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Volume 272, Number 13, Issue of March 28, 1997 pp. 8653-8659
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of Angiotensin II-binding Domains in the Rat AT₂ Receptor with Photolabile Angiotensin Analogs

(Received for publication, August 13, 1996, and in revised form, December 20, 1996)

From the Département de Pharmacologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

To identify binding domains between angiotensin II (AngII) and its type 2 receptor (AT₂), 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 ¹²⁵I-[Bpa¹]AngII·AT₂ 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 ¹²⁵I-[Bpa¹]AngII within amino acids 3-30 of AT₂ receptor. Treatment of ¹²⁵I-[Bpa⁸]AngII·AT₂ receptor complex with cyanogen bromide produced two major receptor fragments of 3.6 and 2.6 kDa. Cyanogen bromide hydrolysis of a mutant AT₂ receptor produced two major fragments of 12.6 kDa and 2.6 kDa defining the labeling site of ¹²⁵I-[Bpa⁸]AngII within residues 129-138 of AT₂ receptor. Our results indicate that the amino-terminal tail of the AT₂ receptor interacts with the amino-terminal end of AngII, whereas the inner half of the third transmembrane domain of AT₂ receptor interacts with the carboxyl-terminal end of AngII.

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