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Received for publication May 17, 2006.
Revised July 29, 2006.
Accepted for publication July 31, 2006.
opioid receptor (hKOR) by activation-induced down-regulation and pharmacological chaperone-mediated enhancement: differential effects of non-peptide and peptide agonists
Two peptide agonists, 8 non-peptide agonists, and 5 non-peptide antagonists were evaluated for their capacity to regulate FLAG-tagged hKOR stably expressed in CHO cells following incubation for 4 h with a ligand at a concentration ~1000-fold of its EC50 (agonist) or Ki (antagonist) value. Dynorphin A and B decreased the fully glycosylated mature form (55-kDa) of FLAG-hKOR by 70%, whereas non-peptide full agonists (U50,488H, TRK820, ethylketocyclazocine, bremazocine, asimadoline and ICI204,448) caused 10~30% decreases. In contrast, pentazocine (partial agonist) and etorphine (full agonist) up-regulated by ~15% and 25%, respectively. The antagonists naloxone and Nor-BNI also significantly increased the 55-kDa receptor, whereas selective µ, 
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 ER as a pharmacological chaperone. Following treatment with BFA, all non-peptide agonists up-regulated the 51-kDa form, whereas dynorphins A and B did not, indicating that non-peptide agonists act as pharmacological chaperones, but peptide agonists do not. BFA treatment enhanced down-regulation of cell surface receptor induced by non-peptide agonists, but not by the 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 effect and agonist-promoted down-regulation and the net effect reflects the algebraic sum of the two.
Key words:
Biosynthesis, Cell surface expression, ER-to-Golgi transport, Intracellular trafficking, Post-activation receptor adaptation, Receptor maturation
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