Ligands Regulate Cell Surface Level of the Human kappa Opioid Receptor by Activation-Induced Down-Regulation and Pharmacological Chaperone-Mediated Enhancement:Differential Effects of Nonpeptide and Peptide Agonists

摘要

Two peptide agonists,eight nonpeptide agonists,and five nonpeptide antagonists were evaluated for their capacity to regulate FLAG(DYKDDDDK)-tagged human kappa opioid receptors(hKORs)stably expressed in Chinese hamster ovary cells after incubation for 4 h with a ligand at a concentration approx 1000-fold of its EC_(50)(agonist)or K_i(antagonist)value.Dynorphins A and B decreased the fully glycosylated mature form(55-kDa)of FLAG-hKOR by 70%,whereas nonpeptide full agonists [2-(3,4-dichlorophenyl)-N-methyl-N-[(2R)-2-pyrrolidin-1-ylcyclohexyl-]acetamide(U50,488H),17-cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[N-methyl-trans-3-(3-furyl)acrylamido] morphinan hydrochloride(TRK-820),ethylketocyclazocine,bremazocine,asimadoline,and(RS)-[3-[1-[[(3,4-dichlorophenyl)acetyl]-methylamino]-2-(1-pyrrolidinyl)etnyl]phenoxy] acetic acid hydrochloride(ICI 204,448)caused 10-30% decreases.In contrast,pentazocine(partial agonist)and etorphine(full agonist)up-regulated by approx 15 and 25%,respectively.The antagonists naloxone and norbinaltorphimine also significantly increased the 55-kDa receptor,whereas selective mu,delta,and D_1 receptor antagonists had no effect.Naloxone up-regulated the receptor concentration-and time-dependently and enhanced the receptor maturation extent,without affecting its turnover.Treatment with brefeldin A(BFA),which disrupts Golgi,resulted in generation of a 51-kDa form that resided intracellularly.Naloxone up-regulated the new species,indicating that its action site is in the endoplasmic reticulum as a pharmacological chaperone.After treatment with BFA,all nonpeptide agonists up-regulated the 51-kDa form,whereas dynorphins A and B did not,indicating that nonpeptide agonists act as pharmacological chaper-ones,but peptide agonists do not.BFA treatment enhanced down-regulation of the cell surface receptor induced by nonpeptide agonists,but not that by peptide agonists,and unmasked etorphine-and pentazocine-mediated receptor down-regulation.These results demonstrate that ligands have dual effects on receptor levels:enhancement by chaperone-like effects and agonist-promoted down-regulation,and the net effect reflects the algebraic sum of the two.