Mutagenesis and the molecular modeling of the rat angiotensin II receptor (AT1).
The molecular interaction involved in the ligand binding of the rat angiotensin II receptor (AT1A) was studied by site-directed mutagenesis and receptor model building. The three-dimensional structure of AT1A was constructed on the basis of a multiple amino acid sequence alignment of seven transmembrane domain receptors and angiotensin II receptors and after the beta 2 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, His166, Arg167, Glu173, His183, Glu185, Lys199, Trp253, His256, Phe259, Thr260, and Asp263, 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 Lys199-->Ala, Trp253-->Ala, Phe259-->Ala, Asp263-->Ala, and Arg167-->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 AT1A. Binding of EXP985, a nonpeptide angiotensin II antagonist, was found to be involved with Arg167 but not Lys199.