MUTATION OF ASP(74) OF THE RAT ANGIOTENSIN-II RECEPTOR CONFERS CHANGES IN ANTAGONIST AFFINITIES AND ABOLISHES G-PROTEIN COUPLING

Aspartic acid in the second transmembrane domain is a highly conserved amino acid among the G protein-coupled receptors and is functionally important for agonist binding and G-protein coupling in beta2-adrenerg ic and luteinizing hormone receptors. To determine whether this aspart ic acid is also involved in the function of the rat vascular angiotens in II receptor subtype 1 (AT1a), Asp74 was replaced either by asparagi ne or by glutamic acid. When expressed in CHO cells, the two mutants a nd the wild-type receptor displayed similarly high affinities for the agonist [Sar1, Tyr(I-125)4]angiotensin II [where Sar is sarcosine and Tyr(I-125) is monoiodinated tyrosine] and the other agonists: ([Sar1]a ngiotensin II > angiotensin II > angiotensin III >> angiotensin I). Ho wever, the Asn74 mutant shows striking differences in its affinity for some antagonists when compared with the wild-type receptor: the affin ity for DUP753 was decreased 10-fold, whereas it was increased 6-fold for [Sar1,Ala8]angiotensin II and 20-fold for CGP42112A. These pharmac ological changes were associated with a major defect in transmembrane signaling, since angiotensin II was unable to stimulate inositol phosp hate production and increase cytosolic Ca2+ concentration through the two mutated receptors, whereas a clear dose-dependent stimulation was observed in cells expressing the wild-type receptor. Angiotensin II wa s able to promote DNA synthesis through the wild type but not through the mutated receptors. These data indicate that the conserved Asp74 re sidue of the AT1a receptor is important for the binding of angiotensin II antagonists and is essential for the transmembrane signaling casca de.