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J. Biol. Chem., Vol. 282, Issue 8, 5116-5124, February 23, 2007

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Conformation State-sensitive Antibodies to G-protein-coupled Receptors^*

Achla Gupta, Fabien M. Décaillot, Ivone Gomes, Oleg Tkalych, Andrea S. Heimann, Emer S. Ferro^¶, and Lakshmi A. Devi¹

From the Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029, Proteimax S/C, Via das Margaridas 413, 06700-020 Cotia, São Paulo, Brazil, and the ^¶Department of Cell Biology and Development, University of São Paulo, 05504-900 São Paulo, Brazil

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of µ-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of µ receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize µ receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native µ receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G_i-coupled (-opioid, CB1 cannabinoid, _2A-adrenergic) as well as G_s-(₂-adrenergic) and G_q-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.

Received for publication, September 29, 2006 , and in revised form, November 24, 2006.

^* This work was supported by National Institutes of Health Grants DA08863 and DA19521 (to L. A. D.) and by São Paulo State Research Foundation Grants 04/04933-2 (to E. S. F.) and 04/14258-0 (to A. S. H.). 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 Fig. 1 and Tables 1–5.

¹ To whom correspondence should be addressed: Dept. of Pharmacology and Biological Chemistry, 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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