Transmembrane elements 3,6 and 7 form the ligand binding pocket of the type 1 angiotensin II receptor(AT_1)

摘要

The cognate receptors of angiotensin II(AngII)are the seven transmembranes G-protein coupled receptors(GPCR)AT_1 and AT_2.We have shown recently that the Angll peptide is bound in a transmembrane geometry,parallel to the transmembrane domains(TMD)of those receptors,with the C-terminal end of Angll towards the cytoplasmic side and the N-terminal end in contact with the extracellular elements of the receptors.A more refined analysis of the C-terminal binding pocket in hAT_1 was initiated by applying the methionine proximity assay(MPA)which took advantage of the Methionine(Met)selectivity of the photoactivated benzophenone moiety.An extensive Met-walk was undertaken through all 7 TMD on the hATl receptor in order to define the three-dimensional binding environment of the C-terminal amino acid of Angll.Those Met-mutants were characterized and then photolabeled with I[Sar,Val,Bpa ]AngII.Labelled receptors were subjected to CNBr digestion(selective for Met residues)and SDS-PAGE analysis.Label release or new fragments in the observed labelling pattern of the methionine mutants compared to those of native hATl indicated such contacts through the 6 A reach of Bpa towards Met residues.