Endothelin-converting enzyme 2 differentially regulates kappa opioid receptor trafficking and function

Following activation by endogenous opioid peptides, mu and delta opioid receptors have been shown to undergo differential internalization and recycling; the rate and extent of recycling but not internalization was found to be regulated by the endocytic peptide converting enzyme, ECE2. This study focuses on kappa opioid receptors (KOR) and the ability of endogenous opioid peptides released by post-translational processing of prodynorphin and proenkephalin to induce KOR and ECE2 internalization/recycling, and how ECE2 modulates these processes. Using a proximity-based ligation assay we show that KOR and ECE2 are in close proximity to facilitate co-internalization. In addition, we find that treatment with longer opioid peptides induces fast and robust internalization and recycling of ECE2 at a rate and extent comparable to that of KOR. Next, we directly examined the role of ECE2 in modulating KOR recycling. We find that ECE2 inhibition significantly attenuates KOR recycling. Finally, we examined the role of ECE2 in modulating KOR signaling and find that resensitization of KOR by peptides that are substrates of ECE2 are attenuated by ECE2 inhibition. Taken with the differential expression of ECE2 in the brain (relatively high expression in midbrain & hypothalamus and low expression in the striatum & hippocampus), these results highlight a pivotal role for ECE2 in differentially modulating KOR function. Significance StatementThis study highlights a role for ECE2 in agonist mediated regulation of KOR function by select prodynorphin and proenkephalin derived peptides. Collectively our studies suggest that ECE2 inhibitors could be developed as therapeutics for pathologies involving dysregulations in KOR signaling.