STRUCTURAL ELEMENTS FOR SPECIFIC BINDING OF ANGIOTENSIN II TO ITS TYPE I RECEPTOR BY SITE-DIRECTED MUTAGENESIS

Abstract

To identify structural components essential for ligand binding in AT₁ receptor, we mutated and transiently expressed the modified rat AT₁ receptors in COS7 cells and examined changes in ligand binding activity. We had already reported that the replacement of any one of 4 Cys with Gly in the extracellular domains markedly reduced the binding affinity. In the present study, we mutated Glu(173) with Gln in the second extracellular loop, and Asp (263) with Asn in the sixth transmembrane domains. The replacement of Asp(263) profundly reduced the binding affinity, but not the maximum binding, whereas the replacement of Glu(173) had a weak effect in the binding affinity. We propose that two extracellular disulfied bridges are essential for the maintenance of active stereo-structure of the receptor, and the charged residuc(Asp 263) in the sixth transmembrane region is important for the binding of the positive charged structure of angiotensin H.

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