Critical role of a conserved intramembrane tyrosine residue in angiotensin II receptor activation.

The rat type 1a (AT1a) angiotensin II (Ang II) receptor contains a highly conserved tyrosine residue in the fifth transmembrane region that is present in most G protein-coupled receptors. The role of this amino acid in AT1 receptor activation was analyzed in a mutant receptor (Y215F) created by replacing Tyr215 with phenylalanine. The mutant receptor was highly expressed in transfected COS-7 cells, and its binding affinity for the peptide antagonist [Sar1,Ile8]Ang II was similar to that of the wild type receptor. Although the structural integrity of the peptide ligand binding domain was preserved in the Y215F mutant receptor, its affinity for the native agonist, Ang II, was significantly reduced. Also, whereas guanosine 5'-3-O-(thio)triphosphate markedly reduced Ang II binding to the wild type receptor, it had little effect on agonist binding to the mutant receptor. Agonist-induced internalization of the mutant receptor was also impaired, and its ability to mediate inositol phosphate responses to Ang II stimulation was abolished. The concomitant decreases in receptor internalization and G protein-mediated signaling of the Y215F mutant receptor indicate that Tyr215 has a critical role in AT1 receptor activation. In view of its conservation among members of the seven transmembrane domain receptor superfamily, this residue is likely to be of general importance in signal transduction from G protein-coupled receptors.