Regulation of Opioid Receptor Trafficking and Signaling by Opioid Peptides

Abstract

All opioids - whether they are addictive opioid analgesics or endogenous opioids like endorphins produced by our body - activate one of three receptors in our brain. However, different opioids cause different effects, including effects on pain and addiction. How opioids generate this variety in signaling has been a long-standing question in the field. My thesis tests the exciting new idea that specific opioids traffic receptors to different compartments of the cell, from where they can signal in a compartment-specific manner. In this dissertation I review the selective mechanisms that regulate G protein-coupled receptor (GPCR) trafficking, which highlight the complex framework underlying spatial regulation of receptor function. Lipid membrane dynamics, post-translational modifications, cell-specific protein interactors, and agonist-selectivity are all key determinants for selective GPCR trafficking. Specifically, I focus on how opioid peptides may cause distinct functional outcomes through activation of the same receptor. To address this question, I use high-resolution imaging assays to examine how different dynorphin peptides regulated kappa opioid receptor (KOR) trafficking and post-endocytic sorting. Interestingly, we observe that highly related dynorphin peptides caused distinct opioid receptor trafficking fates (recycling vs. degradation) as well as compartment-specific signaling from late endosomes and lysosomes, depending on the peptide ligand that activated the receptor. Specifically, we show that KOR activation by dynorphin A leads to receptor degradation and activation from late endosomal compartments, while KOR activation by the highly related dynorphin B leads to receptor recycling from Rab5 and Rab11 compartments, without endosomal signaling. Further, I investigate the dynamic relationship between receptor signaling and receptor trafficking. Initially, we observed that agonist washout caused a decrease in mu opioid receptor (MOR) recycling. This finding led us to hypothesize that signaling downstream of receptor activation regulated receptor trafficking back to the cell surface, which regulates resensitization. To test this, I used total internal reflection fluorescence (TIRF) microscopy to detect real-time exocytic fusion events in living cells. I tested various components of known signaling pathways downstream of MOR activation using pharmacological inhibitors and phosphodeficient and phosphomimetic receptor mutants to quantitate differences in MOR recycling. Our results indicate that MOR recycling is regulated via an agonist-dependent Gβγ signaling pathway, resulting in the phosphorylation of the receptor’s C-terminal tail to increase receptor recycling. I follow up on this work by further testing the role of receptor phosphorylation in regulating MOR trafficking and signaling. By understanding the signaling pathways that regulate receptor trafficking and resensitization, this work opens new avenues for potential druggable targets. Collectively, this work increases our understanding of opioid physiology. Further, because the opioid receptor is a prototype for GPCRs - the largest and highly conserved family of signaling receptors in humans - the principles could be relevant across many physiologically relevant members of this family.