No abstract

Activation of κ opioid receptors (KORs) produces analgesia and aversion via distinct intracellular signaling pathways, but whether G protein-biased KOR agonists can be designed to have clinical utility will depend on a better understanding of the signaling mechanisms involved. We found that KOR activation produced conditioned place aversion and potentiated CPP for cocaine in male and female C57BL/6N mice. Consistent with this, males and females both showed arrestin-mediated increases in phospho-p38 MAPK following KOR activation. Unlike in males, however, KOR activation had inconsistent analgesic effects in females and KOR increased Gβγ-mediated ERK phosphorylation in males, but not females. KOR desensitization was not responsible for the lack of response in females because neither nor gene knock-out enhanced analgesia. Instead, responsiveness was estrous cycle dependent because KOR analgesia was evident during low estrogen phases of the cycle and in ovariectomized (OVX) females. Estradiol treatment of OVX females suppressed KOR-mediated analgesia, demonstrating that estradiol was sufficient to blunt Gβγ-mediated KOR signals. G protein-coupled receptor kinase 2 (GRK2) is known to regulate ERK activation, and we found that the inhibitory, phosphorylated form of GRK2 was significantly higher in intact females. GRK2/3 inhibition by CMPD101 increased KOR stimulation of phospho-ERK in females, decreased sex differences in KOR-mediated inhibition of dopamine release, and enhanced mu opioid receptor and KOR-mediated analgesia in females. In OVX females, estradiol increased the association between GRK2 and Gβγ. These studies suggest that estradiol, through increased phosphorylation of GRK2 and possible sequestration of Gβγ by GRK2, blunts G protein-mediated signals. Chronic pain disorders are more prevalent in females than males, but opioid receptor agonists show inconsistent analgesic efficacy in females. κ opioid receptor (KOR) agonists have been tested in clinical trials for treating pain disorders based on their analgesic properties and low addictive potential. However, the molecular mechanisms underlying sex differences in KOR actions were previously unknown. Our studies identify an intracellular mechanism involving estradiol regulation of G protein-coupled receptor kinase 2 that is responsible for sexually dimorphic analgesic responses following opioid receptor activation. Understanding this mechanism will be critical for developing effective nonaddictive opioid analgesics for use in women and characterizing sexually dimorphic effects in other inhibitory G protein-coupled receptor signaling responses.

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.

Local translation of nuclear-encoded mitochondrial mRNAs is essential for mitochondrial activity, yet there is little insight into the role that axonal trafficking of these transcripts play in neuronal function and behavior. Previously, we identified a 38 nucleotide stem-loop structure (zipcode) in the 3′ untranslated region of the Cytochrome C oxidase IV (COXIV) mRNA that directs the transport of a reporter mRNA to the axon of superior cervical ganglion neurons (SCG). Over-expression of a chimeric reporter mRNA with the COXIV zipcode competed with the axonal trafficking of endogenous COXIV mRNA, and led to attenuated axon growth in SCG neurons. Here, we show that exogenous expression of the COXIV zipcode in cultured SCG neurons also results in the reduction of local ATP levels and increases levels of reactive oxygen species (ROS) in the axon. We took advantage of this “competition” phenotype to investigate the in vivo significance of axonal transport of COXIV mRNA. Towards this end, we generated transgenic mice expressing a fluorescent reporter fused to COXIV zipcode under a forebrain-specific promoter. Immunohistological analyses and RT-PCR analyses of RNA from the transgenic mouse brain showed expression of the reporter in the deep layer neurons in the pre-frontal and frontal cortex. Consistent with the in vitro studies, we observed increased ROS levels in neurons of these transgenic animals. A battery of behavioral tests on transgenic mice expressing the COXIV zipcode revealed an “anxiety-like” behavioral phenotype, suggesting an important role for axonal trafficking of nuclear-encoded mitochondrial mRNAs in neuronal physiology and animal behavior.