to signal by affecting opioid affinity and probably G protein binding. The majority of the third intracellular loop (ICL3) variants had detrimental effect in receptor signalling and regulation which indicates the important role this region plays in G protein activation. In addition the multiple phosphorylation mutants also affected membrane expression which was related to endoplasmic reticulum sequestration and possible changes in receptor stability. Finally, deleting all the putative phosphorylation sites in the human MOPr C-terminal domain did not greatly influence homologous desensitisation of the membrane potential signal, yet completely abolished internalisation as expected. In contrast heterologous desensitisation was deeply compromised in some mutants of the ICL3 while total phosphorylation deletion of the C-terminal was the only variant to increase desensitisation of somatostatin signalling. Interestingly buprenorphine induced signalling had a quite different profile across the variants, and morphine and methodone signalling were more affected by the ICL3 changes when compared to opioid peptides and buprenorphine. Overall, these results support the hypothesis of multiple mechanisms involved in regulation of MOPr, where ICL2 and ICL3 are crucial for G protein signalling, and receptor phosphorylation is not necessary for receptor desensitisation. In addition, this work highlights the profound effect of rare polymorphisms on opioid response at the molecular level, which is likey to contribute to the significant inter-individual variability observed with opioid therapy.