THE CONFORMATIONAL CHANGE RESPONSIBLE FOR AT(1) RECEPTOR ACTIVATION IS DEPENDENT UPON 2 JUXTAPOSED ASPARAGINE RESIDUES ON TRANSMEMBRANE-HELICES-III AND TRANSMEMBRANE-HELICES-VII

A model of the angiotensin AT(1) receptor and site-directed mutagenesi s were used to identify key residues involved in ligand binding. Recep tors were stably expressed in human embryonic kidney 293 cells, and th eir binding properties compared. Wild type receptors exhibited low and high affinity binding sites for peptides, Substitution of Asn(111), s ituated in the third transmembrane helix, resulted in a significant al teration in ligand binding with only high affinity binding of the pept ides, angiotensin II, angiotensin III, and [p-amino-Phe(6)]angiotensin II and a marked loss in the binding affinity of the AT(1) receptor se lective non-peptide antagonist losartan. From our model it was apparen t that Asn(111) was in close spatial proximity to Asn(295) in the seve nth transmembrane helix. Substitution of Asn(295), produced identical changes in the receptor's pharmacological profile. Furthermore, the Se r(111)AT(1)A and Ser(295)AT(1A) mutants did not require the associatio n of a G-protein for high affinity agonist binding. Finally, the Ser(2 96)AT(1A) mutant maintained higher basal generation of inositol trisph osphate than the wild type, indicating constitutive activation. We pro pose that substitution of these residues causes the loss of an interac tion between transmembrane helices III and VII, which allows the AT(1) receptor to ''relax'' into its active conformation.