Determining the environment of the ligand binding pocket of the human angiotensin II type I (hAT1) receptor using the methionine proximity assay.
The peptide hormone angiotensin II (AngII) binds to the AT0 (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.
Clément
Martin
M
Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.
Martin
Stéphane S
SS
Beaulieu
Marie-Eve
ME
Chamberland
Caroline
C
Lavigne
Pierre
P
Leduc
Richard
R
Guillemette
Gaétan
G
Escher
Emanuel
E
D019943:Amino Acid Substitution; D000804:Angiotensin II / Q000378:metabolism; D001577:Benzophenones; D001665:Binding Sites; D006801:Humans; D008024:Ligands; D008715:Methionine / Q000235:genetics*; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*
D001577:Benzophenones; D008024:Ligands; D044140:Receptor, Angiotensin, Type 1; D000804:Angiotensin II; C047723:benzophenone; D008715:Methionine