Endothelin-converting enzyme 2 regulates κ opioid receptor trafficking and function

Previous studies show that endothelin-converting enzyme 2 (ECE2), an enzyme involved in nonclassical processing of neuropeptide precursors, regulates the rate and extent of μ and δ opioid receptor recycling, but not internalization following activation by select synthetic and endogenous peptidic agonists. This study focuses on κ opioid receptors (KORs) and examines how prodynorphin and proenkephalin-derived peptides that are substrates of ECE2 modulate the internalization and recycling of both KOR and ECE2; as controls we use peptides that are not ECE2 substrates. First, we use a proximity-based ligation assay to show that KOR and ECE2 are in close proximity to directly interact and cointernalize. Treatment with the peptides induces internalization and recycling of ECE2 at a rate and extent comparable to that of KOR with longer opioid peptides inducing fast and robust internalization and recycling of both ECE2 and KOR compared with shorter peptides. We find that in recombinant cell lines and naïve cells expressing endogenous receptors, a small molecule ECE2 inhibitor attenuates KOR recycling as well as signaling by only peptides that are ECE2 substrates. Taken with the differential expression of ECE2 in the brain (relatively high expression in midbrain and dentate gyrus of the hippocampus and low expression in the striatum), these results highlight a pivotal role for ECE2 in differentially modulating KOR function. SIGNIFICANCE STATEMENT: This study highlights a role for endothelin-converting enzyme 2 in agonist mediated regulation of κ opioid receptor function by select prodynorphin and proenkephalin-derived peptides. Collectively, studies by authors suggest that endothelin-converting enzyme 2 inhibitors could be developed as therapeutics for pathologies involving dysregulations in κ opioid receptor signaling.