Mutagenesis and the Molecular Modeling of the Rat Angiotensin II Receptor (AT)

doi:10.1074/jbc.270.23.14024

Mutagenesis and the Molecular Modeling of the Rat Angiotensin II Receptor (AT) (*)

From the (1) Laboratory of Metabolic Biochemistry, Faculty of Agriculture, Tottori University, Tottori 680, Japan, the Department of Pediatrics, Yamanashi Medical School, Yamanashi 409-38, Japan, the
(2) Green Cross Corp., Shodai-Ohtani, 2-25-1, Hirakata, Osaka 573, Japan, and the
(3) Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232

^§ To whom correspondence should be addressed. Tel.: 615-322-4347; Fax: 615-343-5244.

Abstract

The molecular interaction involved in the ligand binding of the rat angiotensin II receptor (AT) was studied by site-directed mutagenesis and receptor model building. The three-dimensional structure of AT was constructed on the basis of a multiple amino acid sequence alignment of seven transmembrane domain receptors and angiotensin II receptors and after the β2 adrenergic receptor model built on the template of the bacteriorhodopsin structure. These data indicated that there are conserved residues that are actively involved in the receptor-ligand interaction. Eleven conserved residues in AT¹, His, Arg, Glu, His, Glu, Lys, Trp, His, Phe, Thr, and Asp, were targeted individually for site-directed mutation to Ala. Using COS-7 cells transiently expressing these mutated receptors, we found that the binding of angiotensin II was not affected in three of the mutations in the second extracellular loop, whereas the ligand binding affinity was greatly reduced in mutants Lys Ala, Trp Ala, Phe Ala, Asp Ala, and Arg Ala. These amino acid residues appeared to provide binding sites for Ang II. The molecular modeling provided useful structural information for the peptide hormone receptor AT. Binding of EXP985, a nonpeptide angiotensin II antagonist, was found to be involved with Arg but not Lys.

Footnotes

↵* This work was supported by a scientific grant from the Ministry of Education, Science and Culture, the Uehara Memorial Foundation, Japan, and by United States Public Health Service Research Grants HL-14192 and HL-35323 from the National Institute of Health. 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.
↵¹ The abbreviations used are:

Ang II

angiotensin II

AT¹

angiotensin type 1 receptor

AT²

angiotensin type 2 receptor

GPCR

G-protein coupled receptor

TM

transmembrane domain

bRh

bacteriorhodopsin

β2-AR

β2-adrenergic receptor

Pen

penicillamine

SH2

src homology-2

kb

kilobase(s).
↵²Y. Inoue, Y. Yamamura, N. Nakamura, Y. Yamano, K. Ohyama, T. Inagami, A. Scheiner, M. Wrinn, and J. Andzelm, manuscript in preparation.