The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase known for its oncogenic potential that is involved in the development of the peripheral and central nervous system. ALK receptor ligands ALKAL1 and ALKAL2 were recently found to promote neuronal differentiation and survival. Here, we show that inflammation or injury enhanced ALKAL2 expression in a subset of TRPV1⁺ sensory neurons. Notably, ALKAL2 was particularly enriched in both mouse and human peptidergic nociceptors, yet weakly expressed in nonpeptidergic, large-diameter myelinated neurons or in the brain. Using a coculture expression system, we found that nociceptors exposed to ALKAL2 exhibited heightened excitability and neurite outgrowth. Intraplantar CFA or intrathecal infusion of recombinant ALKAL2 led to ALK phosphorylation in the lumbar dorsal horn of the spinal cord. Finally, depletion of ALKAL2 in dorsal root ganglia or blocking ALK with clinically available compounds crizotinib or lorlatinib reversed thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury, respectively. Overall, our work uncovers the ALKAL2/ALK signaling axis as a central regulator of nociceptor-induced sensitization. We propose that clinically approved ALK inhibitors used for non–small cell lung cancer and neuroblastomas could be repurposed to treat persistent pain conditions.

Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for 5 days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking phospholipase C (PLC) as well as protein kinase C beta (PKCβ), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCβ-mediated reduction of TRPM8 desensitization. This MOR-PKCβ dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCβ as important targets for opioid-induced cold hypersensitivity.

Transient receptor potential vanilloids (TRPV1) are non-selective cation channels that sense and transduce inflammatory pain signals. We previously reported that activation of TRPV1 induced the translocation of β-arrestin2 (ARRB2) from the cytoplasm to the nucleus, raising questions about the functional role of ARRB2 in the nucleus. Here, we determined the ARRB2 nuclear signalosome by conducting a quantitative proteomic analysis of the nucleus-sequestered L395Q ARRB2 mutant, compared to the cytosolic wild-type ARRB2 (WT ARRB2), in a heterologous expression system. We identified clusters of proteins that localize to the nucleolus and are involved in ribosomal biogenesis. Accordingly, L395Q ARRB2 or WT ARRB2 after capsaicin treatment were found to co-localize and interact with the nucleolar marker nucleophosmin (NPM1), treacle protein (TCOF1) and RNA polymerase I (POL I). We further investigated the role of nuclear ARRB2 signaling in regulating neuroplasticity. Using neuroblastoma (neuro2a) cells and dorsal root ganglia (DRG) neurons, we found that L395Q ARRB2 mutant increased POL I activity, inhibited the tumor suppressorp53 (p53) level and caused a decrease in the outgrowth of neurites. Together, our results suggest that the activation of TRPV1 promotes the ARRB2-mediated regulation of ribosomal biogenesis in the nucleolus. The ARRB2-TCOF1-p53 checkpoint signaling pathway might be involved in regulating neurite outgrowth associated with pathological pain conditions.