Opioid receptor function is regulated by post-endocytic peptide processing.
Most neuroendocrine peptides are generated in the secretory compartment by proteolysis of the precursors at classical cleavage sites consisting of basic residues by well studied endopeptidases belonging to the subtilisin superfamily. In contrast, a subset of bioactive peptides is generated by processing at non-classical cleavage sites that do not contain basic residues. Neither the peptidases responsible for non-classical cleavages nor the compartment involved in such processing has been well established. Members of the endothelin-converting enzyme (ECE) family are considered good candidate enzymes because they exhibit functional properties that are consistent with such a role. In this study we have explored a role for ECE2 in endocytic processing of δ opioid peptides and its effect on modulating δ opioid receptor function by using selective inhibitors of ECE2 that we had identified previously by homology modeling and virtual screening of a library of small molecules. We found that agonist treatment led to intracellular co-localization of ECE2 with δ opioid receptors. Furthermore, selective inhibitors of ECE2 and reagents that increase the pH of the acidic compartment impaired receptor recycling by protecting the endocytosed peptide from degradation. This, in turn, led to a substantial decrease in surface receptor signaling. Finally, we showed that treatment of primary neurons with the ECE2 inhibitor during recycling led to increased intracellular co-localization of the receptors and ECE2, which in turn led to decreased receptor recycling and signaling by the surface receptors. Together, these results support a role for differential modulation of opioid receptor signaling by post-endocytic processing of peptide agonists by ECE2.
From the Department of Pharmacology and Systems Therapeutics and.
Achla
A
Gupta
From the Department of Pharmacology and Systems Therapeutics and.
Ivone
I
Gomes
From the Department of Pharmacology and Systems Therapeutics and.
Jonathan
J
Wardman
From the Department of Pharmacology and Systems Therapeutics and the Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029 lakshmi.devi@mssm.edu.
Lakshmi A
LA
Devi
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis / Q000502:physiology*; D000072396:Endothelin-Converting Enzymes; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D010455:Peptides; D017465:Receptors, Opioid, delta; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606926:Ece2 protein, mouse; D000072396:Endothelin-Converting Enzymes
Enkephalin; G Protein-coupled Receptor (GPCR); Morphine; Neprilysin; Opiates; Receptor Desensitization; Receptor Recycling; Receptor Regulation; Resensitization; Signal Transduction