Abstract
Inactivation of opioid receptors limits the therapeutic efficacy of morphine-like analgesics and mediates the long duration of kappa opioid antidepressants by an uncharacterized, arrestin-independent mechanism. Here we use an iterative, discovery-based proteomic approach to show that following opioid administration, peroxiredoxin 6 (PRDX6) is recruited to the opioid receptor complex by c-Jun N-terminal kinase (JNK) phosphorylation. PRDX6 activation generates reactive oxygen species via NADPH oxidase, reducing the palmitoylation of receptor-associated Gαi in a JNK-dependent manner. Selective inhibition of PRDX6 blocks Gαi depalmitoylation, prevents the enhanced receptor G-protein association and blocks acute analgesic tolerance to morphine and kappa opioid receptor inactivation in vivo. Opioid stimulation of JNK also inactivates dopamine D2 receptors in a PRDX6-dependent manner. We show that the loss of this lipid modification distorts the receptor G-protein association, thereby preventing agonist-induced guanine nucleotide exchange. These findings establish JNK-dependent PRDX6 recruitment and oxidation-induced Gαi depalmitoylation as an additional mechanism of Gαi-G-protein-coupled receptor inactivation.
Highlights
Inactivation of opioid receptors limits the therapeutic efficacy of morphine-like analgesics and mediates the long duration of kappa opioid antidepressants by an uncharacterized, arrestinindependent mechanism
An alternative explanation is that Jun N-terminal kinase (JNK) phosphorylation recruits an arrestin-like substrate within the signaling complex that occludes receptor G-protein interaction, and we used quantitative SILAC proteomics[11] to determine whether a protein with arrestin-like properties showed increased association with the opioid receptor after JNK activation with norBNI, a selective kappa opioid receptor (KOR) antagonist with collateral agonist activity at the JNK pathway[4]
This analysis failed to detect an arrestin-like protein whose association with mycKOR was increased by JNK activation, surprisingly the associations of Gαi and Gβ with mycKOR were significantly increased by norBNI treatment (40% and 100%, respectively)
Summary
Inactivation of opioid receptors limits the therapeutic efficacy of morphine-like analgesics and mediates the long duration of kappa opioid antidepressants by an uncharacterized, arrestinindependent mechanism. Selective inhibition of PRDX6 blocks Gαi depalmitoylation, prevents the enhanced receptor G-protein association and blocks acute analgesic tolerance to morphine and kappa opioid receptor inactivation in vivo. Pharmacological inhibition or genetic deletion of specific JNK isoforms blocks acute analgesic tolerance to morphine and prevents long-lasting KOR antagonism, as does pretreatment with KOR antagonists which do not activate JNK4, 5, but how JNK inactivates opioid receptor signaling is not known. These results suggest that JNK may phosphorylate a component of the opioid receptor signaling complex, thereby preventing G-protein activation in an alternative process to arrestin-mediated desensitization. We show that this JNK-PRDX6 pathway results in drug tolerance in vivo
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