The current opioid epidemic highlights the need for a deeper understanding of how opioid receptors are activated in our body. Our knowledge about the downstream events following opioid receptor activation comes mainly from studies on opiates (e.g., morphine), synthetic drugs (e.g., fentanyl), and modified peptides (e.g., DAMGO). While these studies are important, the physiological ligands of the opioid receptors are endogenous opioid peptides such as endorphins, enkephalins, and dynorphins. Over 20 different opioid peptides have been identified in the brain, all of which activate the three types of opioid receptors (µOR, dOR, kOR) albeit with different affinities and efficacies for G protein-mediated versus β-arrestin-mediated signaling pathways. Thus, endogenous opioid peptides show biased agonism, a property previously found for opiates and synthetic opioids. Recent efforts to design safer opioid drugs were focused on optimizing G protein-mediated over β-arrestin- mediated pathways. However, this is overly simplistic because respiratory depression and other negative side effects of opioids have been shown to be G protein-dependent. Also, the model does not consider the possibility that ligands can preferentially activate different subtypes of G proteins and downstream mediators. Another factor that contributes to divergent signaling is agonist-mediated post endocytic signaling after receptor trafficking to endosomes followed either by lysosomal degradation versus recycling to the cell surface. Because endogenous opioid peptides are widely considered to be less toxic than opioid drugs, it is important to know the signaling properties of these peptides in comparison to representative opioid drugs. In this application we describe studies to characterize signaling by opioid peptides at individual receptor types. Based on preliminary data we hypothesize that “each opioid peptide exhibits a distinct signaling profile at each receptor type resulting in the expansion of opioid receptor signaling repertoire”. To test the hypothesis, in Aim 1 we will investigate the specific signaling pathways activated by individual opioid peptides and compare them to a selected set of opioid drugs that range in addiction potential and toxicity. In Aim 2 we will examine receptor trafficking and endosomal signaling upon activation by individual opioid peptides in comparison with a selected set of opioid drugs. In both Aims we utilize a combination of classic and modern techniques to study the effect of opioid peptides on cell lines expressing defined opioid receptors and signaling proteins, followed by electrophysiological analysis of brain regions important for rewarding effects (ventral tegmental area) and toxicity such as respiratory depression (the Kölliker Fuse nucleus). Together, these studies will broaden our understanding of opioid receptor signaling in response to their biological ligands, laying the groundwork for safer novel therapeutics for the treatment of ...