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J. Biol. Chem., Vol. 282, Issue 32, 23171-23183, August 10, 2007

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Opioid Receptor Pharmacological Chaperones Act by Binding and Stabilizing Newly Synthesized Receptors in the Endoplasmic Reticulum^*

From the Biocenter Oulu and Department of Anatomy and Cell Biology, University of Oulu, FI-90014 Oulu, Finland

Accumulating evidence has indicated that membrane-permeable G protein-coupled receptor ligands can enhance cell surface targeting of their cognate wild-type and mutant receptors. This pharmacological chaperoning was thought to result from ligand-mediated stabilization of immature receptors in the endoplasmic reticulum (ER). In the present study, we directly tested this hypothesis using wild-type and mutant forms of the human -opioid receptor as models. ER-localized receptors were isolated by expressing the receptors in HEK293 cells under tightly controlled tetracycline induction and blocking their ER export with brefeldin A. The ER-retained -opioid receptor precursors were able to bind [³H]diprenorphine with high affinity, and treatment of cells with an opioid antagonist naltrexone led to a 2-fold increase in the number of binding sites. After removing the transport block, the antagonist-mediated increase in the number of receptors was detectable at the cell surface by flow cytometry and cell surface biotinylation assay. Importantly, opioid ligands, both antagonists and agonists, were found to stabilize the ER-retained receptor precursors in an in vitro heat inactivation assay and the treatment enhanced dissociation of receptor precursors from the molecular chaperone calnexin. Thus, we conclude that pharmacological chaperones facilitate plasma membrane targeting of -opioid receptors by binding and stabilizing receptor precursors, thereby promoting their release from the stringent ER quality control.

Received for publication, November 27, 2006 , and in revised form, June 4, 2007.

^* This work was supported by the Biocenter Oulu and by Academy of Finland Grant 200732. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Research fellow of the Academy of Finland. To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FI-90014 Oulu, Finland. Tel.: 358-8-537-5193; Fax: 358-8-537-5172; E-mail: Ulla.Petaja-Repo{at}oulu.fi.

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