Abstract

His256 (HisVI:16) of transmembrane segment (TM)-VI of the rat angiotensin type 1 (AT1) receptor was targeted for mutagenesis to investigate its potential involvement in ligand binding. Substitution of His256 with alanine, phenylalanine, glutamine, or isoleucine did not affect the binding of either angiotensin II or nine different biphenylimidazole AT1 antagonists. In contrast, the binding affinity of the prototype imidazoleacrylic acid antagonist SKF-108,566 was reduced 15-fold by the exchange of His256 with alanine. Substitution of His256 with either isoleucine or phenylalanine yielded similar results, whereas a glutamine residue was able to substitute for His256, suggesting that the epsilon-nitrogen of His256 could be involved in the interaction with the imidazoleacrylic acid. To identify the chemical groups on SKF-108,566 that interact with His256 and with Asn295, a previously identified interaction point for nonpeptide antagonists located in TM-VII, we tested the binding of 15 analogs of SKF-108,566 in which different chemical moieties were systematically exchanged. The results indicated that the carboxyphenyl group of SKF-108,566 interacts with the imidazole side chain of His256. The data did not point to any particular contact group on the antagonist for Asn295. It is concluded that the imidazoleacrylic acid antagonists share some interactions in TM-VII of the AT1 receptor with the biphenylimidazole antagonists, but the binding of the imidazoleacrylic acid compounds is uniquely dependent on His256 in TM-VI, possibly through the carboxyphenyl moiety.