The conformational change responsible for AT(1) receptor activation is dependent upon two juxtaposed asparagine residues on transmembrane helices III and VII

A model of the angiotensin AT(1) receptor and site-directed mutagenesis were used to identify key residues involved in ligand binding. Receptors were stably expressed in human embryonic kidney 293 cells, and their binding properties compared. Wild type receptors exhibited low and high affinity binding sites for peptides, Substitution of Asn(111), situated in the third transmembrane helix, resulted in a significant alteration in ligand binding with only high affinity binding of the peptides, angiotensin II, angiotensin III, and [p-amino-Phe(6)]angiotensin II and a marked loss in the binding affinity of the AT(1) receptor selective non-peptide antagonist losartan. From our model it was apparent that Asn(111) was in close spatial proximity to Asn(295) in the seventh transmembrane helix. Substitution of Asn(295), produced identical changes in the receptor's pharmacological profile. Furthermore, the Ser(111)AT(1)A and Ser(295)AT(1A) mutants did not require the association of a G-protein for high affinity agonist binding. Finally, the Ser(296)AT(1A) mutant maintained higher basal generation of inositol trisphosphate than the wild type, indicating constitutive activation. We propose that substitution of these residues causes the loss of an interaction between transmembrane helices III and VII, which allows the AT(1) receptor to ''relax'' into its active conformation.