Identification of angiotensin II-binding domains in the rat AT(2) receptor with photolabile angiotensin analogs

To identify binding domains between angiotensin II (AngII) and its type 2 receptor (AT(2)), two different radiolabeled photoreactive analogs were prepared by replacing either the first or the last amino acid in the peptide with p-benzoyl-L-phenylalanine (Bpa). Digestion of photolabeled receptors with kallikrein revealed that the two photoreactive analogs label the amino-terminal part of the receptor within the first 182 amino acids. Digestion of I-125-[Bpa(1)]AngII . AT(2) receptor complex with endoproteinase Lys-C produced a glycoprotein of 80 kDa. Deglycosylation of this 80-kDa product decreased its apparent molecular mass to 4.6 kDa and further cleavage of this 4.6-kDa product with V8 protease decreased its molecular mass to 3.6 kDa, circumscribing the labeling site of I-125-[Bpa(1)]AngII within amino acids 330 of AT(2) receptor. Treatment of I-125-[Bpa(8)]AngII . AT(2) receptor complex with cyanogen bromide produced two major receptor fragments of 3.6 and 2.6 kDa. Cyanogen bromide hydrolysis of a mutant AT(2) receptor produced two major fragments of 12.6 kDa and 2.6 kDa defining the labeling site of I-125-[Bpa(8)]AngII within residues 129-138 of AT(2) receptor. Our results indicate that the aminoterminal tail of the AT(2) receptor interacts with the amino-terminal end of AngII, whereas the inner half of the third transmembrane domain of AT(2) receptor interacts with the carboxyl-terminal end of AngII.