This proposal addresses an exciting aspect of the biology of the delta opioid receptor (DOR), a prototypical and clinically relevant G protein-coupled receptor (GPCR) that has long been considered a promising target for treating pain and opioid addiction - two highly prevalent and comorbid diseases. Pain, one of the most common symptoms presented at hospitals, is currently managed primarily by opioid analgesics that target the mu-opioid receptor. While effective, their use is highly limited due to extensive side effects and a high potential for addiction. DOR activation is a promising alternate strategy to reduce pain without causing addiction as the receptor is not expressed much in the reward pathway. While the contribution of DOR-expressing neurons to distinct modalities of pain is being heavily explored, the critical problem with this strategy is that effectively targeting DOR in vivo has been difficult. DOR agonists activate the receptor and signal efficiently in isolated systems, but they show poor analgesic responses in vivo. Centrally acting DOR agonists can inhibit pain at high doses in animal models, but they also induce convulsions which preclude their use. This low analgesic effectiveness in vivo, which is a critical limiting factor in developing analgesics targeting DOR, is being studied at various levels including at the pharmacological and neural circuit level. However, we still do not fully understand the mechanisms underlying this low effectiveness. Interestingly, DOR is unique when compared to many other GPCRs in that it localizes mostly to intracellular compartments, with very little expressed on the cell surface. Our proof of mechanism experiments showing that relocating DOR to the neuronal surface increases the effectiveness of DOR agonists, suggesting the exciting idea that this intracellular location is what contributes to the low analgesic potency. Understanding how and why DOR is intracellular in neurons is therefore critical to understand opioid physiology and to develop DOR as a target for pain management. This proposal addresses the mechanisms and consequences of DOR localization to intracellular compartments in neurons. We hypothesize that sequence- specific interactions of DOR mediate retention of newly synthesized DOR in intracellular compartments in neurons and allow compartment-specific signaling. We will test our hypothesis by determining the mechanisms of DOR intracellular retention and testing the role of these mechanisms in compartment-specific DOR signaling. Specifically, we will follow two aims, 1) To determine the mechanisms regulating DOR localization in intracellular compartments, and 2) To investigate compartment-specific DOR signaling from intracellular membranes. We will use focused candidate-based and unbiased genome-wide approaches to pursue these aims. Completion of this project will validate a novel and physiologically relevant model for how DOR is localized to intracellular compartments in neurons...