SUMMARY Opioids are the most effective analgesics but are associated with severe side effects including respiratory depression, tolerance, and addiction. These factors helped cause the opioid abuse epidemic in the US, making drug overdose the leading cause of accidental death in the US. Thus, the identification of safer analgesics with diminished side effects and abuse potential is critical to address the ongoing crisis. Clinically used opioids predominantly exert both their analgesic and adverse effects through their action on the µ-opioid receptor (MOR). While several approaches were taken towards safer analgesics, these efforts are limited by a lack of understanding the complex biochemical networks engaged and activated by MOR in response to ligand binding. This proposal builds on recent evidence suggesting that (1) MOR signaling is dependent on the interplay between subcellular localization and membrane trafficking in a ligand-specific manner and (2) MOR shows ligand- dependent effects on its protein interaction network and the signaling pathways it activates. Thus, delineating the MOR-initiated signaling pathways for endogenous peptides and addictive opioids and how these are coordinated by receptor location and trafficking provides potential new strategies for therapeutic modalities and safer analgesics. The overarching goal of this proposal is to combine quantitative proteomics, functional genomics, and opioid receptor biology to systematically discover and characterize regulators of MOR signaling and trafficking in human induced pluripotent stem cell-derived neurons. We will combine proximity labeling mass spectrometry and quantitative phosphoproteomics to systematically delineate interaction networks that MOR engages and map the signaling pathways it activates. To study the functional role of proteomic targets in MOR signaling and trafficking, we will develop and apply reporter assays for receptor signaling and trafficking in CRISPRi gene regulation screens. Finally, we will test mechanistic hypotheses from proteomic and genetic screens on how novel regulators of trafficking and signaling fine tune the cellular response of MOR activation. Our proposed approach will yield mechanistic insights into MOR-initiated signaling pathways and how these are regulated by receptor trafficking. Identifying key regulators of MOR activation will fill a critical gap for designing safer, pathway selective analgesics and treatments for opioid addiction.