C-Terminal Splice Variants Of The Mu Opioid Receptor Differentially Regulate Signal Transduction And Ligand Bias

Abstract

The opioid field and the quest for the ideal analgesics with limited side effects has accumulated decades of research and thousands of new opioid compounds. However, in spite of the substantial advances in the understanding of opioid receptor pharmacology, the main stays of pain management remain to be mu analgesics such as morphine, fentanyl, and oxycodone, that exhibit the side effect profile - tolerance, dependence, constipation, respiratory depression, and euphoria resulting in abuse and addiction. The opioid response is mediated in conjunction by G protein and β-arrestin signaling pathways, where the G protein pathway is often linked to the therapeutic effect of the drug and the β- arrestin pathway is linked to the side effects. More recently, the field has focused on developing biased agonists that activate only the subset of signaling pathways important for the therapeutic effect while limiting the unwanted side effects. Much of the recent work in the field has explored ligand bias at only the MOR-1 receptor variant. However, the mu receptor gene, Oprm1, shows extensive alternative splicing and more than 60 different splice variants have been identified across different species. In this study, we explored the differences across the C-terminal variants of the mu opioid receptor and how these differences at the tip of the C-terminal tail might impact its ability to stimulate G protein coupling, β-arrestin recruitment, and ultimately, signal bias. In our comparisons across the the C-terminal variants, we were able to identify a C-terminal variant, MOR-1O which showed significant β-arrestin bias relative to MOR-1. The MOR-1O receptor variant contains exons 1, 2 and 3 like MOR-1, with splicing downstream of exon 3 where it has exon 7 instead of exon 4 in MOR-1. We were able to generate an exon 7 knockout mutant mouse model by introducing a stop codon at the 5’-end of exon 7 (E7) to stop translation at the end of exon 3 in variants that contain exon 7. Interestingly, the mE7M mice show a phenotype similar to the β-arrestin- 2 KO mice. These mE7M mice have no substantial difference in analgesic sensitivity or physical dependence but develop no tolerance in response to morphine. This provides a strong support for our in-vitro findings and suggests the functional interaction between the E7-associated C-terminal tails and β-arrestin-2. These findings are critical because they not only provide a potential target for therapeutic intervention to alleviate the morphine effects, but also, provides additional insights regarding the mu opioid receptor variants and their signaling biases to better screen for future new opioid analgesics.