Abstract
Two peptide agonists, eight nonpeptide agonists, and five nonpeptide antagonists were evaluated for their capacity to regulate FLAG (DYKDDDDK)-tagged human κ opioid receptors (hKORs) stably expressed in Chinese hamster ovary cells after incubation for 4 h with a ligand at a concentration ∼1000-fold of its EC50 (agonist) or Ki (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)ethyl]phenoxy] acetic acid hydrochloride (ICI 204,448) caused 10–30% decreases. In contrast, pentazocine (partial agonist) and etorphine (full agonist) up-regulated by ∼15 and 25%, respectively. The antagonists naloxone and norbinaltorphimine also significantly increased the 55-kDa receptor, whereas selective μ, δ, and D1 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 chaperones, 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.
Footnotes
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This work was supported by National Institutes of Health Grants DA17302 and DA04745.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.107987.
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ABBREVIATIONS: KOR, κ opioid receptor; U50, 488H, 2-(3,4-dichlorophenyl)-N-methyl-N-[(2R)-2-pyrrolidin-1-ylcyclohexyl]acetamide; U69, 593, (5α, 7α, 8β)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)benzeneacetamide; ICI 204, 448, (RS)-[3-[1-[[(3,4-dichlorophenyl)acetyl]-methylamino]-2-(1-pyrrolidinyl)ethyl]phenoxy] acetic acid hydrochloride; TRK-820,17-cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[N-methyl-trans-3-(3-furyl) acrylamido] morphinan hydrochloride; Nor-BNI, norbinaltorphimine; EBP50/NERF-1, Ezrin-radixin-moesin-binding phosphoprotein-50/Na+,H+-exchanger regulatory factor-1; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; FLAG, DYKDDDDK; FLAG-hKOR, FLAG-tagged human κ opioid receptor; R(+)-SCH 23,0390, (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; CHO-FLAG-hKOR, CHO cell lines stably expressing FLAG-hKOR; BFA, brefeldin A; PBS, phosphate-buffered saline; Dyn, dynorphin; GPCR, G protein-coupled receptor; ICI 118,551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol.
- Received May 17, 2006.
- Accepted July 31, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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