Supplementary figures and tables Table of content

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1 Supplementary figures: Supplementary figures and tables Table of content Figure S1: The minimal i model written with BIOCHAM reaction rules. Figure S2: Ordinary differential equation (ODE) set of the model. Figure S3: Conservation laws and observed variables of the model. Figure S4: Simulation of the consequences of the absence of HRP2 dephosphorylation, HRP2barr2 dissociation or b dephosphorylation. Figure S5: Quantitative assessment of ERK1/2 and MEK1/2 phosphorylation in HEK293 cells expressing the AT 1A R using a reverse phase protein array (RPPA) approach. Figure S6: Representative Western blots of HEK293 cells transiently co-transfected with wildtype or 13A AT 1A R and either control or -arrestin 2 sirnas. Figure S7: Representative Western blots of HEK293 cells transiently transfected with the different sirna used in this study. Figure S8: Representative Western blots of HEK293 cells transiently co-transfected with wildtype or 13A AT 1A R and either control, GRK2 or GRK5 sirnas. Figure S9: CellDesigner representation of the network structure with rate constants and parameter numbers indicated on the corresponding species and reactions. Figure S10: Principle of the global parameter optimization strategy. Figure S11: Standard errors on the fits generated with parameter sets 1, 2, 3 and 4. Figure S12: Sensitivity analysis for each parameter of the 4 optimized parameter sets. Figure S13: Simulation of the consequences of -arrestin 1 or 2 depletion on total response to increasing angiotensin concentrations at different time points. Figure S14: Simulated kinetics (parameter set number 4) of, DAG and PKC_a at different HR concentrations. Figure S15: Simulated kinetics (parameter set number 4) of G_a in control versus perturbed conditions. Figure S16: Simulated kinetics (parameter set number 4) of at different H concentrations. Figure S17: Simulated kinetics (parameter set number 4) of the different receptor forms in control versus perturbed conditions. Figure S18: Simulated G and b responses at 5 min as a function of angiotensin concentration in control, GRK2/3-depleted, or GRK2/3-depleted and PKC-inhibited conditions. Figure S19: Representative Western blots of HEK293 cells transiently co-transfected with wildtype AT 1A R and either control or GRK2 sirnas. Figure S20: Representative Western blots of HEK293 cells transiently co-transfected with wildtype AT 1A R and either control, GRK5 or GRK6 sirnas. Figure S21: Representative Western blots of HEK293 cells transiently co-transfected with different GPCRs and either control or GRK2 sirnas. Supplementary tables: Table S1: Parameter values for each of the 4 best parameter sets. Table S2: Simulated versus experimental activation/deactivation half-lives measured in independent studies. SBML model file: separate file

2 MA(k0) for G => G_a. MA(k1) for G =[R~{P1}-H]=> G_a. MA(k2) for G =[R-H]=> G_a. MA(k3) for PIP2 =[G_a]=> DAG. MA(k4) for PKC =[DAG]=> PKC_a. MA(k5) for ERK =[PKC_a]=> G. MA(k6) for G_a => G. MA(k7) for DAG => PIP2. MA(k8) for PKC_a => PKC. MA(k9) for G => ERK. MA(k10*GRK23) for R-H => R~{P1}-H. (MA(k11),MA(k13)) for R~{P1}-H + barr1 <=> R~{P1}-H-barr1. (MA(k12),MA(k14)) for R~{P1}-H + barr2 <=> R~{P1}-H-barr2. MA(k15) for R~{P1}-H-barr1 => R-H + barr1. MA(k16) for R~{P1}-H-barr2 => R-H + barr2. MA(k18*GRK56) for R-H => R~{P2}-H. MA(k17) for R~{P2}-H => R-H. (MA(k19),MA(k23)) for barr2 + R-H <=> barr2-r-h. (MA(k20),MA(k24)) for barr2 + R~{P2}-H <=> barr2-h-r~{p2}. MA(k21) for ERK =[barr2-r-h]=> b. MA(k22) for ERK =[barr2-h-r~{p2}]=> b. MA(k25) for b =>ERK. Figure S1: The minimal model written with BIOCHAM reaction rules (

3 Figure S2: Ordinary differential equation (ODE) set of the model.

4 Figure S3: Conservation laws and observed variables of the model.

5 Simulated L) 5 4 k25 = 0 k24 = 0 k17 = 0 (µmol/ Time (min) Figure S4: Simulation of the consequences of the absence of HRP2 dephosphorylation (k17 = 0, pink), HRP2barr2 dissociation (k24 = 0, purple) or b dephosphorylation (k25 = 0, blue).

A. 0.78 1.56 3.12 6.25 12.5 25 Protein concentration [nm] 100 2 pmek1 C.

/pm MEK ratio B. Stimulation time (min) D.

using a reverse phase protein array (RPPA) approach.

anti-pmek pmek primary antibodies. Linear range was determined for both standard curves.

6 A Protein concentration [nm] pmek1 C. pmek 1/8 1/4 1/2 50 ns An ng II + PKC inhibitor Ang II + DMSO Ang II + PKC inhibitor 2 Ang II + DMS SO 1/8 1/4 1/2 /pm MEK ratio B. Stimulation time (min) D. ns Exp # Figure S5: Quantitative assessment of ERK1/2 and MEK1/2 phosphorylation in HEK293 cells expressing the AT1AR using a reverse phase protein array (RPPA) approach. (A) Wide ranges of recombinant 2 and pmek1 dilutions were arrayed in four replicates and revealed in near infrared using anti- anti and anti anti-pmek pmek primary antibodies. Linear range was determined for both standard curves. (B) Cells, pre-treated or not with PKC inhibitor (Ro , 1 µm), were stimulated for 2 or 30 minutes with 100 nm angiotensin. Whole cell lysates corresponding to four independent experiments were arrayed in 3 dilutions (i.e.: 1/2, 1/4, 1/8). (C) The molar quantities of 1/2 and of pmek1/2 were calculated according to each standard curves and the /pmek ratios were determined for each condition. (D) ERK phosphorylation catalyzed by the AT1AR/ -arrestin 2 complex in SBGN.

7 A. Time (min) WT 13A B. Time (min) βarr2 sirna C. Time (min) βarr2 sirna Figure S6: Representative Western blots of HEK293 cells transiently cotransfected with wild-type (WT) or 13A AT 1A R and either control (CTL) or -arrestin 2 (barr2) sirnas. Cells were stimulated with 100 nm AngII. (A) Kinetics of ERK phosphorylation h in WT versus 13A-transfected t cells. (B) Kinetics of ferk phosphorylation in WT-transfected cells depleted or not in -arrestin 2. (C) Kinetics of ERK phosphorylation in 13A-transfected cells depleted or not in -arrestin 2.

8 A. arr1 arr2 B. GRK2 C. GRK5 D. GRK6 Figure S7: Representative Western blots of HEK293 cells transiently transfected with the different sirna used in this study. Endogenous proteins are shown. (A) Control versus -arrestin 2-depleted cells. (B) Control versus GRK2-depleted cells. (C) Control versus GRK5-depleted cells. (D) Control versus GRK6-depleted cells.

9 A. GRK2 sirna Time (min) B. GRK5 sirna Time (min) C. GRK2 sirna Time (min) D. GRK5 sirna Time (min) Figure S8: Representative Western blots of HEK293 cells transiently co-transfected with wild-type (WT) or 13A AT 1A R and either control (CTL), GRK2 or GRK5 sirnas. (A) Effect of GRK2 depletion on the kinetics of ERK phosphorylation upon AngII (100 nm) stimulation in WT-transfected cells. (B) Effect of GRK5 depletion on the kinetics of ERK phosphorylation upon AngII stimulation in WT-transfected cells. (C) Effect of GRK2 depletion on the kinetics of ERK phosphorylation upon AngII stimulation in 13Atransfected cells. (D) Effect of GRK5 depletion on the kinetics of ERK phosphorylation upon AngII stimulation in 13A-transfected cells.

10 Figure S9: CellDesigner representation of the network structure (as presented in figure 1) with rate constants (in red) and parameter numbers (in green) indicated on the corresponding species and reactions.

11 Control PKC inhibition -arr2 sirna GRK2/3 sirna GRK5/6 sirna Conditions, DAG, PKC 47 parameters 44 identical to 44 identical to 44 identical to control + 3 control + 3 control + 3 perturbed perturbed perturbed (10, 11 and 15) (10, 11 and 17) (10, 11 and 32) 44 identical to control + 3 perturbed (10, 11 and 40) Ob bservables Parame eters Control simulated experimental 24 known + 35 unknown parameters ODEs integration Simulated time series PKC inhibition -arr2 sirna GRK2/3 sirna simulated simulated simulated experimental experimental experimental GRK5/6 sirna simulated experimental Dis tance Iterative optimizatio on Pondered global error = of distances Final parameter set Figure S10: Principle of the global parameter optimization strategy. One control and four perturbed data sets (in yellow) were used. Each data set corresponds to different observables (in blue) and to a number of unknown parameters (in pink). Only the parameter targeted by the biological perturbation was modified within the control parameter set, all the others being identical to control. A total of 38 unknown parameters was optimized. The optimization algorithm minimizes the global error (i.e.: the distance of the simulated model with respect to the different experimental curves, either control or perturbed [in green]) through an iterative and autoadaptative optimization algorithm.

Experimental data are represented as mean ± S.E.M M(in black).

12 Data fitting A. B. (arbitrary un nits) (arbitrary un nits) Time (min) C. D. Time (min) (arbit trary units) (arbit rary units) Time (min) E. F. Time (min) DAG (arbitrary units) PKC (arbitrary units) Time (min) Time (min) Figure S11: Standard errors on the fits generated with parameter sets 1, 2, 3 and 4. Experimental data are represented as mean ± S.E.M M(in black). A, control sirna (open circles) versus arr2 sirna (black circles); B, control (open circles) versus PKC inhibition (black circles); C control (open circles) versus GRK2/3 sirna (black circles); D control (open circles) versus GRK5/6 sirna (black circles); E control DAG and F control PKC activity. All the colored curves were generated through model simulations: control conditions are in blue and perturbed conditions in red. The curve widths represent the mean +/- standard error on the four parameter sets.

Global error Global error Global error Global error al error Globa Global error Global

value -9-7 -5-3 -1 1 3 5 Log of parameter value -9-7 -5-3 -1 1 3 5 Log of parameter

each parameter of the 4 optimized parameter sets.

the normalized global error for each of the 4 parameter sets (Set1 in blue, Set2 in

13 Global error Global error Global error Global error al error Globa Global error Global error Log of parameter value Log of parameter value Log of parameter value Log of parameter value Log of parameter value Figure S12: Sensitivity analysis for each parameter of the 4 optimized parameter sets. Each parameter value was scanned across 14 logs (from 10-9 to 10 5 ) and plotted against the normalized global error for each of the 4 parameter sets (Set1 in blue, Set2 in yellow, Set3 in red and Set4 in green). The optimal parameter value is figured by a color dot. Optimized parameter numbers correspond to Table 1.

14 Simulated A. B. (µmol/l) arr1 sirna arr2 sirna (µmol/l) arr1 sirna arr2 sirna log [AngII] log [AngII] C. (µmol/l) arr1 sirna arr2 sirna D. (µmol/l) arr1 sirna arr2 sirna log [AngII] log [AngII] Figure S13: Simulation of the consequences of -arrestin 1 or 2 depletion on total response to increasing angiotensin concentrations at different time points. Control conditions are presented as the blue curves. Simulations of either -arrestin 1 (red curve) or -arrestin 2 (green curve) depletion on phosphorylated ERK response as a function of angiotensin concentration were compared at different time points: (A) 5 minutes, parameter set number 3; (B) 5 minutes, parameter set number 4; (C) 10 minutes, parameter set number 4; (D) 30 minutes, parameter set number 4. At the three time points, the model displays antagonistic actions of both -arrestin isoforms, albeit with different intensities.

15 Simulated A. DAG µmo ol/l µmol/l B. C. PKC_a µmol/l Time (min) Figure S14: Simulated kinetics (parameter set number 4) of (A), DAG (B) and PKC_a (C) at different HR concentrations (between 0.1 and 0.01 µmol/l).

16 Simulated µmol/l Time (min) Figure S15: Simulated kinetics (parameter set number 4) of G_a in control versus perturbed conditions.

17 Simulated µmol/l Time (min) Figure S16: Simulated kinetics (parameter set number 4) of at different H concentrations (between M and 10-6 M).

number 4) of the different receptor forms in control versus perturbed conditions.

18 Simulated A. B. µmol/l C. D. µmol/l E. F. µm mol/l Time (min) Time (min) Figure S17: Simulated kinetics (parameter set number 4) of the different receptor forms in control versus perturbed conditions. (A) Control from 0 to 90 min; (B) PKC inhibition; (C) barr2 sirna; (D) GRK23 sirna; (E) GRK56 sirna; (F) Control from 0 to 10 min.

19 Simulated A. G (% of max. re esponse of in -tran nsfected cells) GRK2/3 sirna GRK2/3 sirna + PKC Ro inhibitoro log g[ [AngII] B. b (% of max. res sponse of in -trans sfected cells) GRK2/3 sirna GRK2/3 sirna + PKC Ro inhibitoro log [AngII] Figure S18: Simulated G (A) and b (B) responses at 5 min as a function of angiotensin concentration in control (blue), GRK2/3-depleted (purple), or GRK2/3- depleted and PKC-inhibited (black) conditions. Simulated total response is shown at Figure 6C.

20 A. Log [AngII] B. ERK ERK GRK2 sirna C. ERK GRK2 sirna + PKC inhibitor Figure S19: Representative Western blots of HEK293 cells transiently co-transfected with wild-type (WT) AT 1A R and either control (CTL) or GRK2 sirnas. (A) ERK phosphorylation as a function of AngII concentration (10-10 to 10-7 M) in control cells stimulated for 2 min. (B) ERK phosphorylation as a function of AngII concentration (10-10 to 10-7 M) in GRK2- depleted cells stimulated for 2 min. (C) ERK phosphorylation as a function of AngII concentration (10-10 to 10-7 M) in GRK2-depleted cells pre-incubated with PKC inhibitor (Ro , 1 µm) and stimulated t for 2 min.

21 A. + vehicle Log [AngII] PKC inhibitor ERK B. + vehicle Log [AngII] PKC inhibitor ERK GRK5 sirna C. + vehicle Log [AngII] PKC inhibitor ERK GRK6 sirna Figure S20: Representative Western blots of HEK293 cells transiently co-transfected with wild-type (WT) AT 1A R and either control (CTL), GRK5 or GRK6 sirnas. (A) ERK phosphorylation as a function of AngII concentration (10-10 to 10-7 M) in control cells pretreated with DMSO or the PKC inhibitor Ro (as indicated) and stimulated for 2 min. (B) ERK phosphorylation as a function of AngII concentration (10-10 to 10-7 M) in GRK5- depleted cells pre-treated with DMSO or PKC inhibitor (as indicated) and stimulated for 2 min. (C) ERK phosphorylation as a function of AngII concentration (10-10 to 10-7 M) in GRK6- depleted d cells pre-treated t with DMSO or PKC inhibitor (as indicated) d) and stimulated t for 2 min.

22 A. B. C. D. Endogenous 2AR V2R FSHR NK1R + vehicle GRK2 sirna + PKA inhibitor + vehicle + PKA inhibitor Iso (min) ERK2 GRK2 sirna + vehicle + PKA inhibitor + vehicle + PKA inhibitor dvd AVP (min) GRK2 sirna ERK2 + vehicle + PKA inhibitor + vehicle + PKA inhibitor FSH (min) ERK1/2 GRK2 sirna + vehicle + PKC inhibitor + vehicle + PKC inhibitor SP (min) ERK2 Figure S21: Representative Western blots of HEK293 cells transiently co-transfected with different GPCRs and either control (CTL) or GRK2 sirnas. (A) Time-course of ERK phosphorylation upon stimulation of endogenously expressed 2 AR with isoproterenol (10-5 M) in cells transfected with CTL or GRK2 sirna and pre-treated with DMSO or with a PKA inhibitor (H-89, 10 µm). (B) Time-course of ERK phosphorylation upon stimulation of transiently transfected V2R with dvd-avp (10-7 M) in cells co-transfected with CTL or GRK2 sirna and pre-treated with DMSO or with a PKA inhibitor (H-89, 10 µm). (C) Time-course of ERK phosphorylation h upon stimulation of transiently tl transfected t FSHR with FSH ( M) in cells co-transfected with CTL or GRK2 sirna and pre-treated with DMSO or with a PKA inhibitor (H-89, 10 µm). (D) Time-course of ERK phosphorylation upon stimulation of transiently transfected NK1R with Substance P (10-7 M) in cells co-transfected with CTL or GRK2 sirna and pre-treated with DMSO or with a PKC inhibitor (Ro , 1 µm).

23 Parameter # Description Name in optimization Unit set4 set3 set2 set1 Initial quantities 1Activated G protein (G_a) µmol.l 1 0 2DAG µmol.l Active PKC (PKC_a) µmol.l HRP1 µmol.l 1 0 5HRP1 ß arrestin 1 (HRP1 barr1) µmol.l 1 0 6HRP1 ß arrestin 2 (HRP1 barr2) µmol.l 1 0 7HRP2 µmol.l 1 0 8HR ß arrestin 2 (HR barr2) µmol.l 1 0 9HRP2 ß arrestin 2 (HRP2 barr2) µmol.l b µmol.l G µmol.l Total quantities 12 G protein (G+G_a) k28 µmol.l Receptor (HR+HRP1+HRP1 barr1+hrp1 barr2+hr barr2+hrp2+hrp2 barr2) µmol.l DAG (PIP2+DAG) k29 µmol.l PKC (PKC+PKC_a) k30 µmol.l ß arrestin 1 (barr1+hrp1 barr1) µmol.l ß arrestin 2 (barr2+hrp1 barr2+hr barr2+hrp2 barr2) µmol.l ERK (ERK + b + G) µmol.l Norms 19 ERK norm L.µmol DAG norm k34 L.µmol PKC_a norm k35 L.µmol Reaction rates 22 G auto activation k0 min e e Activation of G by HRP1 k1 L.µmol 1.min Activation of G by HR k2 L.µmol 1.min Activation of DAG by G_a k3 L.µmol 1.min Activation of PKC by DAG k4 L.µmol 1.min Phosphorylation of ERK by PKC k5 Lµmol 1min 1 L.µmol 1.min Deactivation of G_a k6 min Deactivation of DAG k7 min Deactivation of PKC k8 min Dephosphorylation of G k9 min Phosphorylation of HR by GRK23 x GRK23 quantity k10 min Association of HRP1 with ß arrestin 1 k11 L.µmol 1.min e 5 34 Association of HRP1 with ß arrestin 2 k12 L.µmol 1.min Dissociation of HRP1 barr1 complex k13 min Dissociation of HRP1 barr2 complex k14 min Recycling of HRP1 barr1 complex k15 min e e e Recycling of HRP1 barr2 complex k16 min Dephosphorylation of HRP2 k17 min Phosphorylation of HR by GRK56 x GRK56 quantity k18 min Association of HR with barr2 k19 L.µmol 1.min Association of HRP2 with barr2 k20 L.µmol 1.min Phosphorylation of ERK by HR barr2 k21 L.µmol 1.min 1 4.2e Phosphorylation of ERK by HRP2 barr2 k22 L.µmol 1.min Dissociation of HR barr2 complex k23 min Dissociation of HRP2 barr2 complex k24 min Dephosphorylation of b k25 min Inhibited PKC condition 48 PKC total ttlquantity (PKC + PKC_a) k31 µmol.l 1 ll Initial b quantity µmol.l Initial G quantity µmol.l Inhibited ß arrestin 2 condition 51 ß arrestin 2 total quantity k32 µmol.l e e Initial b quantity µmol.l 1 8.2e 4 53 Initial G quantity µmol.l 1 8.2e 4 Inhibited GRK23 condition 54 Phosphorylation rate of HR by GRK23 x GRK23 quantity k26 min Initial b quantity µmol.l Initial G quantity µmol.l Inhibited GRK56 condition 57 Phosphorylation rate of HR by GRK56 x GRK56 quantity k27 min e e e e 6 58 Initial b quantity µmol.l Initial G quantity µmol.l Table S1: Parameter values for each of the 4 best parameter sets. Parameter set4 has been used throughout the paper. The 4 parameter sets have been compared in sensitivity analyses.

24 Description Calculated from simulated kinetics Independent experimental References Activation of G by HRP1 12 sec Activation of G by HR 3.3 sec sec Lohse et al., 2008; Vilardaga et al Activation of DAG by G_a 7.2 sec 5 10 sec Violin et al., 2006 Activation of PKC by DAG 25 sec 30 sec Violin et al., 2006 Phosphorylation of ERK by PKC 37 sec 60 sec Ahn et al., 2004 Deactivation of G_a 27 sec sec Vilardaga et al., 2010 Deactivation of DAG 183 sec 1 2 min Violin et al., 2006 Deactivation of PKC 204 sec 30 sec Violin et al., 2006 Dephosphorylation of G 204 sec 3 min Ahn et al., 2004 Phosphorylation of HR by GRK23 x GRK23 quantity 10 sec Association of HRP1 with ß arrestin 1 80 sec sec Rajagopal et al., 2006; Lohse et al., 2008 Association of HRP1 with ß arrestin 2 80 sec sec Rajagopal et al., 2006; Lohse et al., 2008 Dephosphorylation of HRP2 17 min min Pöll et al., 2011 Phosphorylation of HR by GRK56 x GRK56 quantity 13 sec Association of HRP2 with barr2 80 sec sec Rajagopal et al., 2006; Lohse et al., 2008 Phosphorylation of ERK by HRP2 barr2 150 sec 3 min Ahn et al., 2004 Dephosphorylation of b 60 min > 60 min Ahn et al., 2004 Table S2: Simulated versus experimental activation/deactivation half-lives measured in independent studies.

Supplemental Fig. 1: PEA-15 knockdown efficiency assessed by immunohistochemistry and qpcr

Supplemental figure legends Supplemental Fig. 1: PEA-15 knockdown efficiency assessed by immunohistochemistry and qpcr A, LβT2 cells were transfected with either scrambled or PEA-15 sirna. Cells were then