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Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors

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Science Signaling  04 Dec 2018:
Vol. 11, Issue 559, eaat1631
DOI: 10.1126/scisignal.aat1631
  • Fig. 1 AT1R downstream signaling pathways and BRET sensors for G protein and β-arrestin activation.

    (A) AT1R stimulates signaling through several different classes of heterotrimeric G proteins and activation of βarr2. cAMP, adenosine 3′,5′-cyclic monophosphate. (B) Illustration of BRET-based Gα-Gβγ and βarr2-DB sensors. Upon receptor activation, dissociation of the Gα subunit from Gβγ and conformational changes in β-arrestin cause the BRET signal to decrease. L, ligand; GDP, guanosine diphosphate. (C) AT1R-induced G protein activation. HEK293 cells were transfected with AT1R along with the indicated Gα-RlucII (Gαq, Gα12, or Gαi2) plus GFP10-Gγ and Gβ. Cells were preincubated in the absence or presence of the selective Gαq inhibitor UBO-QIC or vehicle and then stimulated with AngII for 2 min (Gαq and Gαi2) or 10 min (Gα12). Data represent means ± SEM from at least three independent experiments. **P < 0.01, unpaired Student’s t test. (D) Concentration-response curve of the βarr2-DB sensor upon AngII stimulation of AT1R. Cells were transfected with AT1R along with βarr2-DB and stimulated with the indicated concentrations of AngII for 20 min. Data represent means ± SEM from nine independent experiments.

  • Fig. 2 Generation and validation of BRET-based sensors for activation of PLC, PKC, p63/Gαq, and Rho.

    (A) Schematic diagram of the DAG BRET sensor, which measures the generation of DAG by activated PLC. The recruitment of c1b to the PM by DAG increases the BRET signal. (B) Schematic diagram of the PKC-c1b BRET sensor. Phosphorylation of threonine residues in two PKC consensus sequences (TLKI and TLKD) causes a conformational change in the sensor due to interactions between the phosphothreonines and the phosphothreonine-binding domains (FHA1 and FHA2), producing a BRET signal. (C) Schematic diagram of the p63/Gαq BRET sensor. Upon AT1R stimulation, Gαq-RlucII dissociates from Gβγ and binds to a minimal PH domain of p63RhoGEF (p63BD) fused to GFP10 (p63BD-GFP10). (D) Illustration of the BRET sensor for monitoring Rho activation. The recruitment of the RlucII-tagged Rho-binding domain (RBD) of PKN (PKN-RBD-RlucII) to the PM after Rho activation increases bystander BRET with the membrane-anchored rGFP-CAAX. (E) Pharmacological validation of the DAG sensor. HEK293/AT1R cells expressing the DAG sensor were pretreated in the absence (DMSO) or presence of either the Gαq inhibitor UBO-QIC or the PKC inhibitor Gö6983 and then stimulated with either AngII for 70 s or PMA for 10 min. Data are means ± SEM from at least three independent experiments. DMSO, dimethyl sulfoxide. (F) Pharmacological validation of the PKC-c1b sensor. HEK293/AT1R cells expressing the PKC sensor were treated as in (E). Cells were also stimulated with forskolin (Fsk) for 10 min to activate PKA. Data represent mean ± SEM of at least three independent experiments. (G) Time course of AngII-mediated p63 recruitment to Gαq. HEK293 cells were transfected with AT1R along with p63BD-GFP10 and Gαq-RlucII, preincubated with the vehicle (DMSO) or UBO-QIC before stimulation with AngII (arrow), before BRET measurements. Data represent means ± SEM of triplicate in a representative experiment that was repeated three times with similar results. (H) Rho activation profiles in cells with compromised G protein signaling. Parental HEK293 cells and CRISPR Gq/11 or G12/13 cells (ΔGq/11 and ΔG12/13, respectively) were transfected with PKN-RBD-RlucII and rGFP-CAAX along with AT1R. Cells were incubated with or without UBO-QIC (UBO) and stimulated with the indicated concentrations of AngII before BRET measurements. Data represent means ± SEM of three independent experiments.

  • Fig. 3 Activation of PKC and Rho sensors in VSMCs.

    (A and B) Time course of AngII-mediated activation of virally expressed PKC-c1b (A) and Rho (PKN-RBD-RlucII plus rGFP-CAAX) (B) BRET sensors in VSMCs. Arrow indicates addition of AngII. Data represent means of triplicate in a representative experiment that was repeated three times with similar results. (C and D) Concentration-response curves for activation of the PKC-c1b (C) and Rho (D) BRET sensors in VSMCs by the indicated concentrations of various AngII analogs. BRET signals were normalized to that induced by AngII in the same experiment and expressed as %Emax of AngII and then averaged. Data are means ± SEM of at least three independent experiments. (E and F) Validation of AngII-induced, AT1R-mediated PKC and Rho activation in VSMCs. Cells expressing the PKC-c1b (E) or Rho (F) BRET sensor were preincubated with vehicle, the AngII type 2 receptor (AT2R) antagonist PD 123319, the AT1R antagonist losartan, or the Gαq inhibitor YM-254890 (YM) and then stimulated with or without AngII. Data represent means ± SEM of AngII-mediated changes in the BRET signal (ΔBRET) derived from three to five independent experiments.

  • Fig. 4 Concentration-response curves for G protein and β-arrestin activation by AngII analogs.

    (A to E) HEK293 cells were transiently transfected with DNA encoding the Gαq (A), Gαi2 (B), Gαi3 (C), Gα12 (D), or βarr2 (E) BRET sensor along with AT1R and stimulated with the indicated concentrations of AngII or various AngII analogs. BRET measurements were recorded and normalized to the response of AngII in the same experiment and expressed as %Emax of AngII. Data are means ± SEM of at least three independent experiments.

  • Fig. 5 Heat map of AT1R signaling signature of various AngII analogs.

    (A and B) The transduction coefficients [log(τ/KA)] of each AngII analog were calculated from the concentration-response curves. The relative activity of each ligand [Δlog(τ/KA)] represents the difference between the calculated transduction coefficient [log(τ/KA)] for each ligand and the transduction coefficient of reference ligand (AngII). The relative activity [Δlog(τ/KA)] of AngII analogs in each signaling pathway was expressed as a heat map for (A) G protein signaling and β-arrestin activation and (B) Gαq signaling and activation of its downstream effectors p63RhoGEF (p63/Gαq), PLC (DAG generation), and PKC.

  • Fig. 6 Rho activation upon AT1R stimulation by various AngII analogs.

    (A to D) Concentration-response curves for Rho activation. HEK293 cells expressing the Rho BRET sensor (PKN-RBD-RLucII plus rGFP-CAAX) along with AT1R were pretreated with vehicle or UBO-QIC (UBO) and then stimulated with the indicated concentrations of AngII analogs. BRET signals were normalized to that of AngII in the absence of UBO-QIC and expressed as %Emax of AngII. Data represent means ± SEM from three to four independent experiments. (E) The relative activity of each ligand [Δlog(τ/KA)] represents the difference between the calculated transduction coefficient [log(τ/KA)] for each ligand and pretreatment conditions (vehicle or UBO-QIC) and the transduction coefficient of AngII with vehicle pretreatment. Data represent means ± SEM from three to four independent experiments. *P < 0.05 and **P < 0.01, unpaired Student’s t test. (F) Scatterplot of Δlog(τ/KA) of Rho activation in the presence of UBO-QIC versus Δlog(τ/KA) of Gα12 activation by the AngII analogs. R2 analysis was determined from a linear regression.

  • Fig. 7 Signaling profile of AT1R variants relative to the wild-type receptor.

    (A) Serpentine structure of human AT1R (obtained from www.gpcrdb.org) with the variant residues highlighted. (B) Scatterplot of the Δlog(relative activity) of wild-type and mutant AT1R for the indicated signaling outputs. Relative activities (Emax/EC50) were obtained from the AngII concentration-response curves and normalized to that of the wild-type receptor. Data represent means ± SEM from three to five independent experiments. Tukey’s post hoc multiple comparisons tests were used to compare wild-type and mutant receptors across the panel of assays (**P < 0.01), as well as to compare the signaling pathways downstream of each receptor (φP < 0.01).

  • Table 1 Potency and relative efficacy (Emax) of AngII and AngII analogs for activating G protein and βarr2 signaling.

    HEK293/AT1R cells expressing each indicated BRET sensor were stimulated with various concentrations of AngII and AngII analogs. BRET signals from each sensor were normalized to the maximal response of AngII (%Emax of AngII) and then averaged. pEC50 and Emax were obtained from the nonlinear regression curve of the averaged data. Data represent means ± SEM of three to eight independent experiments. n.d., not determined due to lack of responses.

    Ligandqi2i312βarr2
    pEC50EmaxpEC50EmaxpEC50EmaxpEC50EmaxpEC50Emax
    %AngII%AngII%AngII%AngII%AngII
    AngII8.55 ± 0.041008.34 ± 0.061008.56 ± 0.041008.19 ± 0.051008.66 ± 0.08100
    AngIII8.30 ± 0.08102.0 ± 3.48.10 ± 0.1186.5 ± 4.18.39 ± 0.0898.5 ± 2.97.68 ± 0.0795.1 ± 2.78.22 ± 0.13101.0 ± 5.2
    [Val4]-AngIII8.31 ± 0.1193.8 ± 4.28.50 ± 0.0786.7 ± 2.58.49 ± 0.1199.7 ± 4.37.78 ± 0.0789.7 ± 2.88.44 ± 0.19102.4 ± 7.6
    hSarmesin7.53 ± 0.1239.6 ± 1.97.34 ± 0.2942.0 ± 5.17.76 ± 0.2340.2 ± 3.97.38 ± 0.2053.8 ± 4.48.03 ± 0.1993.2 ± 7.5
    [Val5]-Sarmesin7.46 ± 0.2149.2 ± 4.27.15 ± 0.2043.2 ± 3.97.27 ± 0.2040.6 ± 3.47.56 ± 0.1454.6 ± 3.18.02 ± 0.2384.3 ± 8.2
    SBpa7.53 ± 0.0842.5 ± 1.47.98 ± 0.0873.2 ± 2.58.06 ± 0.1255.8 ± 3.08.17 ± 0.1264.0 ± 3.28.05 ± 0.1589.9 ± 5.9
    SVdF7.10 ± 0.0941.7 ± 1.87.83 ± 0.1259.0 ± 3.07.69 ± 0.1353.7 ± 3.07.88 ± 0.0967.5 ± 2.58.16 ± 0.1582.6 ± 5.1
    SI7.22 ± 0.5516.5 ± 4.08.45 ± 0.3527.1 ± 3.78.59 ± 0.2725.6 ± 2.77.83 ± 0.0948.4 ± 1.98.26 ± 0.1894.1 ± 6.8
    Saralasin6.75 ± 0.4811.3 ± 2.68.30 ± 0.3925.6 ± 4.08.66 ± 0.3430.1 ± 4.17.90 ± 0.0960.1 ± 2.38.22 ± 0.2574.8 ± 7.8
    TRVn.d.2.1 ± 2.0*8.18 ± 0.1540.0 ± 2.58.23 ± 0.1829.7 ± 2.17.61 ± 0.1064.0 ± 2.68.30 ± 0.2881.4 ± 9.2
    DVGn.d.4.6 ± 1.3*7.84 ± 0.2236.1 ± 3.37.88 ± 0.2132.0 ± 2.87.44 ± 0.1060.9 ± 2.47.62 ± 0.1782.4 ± 5.6
    Ang(1–7)n.d.1.5 ± 2.3*6.36 ± 0.3731.7 ± 5.86.99 ± 0.2922.9 ± 3.15.07 ± 0.2546.7 ± 6.86.54 ± 0.2353.4 ± 5.7
    SIIn.d.7.3 ± 1.2*7.06 ± 0.2227.1 ± 2.77.66 ± 0.2924.4 ± 3.06.88 ± 0.1743.3 ± 3.46.69 ± 0.2273.9 ± 7.4
    SIII5.13 ± 0.3924.1 ± 7.46.44 ± 0.4323.6 ± 4.96.46 ± 0.4925.9 ± 6.06.69 ± 0.2136.1 ± 3.56.21 ± 0.3272.5 ± 12.3

    *Emax was obtained from the response of the ligand at a concentration of 10 μM.

    • Table 2 Transduction ratio and relative effectiveness of AngII and AngII analogs for activating AT1R downstream pathways.

      Concentration-response data for each ligand were analyzed by nonlinear regression using the operational model equation in GraphPad Prism with AngII as the reference ligand, as described previously (29). ΔLog(τ/KA)s were calculated by subtracting the log(τ/KA) value of AngII in each pathway (Eq. 1). The SEs of Δlog(τ/KA) were estimated by Eq. 3 as described in Materials and Methods. Data represent means ± SEM of three to eight independent experiments. Relative effectiveness (RE) of the ligand toward each pathway, relative to AngII, was determined using Eq. 2.

      LigandLog(τ/KA)ΔLog(τ/KA)RE
      qi2i312βarr2qi2i312βarr2qi2i312βarr2
      AngII8.55 ± 0.038.41 ± 0.058.58 ± 0.048.22 ± 0.048.68 ± 0.70.00 ± 0.050.00 ± 0.070.00 ± 0.060.00 ± 0.060.00 ± 0.1111111
      AngIII8.32 ± 0.068.00 ± 0.078.36 ± 0.077.65 ± 0.068.22 ± 0.11−0.22 ± 0.07−0.41 ± 0.09−0.22 ± 0.07−0.57 ± 0.07−0.47 ± 0.130.59840.39170.60390.26850.3428
      [Val4]-AngIII8.23 ± 0.067.98 ± 0.068.50 ± 0.077.66 ± 0.068.47 ± 0.12−0.31 ± 0.07−0.43 ± 0.08−0.08 ± 0.07−0.57± 0.08−0.21 ± 0.140.48530.37240.83180.27230.6152
      Sarmesin7.08 ± 0.147.19 ± 0.207.49 ± 0.167.10 ± 0.128.01 ± 0.12−1.47 ± 0.14−1.22 ± 0.21−1.09 ± 0.16−1.13 ± 0.14−0.68 ± 0.140.03410.06030.08130.07480.2113
      [Val5]-Sarmesin7.12 ± 0.136.98 ± 0.167.02 ± 0.177.26 ± 0.138.01 ± 0.16−1.43 ± 0.13−1.43 ± 0.18−1.56 ± 0.17−0.95 ± 0.14−0.68 ± 0.180.03700.03720.02770.11120.2109
      SBpa7.13 ± 0.137.94 ± 0.097.90 ± 0.107.95 ± 0.108.04 ± 0.13−1.42 ± 0.14−0.46 ± 0.11−0.68 ± 0.10−0.27 ± 0.11−0.64 ± 0.150.03790.34590.21040.5370.2296
      SVdF6.69 ± 0.127.75 ± 0.137.52 ± 0.127.69 ± 0.108.14 ± 0.15−1.86 ± 0.12−0.65 ± 0.14−1.06 ± 0.12−0.53 ± 0.11−0.55 ± 0.170.01370.22180.08770.29850.2851
      SI6.74 ± 0.338.07 ± 0.318.13 ± 0.267.48 ± 0.148.25 ± 0.16−1.81 ± 0.33−0.34 ± 0.32−0.45 ± 0.26−0.74 ± 0.15−0.43 ± 0.170.01570.45810.35650.18320.3715
      Saralasin5.63 ± 0.447.93 ± 0.318.33 ± 0.237.65 ± 0.108.18 ± 0.20−2.92 ± 0.45−0.48 ± 0.32−0.24 ± 0.23−0.57 ± 0.11−0.51 ± 0.210.00120.33110.57020.26920.3105
      TRVn.d.7.97 ± 0.177.85 ± 0.207.39 ± 0.118.26 ± 0.18−4.2*−0.44 ± 0.18−0.73 ± 0.20−0.82 ± 0.12−0.43 ± 0.200.00000.36730.18660.150.3758
      DVGn.d.7.58 ± 0.227.52 ± 0.227.20 ± 0.127.61 ± 0.15−4.2*−0.82 ± 0.23−1.05 ± 0.22−1.03 ± 0.12−1.07 ± 0.170.00000.150.08830.09380.0853
      Ang(1–7)n.d.6.05 ± 0.266.23 ± 0.214.93 ± 0.156.51 ± 0.26−4.2*−2.36 ± 0.26−2.34 ± 0.21−3.29 ± 0.16−2.18 ± 0.270.00000.00440.00450.00050.0066
      SIIn.d.6.75 ± 0.257.35 ± 0.246.44± 0.166.66 ± 0.17−4.2*−1.66 ± 0.26−1.22 ± 0.24−1.78 ± 0.17−2.03 ± 0.190.00000.02180.05970.01680.0094
      SIII4.49 ± 0.296.08 ± 0.345.76 ± 0.216.19 ± 0.206.18 ± 0.24−4.05 ± 0.29−2.32 ± 0.35−2.82 ± 0.21−2.03 ± 0.21−2.51 ± 0.260.00010.00470.00150.00930.0031

      *A value of −4.2 for Δlog(τ/KA) was arbitrary given for calculation of bias.

      • Table 3 Binding affinity, potency, and relative efficacy of wild-type and variant AT1Rs for activating Gαq, Gαi2, Gαi3, Gα12, PKC, Rho, and βarr2.

        HEK293 cells were transfected with wild-type (WT) or variant AT1R or along with the indicated BRET sensor (Gαq, Gαi2, Gαi3, Gα12, PKC-c1b, Rho, βarr2-PM, or βarr2-EE) and stimulated with various concentrations of AngII. BRET signals for each pathway were normalized to the maximal response of wild-type (%Emax of WT) and then averaged. pEC50 and Emax were obtained from the nonlinear regression curve of the averaged data. Data represent means ± SEM of three to five independent experiments. AngII binding affinities were obtained from [125I]-AngII saturation binding assays. Data represent means ± SD of two to three independent experiments performed in duplicate.

        ReceptorKd (nM)qi2i312
        pEC50EmaxpEC50EmaxpEC50EmaxpEC50Emax
        %Emax of WT%Emax of WT%Emax of WT%Emax of WT
        WT0.53 ± 0.148.13 ± 0.051007.71 ± 0.051007.97 ± 0.081007.97 ± 0.07100
        A163T1.03 ± 0.528.19 ± 0.08102.3 ± 3.37.63 ± 0.08102.9 ± 3.37.89 ± 0.1392.4 ± 4.98.00 ± 0.14108.9 ± 5.9
        T282Mn.d.7.48 ± 0.0998.7 ± 3.56.78 ± 0.0997.0 ± 3.97.44 ± 0.1573.9 ± 4.76.34 ± 0.1087.4 ± 4.3
        C289Wn.d.7.86 ± 0.0798.5 ± 3.16.98 ± 0.1184.4 ± 4.47.30 ± 0.1373.4 ± 4.07.37 ± 0.10103.3 ± 4.4
        I103T0.24 ± 0.038.18 ± 0.0894.2 ± 3.77.58 ± 0.1973.0 ± 7.28.04 ± 0.2685.5 ± 10.78.37 ± 0.2465.9 ± 6.4
        A244S0.29 ± 0.098.17 ± 0.0897.7 ± 3.77.66 ± 0.2467.9 ± 8.48.20 ± 0.2494.4 ± 10.98.48 ± 0.1575.5 ± 4.4
        ReceptorPKCRhoβarr2-PMβarr2-EE
        pEC50EmaxpEC50EmaxpEC50EmaxpEC50Emax
        %Emax of WT%Emax of WT%Emax of WT%Emax of WT
        WT9.02 ± 0.071008.60 ± 0.071008.16 ± 0.071008.59 ± 0.09100
        A163T8.85 ± 0.07104.1 ± 3.08.46 ± 0.10109.1 ± 4.47.97 ± 0.11100.7 ± 4.98.54 ± 0.1096.2 ± 3.7
        T282M8.31 ± 0.1294.9 ± 4.77.91 ± 0.14100.8 ± 5.86.94 ± 0.0586.0 ± 2.07.47 ± 0.0934.6 ± 1.3
        C289W8.49 ± 0.10104.6 ± 4.27.96 ± 0.11101.5 ± 4.87.18 ± 0.0885.4 ± 3.27.90 ± 0.1093.1 ± 4.0
        I103T8.98 ± 0.0999.3 ± 4.48.57 ± 0.0789.3 ± 3.08.25 ± 0.0574.8 ± 1.78.55 ± 0.0862.6 ± 2.4
        A244S9.03 ± 0.0795.2 ± 3.48.60 ± 0.0586.7 ± 2.18.26 ± 0.0773.9 ± 2.68.52 ± 0.0582.5 ± 1.9
      • Table 4 Relative activity of AT1R and mutant receptors for activating each signaling pathway.

        Top: Relative activity [log(Emax/EC50)] of each AT1R variant obtained from the concentration-response curves from each individual experiment. Bottom: ΔLog(RA)s were calculated by subtracting the log(RA) value of the wild-type receptor for activating the same pathway (Eq. (5)). Data represent means ± SEM of three to five independent experiments.

        ReceptorLog(Emax/EC50)
        qi2i312PKCRhoβarr2-PMβarr2-EE
        WT8.13 ± 0.057.71 ± 0.037.96 ± 0.068.00 ± 0.109.03 ± 0.108.60 ± 0.118.16 ± 0.088.60 ± 0.14
        A163T8.20 ± 0.097.64 ± 0.097.82 ± 0.168.10 ± 0.128.87 ± 0.068.52 ± 0.207.97 ± 0.078.57 ± 0.15
        T282M7.44 ± 0.096.72 ± 0.157.31 ± 0.276.30 ± 0.118.26 ± 0.127.86 ± 0.266.88 ± 0.037.00 ± 0.12
        C289W7.82 ± 0.106.84 ± 0.147.19 ± 0.177.35 ± 0.108.49 ± 0.077.96 ± 0.177.11 ± 0.097.87 ± 0.16
        I103T8.18 ± 0.077.84 ± 0.027.93 ± 0.168.20 ± 0.068.95 ± 0.138.49 ± 0.108.09 ± 0.048.22 ± 0.10
        A244S8.17 ± 0.077.89 ± 0.078.02 ± 0.118.35 ± 0.069.09 ± 0.118.42 ± 0.068.09 ± 0.048.39 ± 0.04
        ReceptorΔLog(Emax/EC50)
        qi2i312PKCRhoβarr2-PMβarr2-EE
        WT00000000
        A163T0.068 ± 0.093−0.078 ± 0.067−0.141 ± 0.1000.099 ± 0.157−0.161 ± 0.106−0.076 ± 0.110−0.192 ± 0.085−0.025 ± 0.015
        T282M−0.691 ± 0.085−0.995 ± 0.136−0.654 ± 0.215−1.698 ± 0.156−0.770 ± 0.105−0.732 ± 0.214−1.285 ± 0.086−1.598 ± 0.030
        C289W−0.314 ± 0.114−0.874 ± 0.149−0.772 ± 0.233−0.648 ± 0.111−0.542 ± 0.045−0.641 ± 0.075−0.968 ± 0.097−0.728 ± 0.076
        I103T0.034 ± 0.079−0.051 ± 0.080−0.073 ± 0.090−0.0234 ± 0.112−0.189 ± 0.0940.172 ± 0.16−0.205 ± 0.034−0.348 ± 0.077
        A244S0.025 ± 0.049−0.007 ± 0.1270.015 ± 0.0720.131 ± 0.127−0.049 ± 0.088−0.402 ± 0.228−0.202 ± 0.022−0.187 ± 0.022

      Supplementary Materials

      • www.sciencesignaling.org/cgi/content/full/11/559/eaat1631/DC1

        Fig. S1. Responses of G protein and βarr2 BRET sensors in naïve and AT1R-expressing HEK293 cells.

        Fig. S2. Characterization of the DAG BRET sensor.

        Fig. S3. Characterization of the PKC BRET sensors.

        Fig. S4. Characterization of the p63RhoGEF (p63/Gαq) sensor.

        Fig. S5. Characterization of the Rho BRET sensor.

        Fig. S6. Heat map signature of AT1R signaling induced by AngII analogs.

        Fig. S7. Concentration-response curves for the activation of the p63RhoGEF (p63/Gαq), PKC, and PLC (DAG) BRET sensors by AngII analogs.

        Fig. S8. Heat map signaling signatures of Gαq and downstream effectors by AngII analogs.

        Fig. S9. Correlation plot analysis of Δlog(τ/KA) for G proteins and β-arrestin sensors against downstream signaling effectors.

        Fig. S10. Heat map and correlation plot of Δlog(τ/KA) for Rho and Gα12 activation by AngII analogs.

        Fig. S11. Concentration-response curves for G proteins, PKC, Rho, and β-arrestin activation by wild-type and mutant AT1Rs.

        Fig. S12. Assessment of cell surface abundance and AngII affinities for wild-type and mutant AT1Rs.

        Table S1. Sequences of AngII and AngII analogs.

        Table S2. Potency and relative efficacy of AngII and AngII analogs for activating the p63RhoGEF (p63/Gαq), PKC, and PLC (DAG) sensors.

        Table S3. Transduction ratio and relative effectiveness of AngII and AngII analogs for activating the p63RhoGEF (p63/Gαq), PKC, and PLC (DAG) sensors.

        Table S4. Potency and relative efficacy of AngII and AngII analogs for activating the Rho sensor in the absence or presence of Gαq/11 inhibition.

        Table S5. Transduction ratio and relative effectiveness of AngII and AngII analogs for activating the Rho sensor.

        Data file S1. Statistical analysis of Δlog(τ/KA) of AngII analogs between signaling pathways.

      • The PDF file includes:

        • Fig. S1. Responses of G protein and βarr2 BRET sensors in naïve and AT1R-expressing HEK293 cells.
        • Fig. S2. Characterization of the DAG BRET sensor.
        • Fig. S3. Characterization of the PKC BRET sensors.
        • Fig. S4. Characterization of the p63RhoGEF (p63/Gαq) sensor.
        • Fig. S5. Characterization of the Rho BRET sensor.
        • Fig. S6. Heat map signature of AT1R signaling induced by AngII analogs.
        • Fig. S7. Concentration-response curves for the activation of the p63RhoGEF (p63/Gαq), PKC, and PLC (DAG) BRET sensors by AngII analogs.
        • Fig. S8. Heat map signaling signatures of Gαq and downstream effectors by AngII analogs.
        • Fig. S9. Correlation plot analysis of Δlog(τ/KA) for G proteins and β-arrestin sensors against downstream signaling effectors.
        • Fig. S10. Heat map and correlation plot of Δlog(τ/KA) for Rho and Gα12 activation by AngII analogs.
        • Fig. S11. Concentration-response curves for G proteins, PKC, Rho, and β-arrestin activation by wild-type and mutant AT1Rs.
        • Fig. S12. Assessment of cell surface abundance and AngII affinities for wild-type and mutant AT1Rs.
        • Table S1. Sequences of AngII and AngII analogs.
        • Table S2. Potency and relative efficacy of AngII and AngII analogs for activating the p63RhoGEF (p63/Gαq), PKC, and PLC (DAG) sensors.
        • Table S3. Transduction ratio and relative effectiveness of AngII and AngII analogs for activating the p63RhoGEF (p63/Gαq), PKC, and PLC (DAG) sensors.
        • Table S4. Potency and relative efficacy of AngII and AngII analogs for activating the Rho sensor in the absence or presence of Gαq/11 inhibition.
        • Table S5. Transduction ratio and relative effectiveness of AngII and AngII analogs for activating the Rho sensor.
        • Legend for data file S1

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        Other Supplementary Material for this manuscript includes the following:

        • Data file S1 (Microsoft Excel format). Statistical analysis of Δlog(τ/KA) of AngII analogs between signaling pathways.

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