Project Summary/Abstract G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and can signal through multiple transducers, including heterotrimeric G protein and β-arrestins. Opioid receptors are GPCRs whose role in pain sensation has made them primary drug targets for pain medications such as oxycodone and morphine. However, opioids have exceptionally high abuse potential and often cause fatal side effects such as respiratory arrest and death. The magnitude of these problems has led to a search for opioid alternatives to treat pain and related conditions. Activation of opioid receptors activates downstream effectors, including multiple G proteins (Gi1, Gi2, Gi3, GoA, GoB, Gz, and Ggustducin) and β-arrestins (β-arrestin1 and β-arrestin2). A major gap is an incomplete understanding of how opioid receptors recognize different transducers and the functional effects of signaling through each pathway. Kappa opioid receptor (KOR) has displayed promising therapeutic potential because of its novel analgesic activity –drugs that target KOR do not lead to addiction or cause death due to overdose as observed from mu opioid receptor agonists. Selective KOR antagonists have also been pursued in clinical trials for the treatment of addiction and depression. The research in my lab is driven by the overall hypothesis that large-scale structural determination studies of receptor-ligand/transducer complexes will provide molecular-level insights into opioid receptor signaling, and facilitate the design and optimization of novel ligand scaffolds that could be further developed into new drugs with desired behavior profile. By combining X- ray crystallography, Cryo-EM, and molecular pharmacology, we will elucidate fundamental mechanisms of KOR ligand selectivity, receptor activation and signaling. To do so, we will pursue the following main directions: (i) identify structural determinants of ligand selectivity between different opioid receptors types, (ii) identify the molecular basis for different G protein subtypes recognition, and (iii) identify structural features responsible for arrestin-bound activating states. The long-term goal is to develop receptor-specific and pathway-selective probes using structure-based drug discovery and study the function of individual opioid receptor signaling in vivo.