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J. Biol. Chem., Vol. 283, Issue 16, 10735-10744, April 18, 2008

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Post-activation-mediated Changes in Opioid Receptors Detected by N-terminal Antibodies^*

Achla Gupta, Raphael Rozenfeld, Ivone Gomes, Kirsten M. Raehal¹, Fabien M. Décaillot, Laura M. Bohn, and Lakshmi A. Devi²

From the Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York 10029 and the Department of Pharmacology and Psychiatry, The Ohio State University College of Medicine & Public Health, Columbus, Ohio 43210

The majority of studies examining activity-induced conformational changes in G protein-coupled receptors have focused on transmembrane helices or intracellular regions. Relatively few studies have examined the involvement of the extracellular region in general and the N-terminal region in particular in this process. To begin to address this, we generated a series of antibodies to the N-terminal region of opioid receptors. Characterization of these antibodies revealed that they differentially recognize activated receptors. Recently, we generated monoclonal antibodies that recognize regions proximal to glycosylation sites in the receptor N terminus. Characterization of these antibodies revealed that agonist treatment leads to a decrease in epitope recognition by the antibody presumably because of a movement of the region of the N terminus proximal to glycosylation sites. The time course of the decrease in antibody recognition suggested that it could be due to a post-activation-mediated event. Examination of the involvement of receptor residues in the C-tail and β-arrestin binding using site-directed mutagenesis and cells or tissues lacking β-arrestin 2 suggests a role for these desensitization-related mechanisms in governing antibody binding to the receptor. Thus, these N-terminally directed antibodies can differentially recognize post-activation-mediated changes in the C-terminal (intracellular) region of the receptor. Therefore, these conformation-sensitive antibodies represent powerful reagents to probe receptor activation states and provide a potential tool for identifying and characterizing new compounds of therapeutic interest.

Received for publication, November 19, 2007 , and in revised form, February 1, 2008.

^* This work was supported in part by National Institutes of Health Grants DA08863, DA19521, and NS053751 (to L. A. D.) and DA14600 and DA18860 (to L. M. B.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5.

¹ Recipient of National Institutes of Health Ruth L. Kirschstein NRSA Grant DA021952.

² To whom correspondence should be addressed: Dept. of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, 19-84 Annenberg Bldg., One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-8345; Fax: 212-996-7214; E-mail: lakshmi.devi{at}mssm.edu.

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