Secondary structure model of the rat AT_1A receptor mutant DRY/AAY (A) and sequences of Ang II peptide and its analog [Sar¹,Ile⁴,Ile⁸]Ang II (B). (A) Change of DRY to AAY is indicated by arrows. Residues shown in filled circles are conserved in many 7MSRs. DRY/AAY mutant expressed equally well as the wild-type AT_1A receptor in all experiments performed. (B) The K_d values of Ang II and [Sar¹,Ile⁴,Ile⁸]Ang II for the wild-type AT_1A receptor have been reported by Holloway et al. (3). The K_d value for the DRY/AAY mutant has been reported to be 2 nM by Gaborik et al. (11). All of these findings have been verified in this study.

[Sar¹,Ile⁴,Ile⁸]Ang II-bound AT_1A receptors and Ang II-bound DRY/AAY mutant receptors are able to induce translocation of β-arrestin 2-GFP into endocytic vesicles. HEK-293 cells were transiently transfected with expression vectors encoding β-arrestin 2-GFP and wild-type (B and D) or DRY/AAY mutant AT_1A receptors (A and C). Cells were not stimulated (A), stimulated with 160 nM Ang II (B and C), or stimulated with 30 μM [Sar¹,Ile⁴,Ile⁸]Ang II (D) for 30 min. Cells were fixed with 5% formaldehyde, and the recruitment of β-arrestin 2-GFP was examined by confocal microscopy. The distribution pattern of β-arrestin 2-GFP in cells that were not stimulated is the same between cells expressing the wild-type and DRY/AAY mutant AT_1A receptors. The results shown are representative of three experiments.

Effect of the PKC inhibitor Ro-31-8425 on Ang II- and [Sar¹,Ile⁴,Ile⁸]Ang II (SII)-induced ERK1/2 activation. (A) HEK-293 cells were transfected with control (CTL) siRNA or β-arrestin 2 siRNA, and expression vectors encoding wild-type AT_1A receptors. Cells were pretreated with or without Ro-31-8425 for 10 min followed by 5-min stimulation by Ang II or [Sar¹,Ile⁴,Ile⁸]Ang II. NS, no stimulation. As a control for the effectiveness of the Ro-31-8425, HEK-293 cells were pretreated with or without Ro-31-8425 for 10 min followed by 5-min stimulation by PMA. The activation of ERK1/2 was determined by immunoblotting with a phospho-ERK1/2-specific antibody (p-Erk1/2). (B) The effects of β-arrestin 2 siRNA and Ro-31-8425 on Ang II-induced ERK1/2 activation were compared by normalizing each phospho-ERK1/2 signal to the response induced by Ang II in non-inhibitor-treated cells transfected with control siRNA (n = 4). *, P < 0.01, compared with the ERK1/2 activation induced by Ang II-stimulated wild-type AT_1A receptors; **, P < 0.01, compared as indicated.

[Sar¹,Ile⁴,Ile⁸]Ang II-bound AT_1A receptors and Ang II-bound DRY/AAY mutant receptors fail to induce IP accumulation and [³⁵S]GTPγS binding to cell membranes. (A) HEK-293 cells were transiently transfected with expression vectors encoding wild-type or DRY/AAY AT_1A mutant receptors. Cells were then labeled with myo-[³H]inositol for 20 h, and the dose–response of Ang II- and [Sar¹,Ile⁴,Ile⁸]Ang II-induced accumulation of IP was measured. Each value was then normalized to a percentage of the maximum production of IP mediated by the wild-type receptor on Ang II treatment in each experiment (n = 4). (B) Cell membranes were prepared, and the agonist-stimulated binding of [³⁵S]GTPγS to cell membranes was measured in the presence of the indicated concentrations [Conc (μM)] of agonists. Data are expressed as percentage increase over the basal values obtained in the absence of agonists (n = 4).

β-Arrestin 2 mediates ERK1/2 activation induced by [Sar¹,Ile⁴,Ile⁸]Ang II-bound AT_1A receptors and the Ang II-bound DRY/AAY mutant receptors. (A) HEK-293 cells were transfected with control siRNA and expression vectors encoding wild-type or DRY/AAY-mutant AT_1A receptors. Three days after transfection, cells were cultured in serum-free medium for 4 h and stimulated with 160 nM Ang II or 30 μM [Sar¹,Ile⁴,Ile⁸]Ang II for 5 min. The activation of ERK1/2 was determined by immunoblotting with a phospho-ERK1/2-specific antibody. The levels of phospho-ERK1/2 were then quantified and normalized to the phospho-ERK1/2 signal induced by Ang II-stimulated wild-type AT_1A receptors in each experiment. **, P < 0.01, compared with the phospho-ERK1/2 induced by Ang II-stimulated wild-type AT_1A receptors (n = 4). (B–G) HEK-293 cells were transfected with control siRNA (CTL siRNA) or β-arrestin 2 siRNA and expression vectors encoding wild-type (WT) (B–E) or DRY/AAY-mutant (F and G)AT_1A receptors. The activation of ERK1/2 in cells stimulated with different concentrations of Ang II (B, C, F, and G) or [Sar¹,Ile⁴,Ile⁸]Ang II (D and E) was determined as described in A. The amounts of total ERK1/2 and β-arrestin 2 were determined by stripping the membrane and immunoblotting for total ERK1/2 and β-arrestin 2. The effect of β-arrestin 2 siRNA on dose-dependent ERK1/2 activation induced by Ang II (C, n = 4)- or [Sar¹,Ile⁴,Ile⁸]Ang II-stimulated wild-type AT_1A receptors (E, n = 6), or the Ang II-stimulated DRY/AAY mutant receptors (G, n = 4) was plotted by normalizing to the maximum phospho-ERK1/2 signal in each experiment. *, P < 0.05; **, P < 0.01, compared with the phospho-ERK1/2 signal in the corresponding CTL siRNA-transfected cells.