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J. Biol. Chem., Vol. 278, Issue 49, 49487-49494, December 5, 2003

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 278/49/49487    most recent M308010200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Petrel, C. Articles by Ruat, M. Search for Related Content PubMed PubMed Citation Articles by Petrel, C. Articles by Ruat, M. Social Bookmarking                      What's this?

Modeling and Mutagenesis of the Binding Site of Calhex 231, a Novel Negative Allosteric Modulator of the Extracellular Ca²⁺-sensing Receptor^*

Christophe Petrel, Albane Kessler, Fouzia Maslah¶, Philippe Dauban, Robert H. Dodd, Didier Rognan¶, and Martial Ruat||

From the Institut de Neurobiologie Alfred Fessard, Institut Fédératif de Recherche 2118 CNRS, and the Laboratoire de Neurobiologie Cellulaire et Moléculaire, Unité Propre de Recherche 9040 CNRS, and the Institut de Chimie des Substances Naturelles, UPR 2301 CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France and the ^¶Laboratoire de Pharmacochimie de la Communication Cellulaire, Unité Mixte de Recherche 7081 CNRS, 74 route du Rhin, 67401 Illkirch, France

A model of the Ca²⁺-sensing receptor (CaSR) seven transmembrane domains was constructed based on the crystal structure of bovine rhodopsin. This model was used for docking (1S,2S,1'R)-N¹-(4-chlorobenzoyl)-N²-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane (Calhex 231), a novel potent negative allosteric modulator that blocks (IC₅₀ = 0.39 µM) increases in [³H]inositol phosphates elicited by activating the human wild-type CaSR transiently expressed in HEK293 cells. In this model, Glu-837^7.39 plays a pivotal role in anchoring the two nitrogen atoms of Calhex 231 and locating the aromatic moieties in two adjacent hydrophobic pockets delineated by transmembrane domains 3, 5, and 6 and transmembrane domains 1, 2, 3, and 7, respectively. To demonstrate its validity, we have mutated selected residues and analyzed the biochemical and pharmacological properties of the mutant receptors transfected in HEK293 cells. Two receptor mutations, F684A^3.32 and E837A^7.39, caused a loss of the ability of Calhex 231 to inhibit Ca²⁺-induced accumulation of [³H]inositol phosphates. Three other mutations, F688A^3.36, W818A^6.48, and I841A^7.43, produced a marked increase in the IC₅₀ of Calhex 231 for the Ca²⁺ response, whereas L776A^5.42 and F821A^6.51 led to a decrease in the IC₅₀. Our data validate the proposed model for the allosteric interaction of Calhex 231 with the seven transmembrane domains of the CaSR. Interestingly, the residues at the same positions have been shown to delimit the antagonist-binding cavity of many diverse G-protein-coupled receptors. This study furthermore suggests that the crystal structure of bovine rhodopsin exhibits sufficient mimicry to the ground state of a very divergent class 3 receptor to predict the interaction of antagonists with the heptahelical bundle of diverse G-protein-coupled receptors.

Received for publication, July 23, 2003 , and in revised form, September 22, 2003.

^* 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 Table 2 and Supplemental Refs. 1-54.

^|| To whom correspondence should be addressed: Lab. de Neurobiologie Cellulaire et Moléculaire, CNRS, Bâtiment 32-33, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France. Tel.: 33-1-6982-3641; Fax: 33-1-6382-3639; E-mail: ruat{at}nbcm.cnrs-gif.fr.

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