Photolabeling Identifies Position 172 of the Human AT1 Receptor as a Ligand Contact Point: Receptor-Bound Angiotensin II Adopts an Extended Structure

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Abstract
An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-l-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT1 receptor. High-affinity binding of the analogue, 125I-[Bpa3]AngII, to the AT1 receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the 125I-[Bpa3]AngII−AT1 complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143−243 of the AT1 receptor. Digestion of the receptor−ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of 125I-[Bpa3]AngII within residues 168−199 of the AT1 receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168−199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand 125I-[Bpa3]AngII forms a covalent bond with the side chain of Met172 of the second extracellular loop of the AT1 receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended β-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.

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