Activation of the AT₁ Angiotensin Receptor Is Dependent on Adjacent Apolar Residues in the Carboxyl Terminus of the Third Cytoplasmic Loop*

The C-terminal region of the third intracellular loop of the AT₁ angiotensin receptor (AT₁-R) is an important determinant of G protein coupling. The roles of individual residues in agonist-induced activation of G_q/11-dependent phosphoinositide hydrolysis were determined by mutational analysis of the amino acids in this region. Functional studies on mutant receptors transiently expressed in COS-7 cells showed that alanine substitutions of the amino acids in positions 232–240 of the third loop had no major effect on signal generation. However, deletion mutations that removed Ile²³⁸or affected its position relative to transmembrane helix VI significantly impaired angiotensin II-induced inositol phosphate responses. Substitution of Ile²³⁸ with an acidic residue abolished the ability of the receptor to mediate inositol phosphate production, whereas its replacement with basic or polar residues reduced the amplitude of inositol phosphate responses. Substitutions of Phe²³⁹ with polar residues had relatively minor effects on inositol phosphate signal generation, but its replacement by aspartic acid reduced, and by positively charged residues (Lys, Arg) significantly increased, angiotensin II-induced inositol phosphate responses. The internalization kinetics of the Ile²³⁸ and Phe²³⁹ mutant receptors were impaired in parallel with the reduction in their signaling responses. These findings have identified Ile²³⁸ and Phe²³⁹ as the critical residues in the C-terminal region of the third intracellular loop of the AT₁-R for receptor activation. They also suggest that an apolar amino acid corresponding to Ile²³⁸ of the AT₁-R is a general requirement for activation of other G protein-coupled receptors by their agonist ligands.