The structural basis for pathway-selective signaling by the µ opioid receptor

Project Summary/Abstract The structural basis for pathway-selective signaling by the µ-opioid receptor. For many patients experiencing acute and chronic pain, opioids like codeine, morphine, and fentanyl have improved their quality of life. Unfortunately, these benefits can be offset by dose-limiting liabilities, like addiction and the respiratory depression responsible for opioid overdose deaths. The µ-opioid receptor (µOR) is responsible for mediating the beneficial and adverse effects of most opioid analgesics. The µOR has complex signaling behavior, activating six different G proteins subtypes (Gi1, Gi2, Gi3, GoA, GoB, Gz) and two arrestin subtypes. Most of the opioid agonists currently used to treat pain activate all of these signaling pathways. Yet there is a growing body of evidence that only a subset of these signaling pathways mediate analgesia, while a different subset may be responsible for the adverse effects. Moreover, it is possible that the Gi/o/z subtypes responsible for alleviating acute pain are different from the subtypes responsible for alleviating chronic pain. We have identified several agonists that preferentially activate subsets of Gi/o/z proteins that retain analgesic efficacy but have fewer adverse effects. The overall goal of the proposal is to determine the structural basis for this pathway-selective G protein signaling. This information will facilitate the development of more pathway- selective agonists. These agonists will provide useful tools for understanding the complex signaling behavior of the µOR and the role of specific pathways in mediating the therapeutic and adverse effects of opioid agonists. The Specific Aims of the overall proposal are: Aim 1A. Determine the structural basis for subtype-selective signaling to Gi/o/z proteins. We will use cryo- electron microscopy to determine structures of the µOR bound to different Gi/o/z subtypes and different pathway selective agonists. Aim1B. Structure-guided synthesis of novel pathway-selective µOR agonists. Aim 2. Characterize the steady-state conformational changes in the cytoplasmic surface of the µOR stabilized by agonists with distinct signaling profiles. We will use double electron-electron resonance (DEER) spectroscopy to map conformational changes in the cytoplasmic surface of the µOR bound to pathway selective and non-selective agonists. Aim 3. Characterize the dynamics of TM6, ICL2, TM7, and the C-terminus in response to the binding of agonists with distinct signaling profiles in the presence and absence of specific G proteins and arrestins. We will use single-molecule Förster Resonance Energy Transfer to monitor conformational changes of these cytoplasmic domains in real-time.