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

Abstract

G protein-coupled receptor desensitization is typically mediated by receptor phosphorylation by G protein 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., 2010 [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, whereas morphine activation of JNK2 was GRK3-independent. In MOR-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 PKC-dependent mechanism, whereas fentanyl activated JNK2 through a Src- GRK3/arrestin2-dependent and PKC-independent 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 therapeutics.

Tolerance to the antinociceptive and hypothermic effects of morphine is mediated by multiple isoforms of c-Jun N-terminal kinase

“…Our results suggest that JNK3 plays the least prominent role in morphine tolerance. These findings are novel in showing that all three forms of JNK contribute to morphine tolerance, and they are consistent with previous work demonstrating that JNK2 can mediate tolerance to morphine [11, 18]. …”

“…Morphine results in an increase in spinal JNK phosphorylation through a protein kinase C (PKC)-dependent process [11]. Furthermore, this effect was abolished in JNK2 KO, but not JNK1 KO or JNK3 KO mice [18]. Both of these studies also demonstrated that JNK2 KO mice have a reduction in acute morphine tolerance.…”

“…Despite these results, it should not be concluded that morphine tolerance is strictly the result of JNK2 phosphorylation by PKC. While other opioids such as fentanyl also cause JNK2 phosphorylation, tolerance to fentanyl is JNK-independent [13, 18]. Moreover, while spinal MOR-desensitization by morphine requires JNK2, the desensitization of MOR in the locus coeruleus appears to be JNK-independent [24].…”

“…However, use of this JNK inhibitor is non-selective for the different JNK isoforms and does not allow determination of which isoforms(s) are responsible for morphine tolerance. It has been suggested by recent studies that JNK2 is required for tolerance to the anti-nociceptive effects of morphine [18]. However, no studies thus far have compared morphine tolerance in JNK1, JNK2, and JNK3 Knock-Out (KO) mice.…”

Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling

“…An alternative strategy is to use mouse embryo fibroblasts derived from arrestin-null animals (7–9), but these cells are challenging to transfect, limiting their usefulness. This has meant that despite the caveats described above and the fact that such arrestin-null mouse embryo fibroblasts have shown that, in this cell type, activation of the extracellular signal-regulated kinases ERK1/2 by certain ligands at the β 2 -adrenoreceptor requires an arrestin (10), much of the basis of underpinning analysis of arrestin signaling has derived from studies in more tractable, transformed cell lines, including human embryonic kidney (HEK) 293 cells where arrestin levels have been reduced but not completely eliminated (11–14). …”