Mutational analysis of the interaction of the N‐ and C‐terminal ends of angiotensin II with the rat AT1A receptor

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Abstract
The role of different residues of the rat AT1A receptor in the interaction with the N‐ and C‐terminal ends of angiotensin II (AngII) was studied by determining ligand binding and production of inositol phosphates (IP) in COS‐7 cells transiently expressing the following AT1A mutants: T88H, Y92H, G196I, G196W and D278E. G196W and G196I retained significant binding and IP‐production properties, indicating that bulky substituents in position 196 did not affect the interaction of AngII's C‐terminal carboxyl with Lys199 located three residues below. Although the T88A mutation did not affect binding, the T88H mutant had greatly decreased affinity for AngII, suggesting that substitution of Thr88 by His might hinder binding through an indirect effect. The Y92H mutation caused loss of affinity for AngII that was much less pronounced than that reported for Y92A, indicating that His in that position can fulfil part of the requirements for binding. Replacing Asp278 by Glu caused a much smaller reduction in affinity than replacing it by Ala, indicating the importance of Asp's β‐carboxyl group for AngII binding. Mutations in residues Thr88, Tyr92 and Asp278 greatly reduced affinity for AngII but not for Sar1 Leu8‐AngII, suggesting unfavourable interactions between these residues and AngII's aspartic acid side‐chain or N‐terminal amino group, which might account for the proposed role of the N‐terminal amino group of AngII in the agonist‐induced desensitization (tachyphylaxis) of smooth muscles. British Journal of Pharmacology (2000) 130, 1263–1268; doi:10.1038/sj.bjp.0703430