Abstract
Activation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) by the Gi/o protein-coupled κ opioid receptor (KOR), μ opioid, and D2 dopamine receptors stimulates peroxiredoxin 6 (PRDX6)-mediated production of reactive oxygen species (ROS). ROS production by KOR-inactivating antagonists norbinaltorphimine (norBNI) and JDTic blocks Gαi protein activation, but the signaling mechanisms and consequences of JNK activation by KOR agonists remain uncharacterized. Binding of arrestins to KOR causes desensitization of G protein signaling and acts as a scaffold to initiate MAPK activation. Here, we found that the KOR agonists U50,488 and dynorphin B stimulated biphasic JNK activation with an early arrestin-independent phase, requiring the small G protein RAC family small GTPase 1 (RAC1) and protein kinase C (PKC), and a later arrestin-scaffolded phase, requiring RAC1 and Ras homolog family member (RHO) kinase. JNK activation by U50,488 and dynorphin B also stimulated PRDX6-dependent ROS production but with an inverted U-shaped dose-response relationship. KOR agonist-induced ROS generation resulted from the early arrestin-independent phase of JNK activation, and this ROS response was suppressed by arrestin-dependent activation of the MAPK p38. The apparent balance between p38 MAPK and JNK/ROS signaling has important physiological implications for understanding of dynorphin activities during the stress response. To visualize these activities, we monitored KOR agonist-mediated activation of ROS in transfected live cells by two fluorescent sensors, CellROX Green and HyPerRed. These findings establish an important aspect of opioid receptor signaling and suggest that ROS induction may be part of the physiological response to KOR activation.
Highlights
Activation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) by the Gi/o protein– coupled opioid receptor (KOR), opioid, and D2 dopamine receptors stimulates peroxiredoxin 6 (PRDX6)-mediated production of reactive oxygen species (ROS)
We found that the KOR agonists U50,488 and dynorphin B stimulated biphasic JNK activation with an early arrestin-independent phase, requiring the small G protein RAC family small GTPase 1 (RAC1) and protein kinase C (PKC), and a later arrestinscaffolded phase, requiring RAC1 and Ras homolog family member (RHO) kinase
KOR agonist-induced ROS generation resulted from the early arrestin-independent phase of JNK activation, and this ROS response was suppressed by arrestin-dependent activation of the MAPK p38
Summary
HEK293 cells stably transfected with KOR respond to the selective agonist U50,488 (10 M) with a biphasic increase in phospho-JNK (Fig. 1, B and C). To confirm that the increase in JNK phosphorylation was mediated through KOR activation of G␣i, cells were pretreated with pertussis toxin (PTX; 10 ng/ml) 3 h prior to treatment with 10 M U50,488 for 15 or 60 min, and cell lysates were immunoblotted for phospho-JNK IR (Fig. 1, D and E). To further confirm agonist-induced JNK activity, we looked at U50,488-mediated phosphorylation of the JNK substrate c-Jun (Fig. 1, F and G). KOR-expressing HEK293 cells were treated for 5– 60 min with 1 M dynorphin B, and cell lysates were immunoblotted for phosphoJNK (Fig. 1, H and I). Similar to U50,488, dynorphin B stimulated a biphasic increase in phospho-JNK IR with significantly increased phospho-JNK IR at 30 min (136 Ϯ 6% of basal, p Ͻ 0.01) and 120 min (133 Ϯ 12% of basal, p Ͻ 0.05), indicating that JNK activation by dynorphin is qualitatively similar to U50,488 but with slightly delayed kinetics. The reason for the slower kinetics of JNK phosphorylation following dynorphin is unclear as no difference in kinetics is observed in dynorphininduced ERK1/2 activation (Fig. 1, J and K)
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