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J. Biol. Chem., Vol. 282, Issue 48, 34968-34976, November 30, 2007

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Allosteric Small Molecules Unveil a Role of an Extracellular E2/Transmembrane Helix 7 Junction for G Protein-coupled Receptor Activation^*

Dorothea Jäger¹, Caroline Schmalenbach¹, Stefanie Prilla, Jasmin Schrobang, Anna Kebig², Matthias Sennwitz, Eberhard Heller^¶, Christian Tränkle, Ulrike Holzgrabe^¶, Hans-Dieter Höltje, and Klaus Mohr³

From the Pharmacology and Toxicology Section, Institute of Pharmacy, Rheinische Friedrich-Wilhelms-University, D-53121 Bonn, Germany, the Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, D-40225 Düsseldorf, Germany, and the ^¶Department of Pharmaceutical Chemistry, Institute of Pharmacy, Julius-Maximilians-University, D-97074 Würzburg, Germany

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M₂ acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M₂Trp⁴²² in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.

Received for publication, July 6, 2007 , and in revised form, September 4, 2007.

^* The work was supported by the Deutsche Forschungsgemeinschaft (Mo821/1-4, Ho1368/7-3). 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–S6.

¹ These authors contributed equally to this work.

² Member of Research Training Group GRK677.

³ To whom correspondence should be addressed: Pharmacology and Toxicology Section, Institute of Pharmacy, Gerhard-Domagk-Str.3, D-53121 Bonn, Germany. Tel.: 49-228-739103; Fax: 49-228-739215; E-mail: k.mohr{at}uni-bonn.de.

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