Journal of Biological Chemistry
Volume 270, Issue 48, 1 December 1995, Pages 28511-28514
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Protein Chemistry and Structure
Interaction of Phe8 of Angiotensin II with Lys199 and His256 of AT1 Receptor in Agonist Activation (*)

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The acidic pharmacophores of selective ligands bind to Lys199 and His256 of the AT1 receptor (Noda, K., Saad, Y., Kinoshita, A., Boyle, T. P., Graham, R. M., Husain, A., and Karnik, S.(1995) J. Biol. Chem. 270, 2284-2289). In this report we examine how interactions between these residues and agonists activate inositol phosphate production in transiently transfected COS-1 cells. [Sar1] angiotensin (Ang II) II and [Sar1]Ang II-amide stimulated a 5-fold inositol phosphate response from wild-type AT1 receptor. The peptide antagonist [Sar1,Ile8]Ang II and the non-peptide agonist L-162,313 produced a partial but saturating response. Stimulation of wild-type receptor by [Sar1]Ang II-amide and the mutant K199Q and K199A receptors by [Sar1]Ang II demonstrates that AT1 receptor activation is not critically dependent on the ion-pairing of the α-COOH group of Ang II with Lys199. The mutation of His256 produced diminished inositol phosphate response without commensurate change in binding affinity of ligands. The His256 side chain is critical for maximal activation of the AT1 receptor, although isosteric Gln substitution is sufficient for preserving the affinity for Phe8-substituted analogues of [Sar1]Ang II. Therefore, AT1 receptor activation requires interaction of Phe8 side chain of Ang II with His256, which is achieved by docking the α-COOH group of Phe8 to Lys199. Furthermore, non-peptide agonists interact with Lys199 and His256 in a similar fashion.

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(*)

This work was supported in part by Specialized Center of Research in Hypertension Grant HL33713 from the National Institutes of Health, a grant-in-aid from the American Heart Association/Northeast Ohio Affiliate Inc. (HANEO), and a HANEO fellowship (to K. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.