Ubiquitin-mediated receptor degradation contributes to development of tolerance to MrgC agonist–induced pain inhibition in neuropathic rats

Abstract

Agonists to subtype C of the Mas-related G-protein-coupled receptors (MrgC) induce pain inhibition after intrathecal (i.t.) administration in rodent models of nerve injury. Here, we investigated whether tolerance develops after repeated MrgC agonist treatments and examined the underlying mechanisms. In animal behavior studies conducted in male rats at 4 to 5 weeks after an L5 spinal nerve ligation (SNL), the ability of dipeptide MrgC agonist JHU58 (0.1 mM, 10 μL, i.t.) to inhibit mechanical and heat hypersensitivity decreased after 3 days of treatment with a tolerance-inducing dose (0.5 mM, 10 μL, i.t., twice/day). In HEK293T cells, acute treatment with JHU58 or BAM8-22 (a large peptide MrgC agonist) led to MrgC endocytosis from the cell membrane and later sorting to the membrane for reinsertion. However, chronic exposure to JHU58 increased the coupling of MrgC to β-arrestin-2 and led to the ubiquitination and degradation of MrgC. Importantly, pretreatment with TAK-243 (0.2 mM, 5 μL, i.t.), a small-molecule inhibitor of the ubiquitin-activating enzyme, during tolerance induction attenuated the development of tolerance to JHU58-induced inhibition of mechanical and heat hypersensitivity in SNL rats. Interestingly, morphine analgesia was also decreased in SNL rats that had become tolerant to JHU58, suggesting a cross-tolerance. Furthermore, i.t. pretreatment with TAK-243, which reduced JHU58 tolerance, also attenuated the cross-tolerance to morphine analgesia. These findings suggest that tolerance can develop to MrgC agonist–induced pain inhibition after repeated i.t. administrations. This tolerance development to JHU58 may involve increased coupling of MrgC to β-arrestin-2 and ubiquitin-mediated receptor degradation.

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“…Conversely, endocytosis of receptors targeting lysosomes can lead to down‐regulation of protein hydrolysis and reduced signal transduction (Dores & Trejo, 2019; Whistler et al, 2002). It has been shown that ubiquitin mediated the endocytic transport and degradation of MrgC (Huang et al, 2021). In our study, treatment of N2a cells with 3MA and CQ, the autophagy‐lysosome inhibitors, inhibited the degradation of MrgC, suggesting that degradation of MrgC after ubiquitination may occur via the autophagy‐lysosome pathway.…”

“…Our previous studies have shown that MrgC plays an important role in the development of BCP, and can inhibit Ca2+ inward flow in spinal cord neurons by regulating the expression and activity of G αi , N ‐methyl‐ d ‐aspartate receptor subunit 2B and neuronal nitric oxide synthase, thus effectively improving the pain behaviour of mouse model with BCP (Sun et al, 2019; Sun, Jiang, et al, 2016; Sun, Zhang, et al, 2016). Other study has shown that MrgC undergoes ubiquitination followed by enhanced receptor endocytosis and down‐regulation of receptor number, thereby down‐regulating signal transduction and agonist actions (Huang et al, 2021). Therefore, reversing the ubiquitination of MrgC receptors may play an integral role in regulating MrgC receptor numbers and signal transduction.…”