Photolabeling identifies position 172 of the human AT(1) receptor as a ligand contact point: Receptor-bound angiotensin II adopts an extended structure

An angiotensin II (AngII) peptidic analogue in which the third residue (val ine) was substituted with the photoreactive p-benzoyl-L-phenyl alanine (Bpa ) was used to identify ligand-binding sites of the human AT(1) receptor. Hi gh-affinity binding of the analogue, I-125-[Bpa(3)]AngII, to the AT(1) rece ptor heterologously expressed in COS-7 cells enabled us to efficiently phot olabel the receptor. Chemical and enzymatic digestions of the I-125-[Bpa(3) ]AngII-AT(1) complex were performed, and receptor fragments were analyzed i n order to define the region of the receptor with which the ligand interact s. Results show that CNBr hydrolysis of the photolabeled receptor gave a gl ycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of th e AT(1) 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 I-125-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which sel ected residues adjacent to residue 168 were replaced by methionine within t he 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage rev ealed that the bound photoligand I-125-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT( 1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformatio n when bound to the receptor and would orient itself within the binding dom ain by having its N-terminal portion interacting with the second extracellu lar loop and its C-terminus interacting with residues of the seventh transm embrane domain.