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J. Biol. Chem., Vol. 280, Issue 29, 27121-27129, July 22, 2005

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Determining the Environment of the Ligand Binding Pocket of the Human Angiotensin II Type I (hAT₁) Receptor Using the Methionine Proximity Assay^*

Martin Clément, Stéphane S. Martin, Marie-Ève Beaulieu, Caroline Chamberland, Pierre Lavigne, Richard Leduc¶, Gaétan Guillemette, and Emanuel Escher, Recipient of the J.C. Edwards Chair in Cardiovascular Research||

From the Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada

The peptide hormone angiotensin II (AngII) binds to the AT₁ (angiotensin type 1) receptor within the transmembrane domains in an extended conformation, and its C-terminal residue interacts with transmembrane domain VII at Phe-293/Asn-294. The molecular environment of this binding pocket remains to be elucidated. The preferential binding of benzophenone photolabels to methionine residues in the target structure has enabled us to design an experimental approach called the methionine proximity assay, which is based on systematic mutagenesis and photolabeling to determine the molecular environment of this binding pocket. A series of 44 transmembrane domain III, VI, and VII X Met mutants photolabeled either with ¹²⁵I-[Sar¹,p'-benzoyl-L-Phe⁸]AngII or with ¹²⁵I-[Sar¹,p''-methoxy-p'-benzoyl-L-Phe⁸]AngII were purified and digested with cyanogen bromide. Several mutants produced digestion patterns different from that observed with wild type human AT₁, indicating that they had a new receptor contact with position 8 of AngII. The following residues form this binding pocket: L112M and Y113M in transmembrane domain (TMD) III; F249M, W253M, H256M, and T260M in TMD VI; and F293M, N294M, N295M, C296M, and L297M in TMD VII. Homology modeling and incorporation of these contacts allowed us to develop an evidence-based molecular model of interactions with human AT₁ that is very similar to the rhodopsin-retinal interaction.

Received for publication, December 3, 2004 , and in revised form, April 4, 2005.

The atomic coordinates and structure factors (code 1ZV0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Canadian Institutes of Health Research and the Quebec chapter of the Canadian Heart and Stroke Foundation. 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 material in the form of a sequence alignment between the hAT₁ receptor and the bovine rhodopsin and a model of the [Sar¹,Bpa⁸]AngII-liganded hAT₁ receptor with Protein Data Bank information as well.

Submitted to fulfill the requirements of a Ph.D. thesis at the Université de Sherbrooke.

Holder of a scholarship from the Fonds Québécois de la recherche sur la nature et les technologies.

^¶ Senior scholar of the Fonds de la recherche en Santé du Québec.

^|| To whom correspondence should be addressed: Dept. of Pharmacology, Faculty of Medicine, Université de Sherbrooke, 3001 12th Ave. N., Sherbrooke, Quebec J1H 5N4, Canada. Tel.: 819-564-5346; Fax: 819-564-5400; E-mail: Emanuel.Escher{at}USherbrooke.ca.

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