The molecular mechanisms and functional significance of mu opioid receptor endosomal sorting

PROJECT SUMMARY Opioid use disorder is a significant public health problem driven by complex biological and sociopolitical factors. People with opioid use disorder often cite the development of tolerance, a set of physiological adaptations that decrease response to opioids, as a reason for continued, escalating use. Given the vast role of tolerance in opioid use disorder, understanding how tolerance develops is of great interest. One factor that contributes to tolerance development is adaptations in regulation of opioid signaling at the level of a single cell. Opioids signal through G protein-coupled receptors (GPCR) called mu opioid receptors (MOR). Cells have evolved mechanisms for regulating GPCR signaling and returning to homeostasis in order to respond to additional signaling events. A major aspect of GPCR regulation is through trafficking: activated GPCRs are selectively internalized into the cell, where they travel through the endosome to the lysosome for degradation. However, some receptors, including MORs, can be rescued from a degradative fate and recycled to the plasma membrane as functional receptors, quickly re-establishing the cell’s ability to continue responding to opioids. The intracellular tail of MOR contains a specific amino acid sequence that is necessary for endosomal recycling. Recently, it was discovered that recycling of MOR through its recycling motif requires an endosomal sorting protein complex known as Retromer. However, the mechanism by which Retromer recycles MOR, and the effects of this process on MOR signaling are still unknown. This proposal seeks to identify the mechanisms by which Retromer-dependent MOR recycling occurs and assess the effects of MOR recycling on MOR signaling. The experiments in this proposal will determine how Retromer interacts with the MOR recycling motif to select MORs for recycling and define the subcellular route that Retromer uses to recycle MORs. This proposal will also test the hypothesis that Retromer is involved in MOR signaling regulation by limiting endosomal signaling using classical electrophysiological techniques and novel location-specific sensors of G protein activation. Overall, this work will uncover the mechanisms behind Retromer-dependent MOR trafficking and its functional consequences for MOR signaling. The long-term goal is to understand how Retromer and MOR recycling contribute to the development of pharmacological tolerance in vivo. The training goals of this proposal are supported by a world- class mentorship team of scientists, physicians, and physician-scientists and include rigorous training in hypothesis generation, advanced cellular and chemical biology techniques, oral and written communication to a wide variety of audiences, addiction medicine from a variety of clinical perspectives, and the professional and leadership skills required for a successful career as a physician-scientist.