Determination of peptide contact points in the human angiotensin II type I receptor (AT1) with photosensitive analogs of angiotensin II

To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT(1)), two different radio-labeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-L-phenylalanine (Bpa). High yield, specific labeling of the AT(1) receptor was obtained with the I-125-[Sar(1),Bpa(8)]AngII analog. Digestion of the covalent I-125-[Sar(1),Bpa(8)]AngII-AT(1) complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar(1),Bpa8]AngII-AT(1) complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of I-125-[Sar(1),Bpa(8)]AngII within amino acids 285 and 295 of the AT(1) receptor. When the AT(1) receptor was photolabeled with I-125-[Bpa(1)]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of I-125-[Bpa(1)]AngII within amino acids 147 and 199 of the Af, receptor. CNBr digestion of the hAT(1) I165M mutant receptor narrowed down the labeling site to the fragment 166-199. Taken together, these results indicate that the seventh transmembrane domain of the AT(1) receptor interacts strongly with the C-terminal amino acid of [Sar(1), Bpa(8)]AngII, whereas the N-terminal amino acid of [Bpa(1)]AngII interacts with the second extracellular loop of the AT(1) receptor.