Inhibition of μ and δ opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin

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We reported recently that the ubiquitin–proteasome pathway is involved in agonist-induced down regulation of μ and δ opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl- l-Leucyl- l-Leucyl- l-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected μ, δ, or κ opioid receptors. The rank order of potency for leupeptin inhibition of [ 3H]bremazocine binding to opioid receptors was μ>δ>κ. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy- l-Leucyl- l-Leucyl- l-Leucinal), had significantly less effect on bremazocine binding to μ, δ, or κ opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions.