Mechanisms of Opioid Receptor Desensitization

Thesis (Ph.D.)--University of Washington, 2015Agonists targeting MOR are effective analgesics, but their clinical use is hindered by side effects, including tolerance and addiction. KOR agonists also produce analgesia, but clinical use of these compounds has remained minimal due to aversive properties in humans. The µ-opioid receptor (MOR) agonists, morphine and fentanyl, both activate c-Jun N-terminal kinase (JNK), which is required for spinally-mediated morphine acute analgesic tolerance, whereas acute analgesic tolerance to fentanyl is blocked by G protein-coupled receptor kinase 3 (GRK3) gene deletion. Similarly, the κ-opioid receptor (KOR) collateral antagonist, norBNI, stimulates phosphorylation of JNK, and JNK1 is specifically required for norBNI’s long duration of antagonism. The durations of action of a broad range of KOR antagonists, including norBNI, positively correlate with the ability of the antagonist to activate JNK1 (Melief et al., 2011), whereas there is no correlation between duration of antagonist action and drug clearance (Munro et al., 2012). A better understanding of the mechanisms that contribute to opioid receptor desensitization is necessary for the development of improved therapeutics that avoid tolerance. This JNK-mediated mechanism of desensitization is likely to be more broadly applicable across the myriad of other GPCR systems. Additionally, elucidating the circuits responsible for p38-dependent, KOR-mediated aversion will assist with the development of KOR analgesics that avoid classic MOR side effects. Therefore, the goal of my thesis project has been to identify ligand-directed signaling mechanisms that contribute to JNK activation following morphine and fentanyl administration and better understand how JNK activation promotes MOR and KOR desensitization. Additionally, this thesis describes efforts to identify G biased KOR compounds with a reduced potential to cause dysphoria. Ultimately, this work provides a deeper understanding of opioid receptor signaling and can provide the basis for future therapeutic development targeting this receptor system

Mu opioid receptor stimulation activates c-Jun N-terminal kinase 2 by distinct arrestin-dependent and independent mechanisms

G protein-coupled receptor desensitization is typically mediated by receptor phosphorylation by G proteincoupled receptor kinase (GRK) and subsequent arrestin binding; morphine, however, was previously found to activate a c-Jun N-terminal kinase (JNK)-dependent, GRK/arrestin-independent pathway to produce mu opioid receptor (MOR) inactivation in spinally-mediated, acute anti-nociceptive responses [Melief et al.] [1]. In the current study, we determined that JNK2 was also required for centrally-mediated analgesic tolerance to morphine using the hotplate assay. We compared JNK activation by morphine and fentanyl in JNK1−/−, JNK2−/−, JNK3−/−, and GRK3−/−mice and found that both compounds specifically activate JNK2 in vivo; however, fentanyl activation of JNK2 was GRK3-dependent, whereasmorphine activation of JNK2 was GRK3-independent. InMOR-GFP expressing HEK293 cells, treatment with either arrestin siRNA, the Src family kinase inhibitor PP2, or the protein kinase C (PKC) inhibitor Gö6976 indicated that morphine activated JNK2 through an arrestin-independent Src- and PKCdependent mechanism, whereas fentanyl activated JNK2 through a Src-GRK3/arrestin-2-dependent and PKCindependent mechanism. This study resolves distinct ligand-directed mechanisms of JNK activation by mu opioid agonists and understanding ligand-directed signaling at MOR may improve opioid therapeutic