Abstract
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.
Highlights
The peptide hormone angiotensin II (AngII) binds to the AT1 receptor within the transmembrane domains in an extended conformation, and its C-terminal residue interacts with transmembrane domain VII at Phe-293/Asn-294
All known physiological effects of AngII are produced through the activation of the hAT1 receptor, which belongs to the class A rhodopsin-like family of the heptahelical G proteincoupled receptor (GPCR) superfamily [1, 2]
We previously identified ligandcontact points within the second extracellular loop (ECL) and the seventh transmembrane domain (TMD) of the hAT1 receptor [12, 15, 16]
Summary
AngII, angiotensin II; hAT1, human angiotensin type 1 (receptor); Bpa, p-benzoyl-L-phenylalanine; ECL, extracellular loop; GPCR, G-protein coupled receptors; MPA, methionine proximity assay; SCAM, substituted cysteine accessibility method; TMD, transmembrane domain; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; wt, wild type. Previous photochemical studies of the benzophenone radical have indicated that it exhibits strong selectivity for thioether groups [20, 21] This selectivity would explain the high ratio of methionine insertion into target proteins through benzophenone photoaffinity labeling. This property can be exploited to introduce Met residues into target structures as “bait” with the goal of identifying other receptor residues that are in close proximity to the ligand label. The immediate molecular environment of this ligand residue can be determined We used this strategy to investigate the binding environment of the C-terminal residue of AngII within the hAT1 receptor. A systematic Met mutagenesis strategy was applied to TMD III, TMD VI, and TMD VII to identify other receptor contacts and define the binding environment of the C-terminal residue of AngII
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