Lysosomal Downregulation of the Mu Opioid Receptor is Opposed by the Retromer Complex

The mu opioid receptor (MOR) is protected from opioid-induced trafficking to lysosomes and proteolytic downregulation by its ability to access the endosomal recycling pathway through its C-terminal recycling motif, LENL. MOR sorting towards the lysosome results in downregulation of opioid signaling while recycling of MOR to the plasma membrane preserves signaling function. However, the mechanisms by which LENL promotes MOR recycling are unknown, and this sequence does not match any known consensus recycling motif. Here we took a functional genomics approach with a comparative genome-wide screen design to identify genes which control opioid receptor expression and downregulation. We identified 146 hits including all three subunits of the endosomal Retromer complex. We show that the LENL motif in MOR is a novel Retromer recycling motif and that LENL is a necessary, sufficient, and conserved mechanism to give MOR access to the Retromer recycling pathway and protect MOR from agonist-induced downregulation to multiple clinically relevant opioids including fentanyl and methadone. Overall design: Single guide RNA (sgRNA) enrichment within sorted cell populations based on fluorogenic reaction readout for opioid receptor abundance after receptor agonist stimulation.