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Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT₁) with Photosensitive Analogs of Angiotensin II

Department of Pharmacology Medical School Université de Sherbrooke Sherbrooke, Québec J1H 5N4 Canada

To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT₁), two different radiolabeled 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 AT₁ receptor was obtained with the ¹²⁵I-[Sar¹,Bpa⁸]AngII analog. Digestion of the covalent ¹²⁵I-[Sar¹,Bpa⁸]AngII-AT₁ complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar¹,Bpa⁸]AngII-AT₁ 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 ¹²⁵I-[Sar¹,Bpa⁸]AngII within amino acids 285 and 295 of the AT₁ receptor. When the AT₁ receptor was photolabeled with ¹²⁵I-[Bpa¹]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 ¹²⁵I-[Bpa¹]AngII within amino acids 147 and 199 of the AT₁ receptor. CNBr digestion of the hAT₁ 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 AT₁ receptor interacts strongly with the C-terminal amino acid of [Sar¹, Bpa⁸]AngII, whereas the N-terminal amino acid of [Bpa¹]AngII interacts with the second extracellular loop of the AT₁ receptor.

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