Molecular Regulation of Opioid Receptor Signaling
Schattauer, Selena Schreiber
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The varied behavioral effects of kappa opioid receptors (KOR) are mediated through different signaling cascades. KOR activation of G protein-dependent signaling results in analgesia, whereas the dysphoric effects are mediated by a different pathway involving G protein-coupled receptor kinase (GRK) and arrestin. Therefore, a partial KOR agonist that does not efficiently activate arrestin-dependent signaling may produce analgesia without dysphoria. No selective KOR partial agonists are currently available, and preclinical assessment is complicated by sequence differences between rodent (r) and human (h) KOR. KOR antagonists are also of therapeutic interest for their potential anxiolytic and antidepressant effects, but many KOR antagonists have long durations of action resulting from selective activation of cJun kinase (JNK). In this thesis, I compared the signaling events initiated by agonist stimulation of hKOR and rKOR. Although a partial agonist at both hKOR and rKOR, pentazocine was more potent at activating p38 MAPK in hKOR than rKOR expressed in HEK293 cells. In contrast, pentazocine was equally potent for arrestin-independent activation of ERK1/2 in hKOR and rKOR. There were no potency differences between hKOR and rKOR in U50,488 activation of ERK1/2 or p38 MAPK. hKOR lacks the Ser369 phosphorylation site in rKOR required for GRK/arrestin-dependent p38 activation, but mutation of the Ser358 to asparagine in hKOR blocked p38 activation without affecting the arrestin-independent activation of ERK1/2. Although pentazocine is dysphoric in humans, an analgesic dose of pentazocine failed to produce KOR-dependent aversion in C57Bl/6 mice, consistent with its lower potency of p38 activation of rKOR. This study shows that hKOR activates p38 MAPK through a phosphorylation and arrestin-dependent mechanism; however ligand directed signaling differences have important implications for preclinical screening of partial KOR agonists. This thesis also includes contributions to other studies on opioid receptor signaling. This includes: characterizing an antibody selective for MOR phosphorylated at Thr370 and Ser357, which are implicated in MOR desensitization; radioligand binding studies demonstrating that norBNI does not change the drug binding site of KOR in vivo; and development of lentiviral vectors expressing KOR which were used to show that KOR expression in the dorsal raphe is sufficient for KOR-mediated analgesia and aversion.
- Pharmacology