Residues 293 and 294 Are Ligand Contact Points of the Human Angiotensin Type 1 Receptor

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Abstract
The human angiotensin II type 1 receptor (hAT1) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, 125I-[Sar1, Val5, p-Benzoyl-L-phenylalanine8]AngII (125I-[Sar1,Bpa8]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285−334) of the C-terminal portion of the hAT1. Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe293 and Asn294 within the seventh transmembrane domain of the hAT1. Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS−PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT1 produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT1 receptor. Taking into account a second receptor−ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry39, 9662−70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.

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