The conformational change responsible for AT1 receptor activation is dependent upon two juxtaposed asparagine residues on transmembrane helices III and VII.

A model of the angiotensin AT1 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 Asn111, 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-Phe6]angiotensin II and a marked loss in the binding affinity of the AT1 receptor selective non-peptide antagonist losartan. From our model it was apparent that Asn111 was in close spatial proximity to Asn295 in the seventh transmembrane helix. Substitution of Asn295, produced identical changes in the receptor's pharmacological profile. Furthermore, the Ser111AT1A and Ser295AT1A mutants did not require the association of a G-protein for high affinity agonist binding. Finally, the Ser295AT1A 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 AT1 receptor to "relax" into its active conformation.