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Research ArticleArticle

Characterization of Angiotensin II Molecular Determinants Involved in AT1 Receptor Functional Selectivity

Ivana Domazet, Brian J. Holleran, Alexandra Richard, Camille Vandenberghe, Pierre Lavigne, Emanuel Escher, Richard Leduc and Gaétan Guillemette
Molecular Pharmacology June 2015, 87 (6) 982-995; DOI: https://doi.org/10.1124/mol.114.097337

Abstract

The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the activation of the AngII type 1 receptor (AT1), a G protein–coupled receptor. The AT1 receptor engages and activates several signaling pathways, including heterotrimeric G proteins Gq and G12, as well as the extracellular signal–regulated kinases (ERK) 1/2 pathway. Additionally, following stimulation, βarrestin is recruited to the AT1 receptor, leading to receptor desensitization. It is increasingly recognized that specific ligands selectively bind and favor the activation of some signaling pathways over others, a concept termed ligand bias or functional selectivity. A better understanding of the molecular basis of functional selectivity may lead to the development of better therapeutics with fewer adverse effects. In the present study, we developed assays allowing the measurement of six different signaling modalities of the AT1 receptor. Using a series of AngII peptide analogs that were modified in positions 1, 4, and 8, we sought to better characterize the molecular determinants of AngII that underlie functional selectivity of the AT1 receptor in human embryonic kidney 293 cells. The results reveal that position 1 of AngII does not confer functional selectivity, whereas position 4 confers a bias toward ERK signaling over Gq signaling, and position 8 confers a bias toward βarrestin recruitment over ERK activation and Gq signaling. Interestingly, the analogs modified in position 8 were also partial agonists of the protein kinase C (PKC)–dependent ERK pathway via atypical PKC isoforms PKCζ and PKCι.

Introduction

The octapeptide hormone angiotensin II (AngII) is the active component of the renin-angiotensin system, responsible for controlling blood pressure and water retention via smooth muscle contraction and ion transport. It exerts a wide variety of physiologic effects, including vascular contraction, aldosterone secretion, neuronal activation, and cardiovascular cell growth and proliferation. Virtually all known physiologic effects of AngII are produced through the activation of the angiotensin II type 1 (AT1) receptor, which belongs to the G protein–coupled receptor (GPCR) superfamily (de Gasparo et al., 2000).

The AT1 receptor interacts with the G protein Gq/11, which activates phospholipase C, which in turn generates inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from the cleavage of phosphatidylinositol 4,5-bisphosphate (Hunyady and Catt, 2006). The second messenger IP3 binds to the calcium channel receptor IP3R present at the surface of the endoplasmic reticulum, thus liberating Ca2+ into the cytosol. G protein–coupled receptor kinases phosphorylate the receptor, leading to βarrestin recruitment and functional uncoupling of G protein signaling. The AT1 receptor also activates the G protein G12 (Ushio-Fukai et al., 1998; Gohla et al., 2000; Sagara et al., 2007; Suzuki et al., 2009). It is known that G12, through the regulation of RhoGEF proteins, leads to the activation of RhoA/Rho kinase and cytoskeleton reorganization (Siehler, 2009). The AT1 receptor also activates extracellular signal–regulated kinases (ERK) 1 and 2 (Tian et al., 1998; Wei et al., 2003). ERK1/2 activation by the AT1 receptor is complex and can be mediated by protein kinase C (PKC; G protein–dependent) or by epidermal growth factor receptor (EGFR) transactivation (G protein–independent) (Luttrell, 2002; Miura et al., 2004).

Recent evidence has demonstrated that different ligands for a GPCR can stabilize the receptor under distinct conformations that promote the activation of some signaling pathways over others (Kenakin, 1995; Galandrin et al., 2007; Shonberg et al., 2014). This phenomenon is referred to as ligand bias or functional selectivity. Biased ligands have been proposed to stabilize receptor conformations that are distinct from those induced by unbiased ligands and selectively change the propensity of GPCR coupling to various effectors, leading to different signaling outcomes. Several biased agonists have been recently described for numerous GPCRs, and such ligands show much therapeutic promise, which could translate to compounds having less off-target effects (Rominger et al., 2014; van der Westhuizen et al., 2014).

The goal of the present study was to better comprehend the molecular basis underlying AT1 receptor functional selectivity. Early structure-activity relationship studies using assays measuring rabbit aorta strip contraction and rat blood pressure have shown that position 8 of AngII is essential for ligand activity, whereas position 4 is critical for ligand affinity, as well as for ligand activity (Regoli et al., 1974). Position 1 of AngII analogs is often substituted with sarcosine (N-methylglycine), which allows the peptides to resist aminopeptidase degradation. Angiotensin III (AngIII), also known as AngII (2-8), is an endogenous AngII peptide where the aspartic acid at position 1 is removed by aminopeptidase degradation. The ligand [Sar1Ile4Ile8]AngII, which is unable to signal via the Gq pathway, is able to selectively recruit βarrestin (Wei et al., 2003). Based on these observations, the goal of the study was to ascertain the impact of positions 1, 4, and 8 of AngII on the signaling profiles of the AT1 receptor and thus gain some insight into whether these molecular determinants of AngII are involved in conferring the property of functional selectivity toward the AT1 receptor. We therefore synthesized and investigated a series of peptide analogs containing amino acid substitutions at positions 1, 4, and 8 and determined their signaling profiles toward six different signaling pathways.

Materials and Methods

All reagents were from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) unless otherwise indicated. Culture media, trypsin, fetal bovine serum, penicillin, and streptomycin were from Wisent (St. Bruno, QC, Canada). OPTI-MEM and RNAiMAX were from Invitrogen Canada Inc. (Burlington, ON, Canada). Polyethyleneimine (PEI) was from Polysciences (Warrington, PA). All small interfering RNAs (siRNAs) were from Sigma-Aldrich (St. Louis, MO). Antibodies against PKCζ and PKCι were from Cell Signaling Technology (Whitby, ON, Canada). Peroxidase-conjugated donkey anti-rabbit IgG was from GE Healthcare (Little Chalfont Buckinghamshire, UK). Western Lightning Chemiluminescence Reagent Plus was from PerkinElmer (Waltham, MA). [125I]AngII (specific radioactivity ∼1000 Ci/mmol) was prepared with Iodo-GEN (Perbio Science, Erembodegem, Belgium) as reported previously.

Constructs.

The cDNA clone for the human AT1 receptor was provided by Dr. Sylvain Meloche (University of Montréal, Montreal, QC, Canada). The AT1-GFP10 construct was built by inserting the GFP10 sequence at the C-terminus of the AT1 construct, joined by the linker GSAGT, using the In-Fusion PCR cloning system (Clontech Laboratories, Mountain View, CA) as recommended by the manufacturer. The Renilla luciferase (RLuc)–βarrestin1, RLuc-βarrestin2, Gα12-RLuc, Gβ1, and Gγ1-GFP10 constructs were provided by Dr. Michel Bouvier (University of Montréal). All constructs were confirmed by automated DNA sequencing by alignment with multiAlin (Corpet, 1988).

Cell Culture and Transfection.

Human embryonic kidney (HEK) 293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. HEK293 cells stably expressing the AT1 receptor were maintained in medium containing 0.5 mg/ml G418. For βarrestin recruitment assays, HEK293 cells (3 ☓ 106 cells) were transiently transfected with 8700 ng of AT1-GFP10 and either 300 ng of RLuc-βarrestin1 or 300 ng of RLuc-βarrestin2 using linear polyethyleneimine (1 mg/ml) (PEI:DNA ratio 4:1). For G12 activation assays, HEK293 cells (3 ☓ 106 cells) were transiently cotransfected with the following constructs: 3000 ng of AT1 receptor, 600 ng Gα12-RLuc, 3000 ng Gγ1-GFP10, and 1800 ng Gβ1, using linear polyethyleneimine (PEI:DNA ratio 4:1). For siRNA transfection, HEK293 cells stably expressing the AT1 receptor were seeded in a 96-well plate (50,000 cells/well), and each well was transfected with 1 pmol of the indicated siRNA using RNAiMAX (0.3 μl).

Binding Experiments.

For binding experiments, broken cells were gently scraped into washing buffer [25 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM MgCl2], centrifuged at 2500g for 15 minutes at 4°C, and resuspended in binding buffer [25 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM MgCl2, 0.1% bovine serum albumin, 0.01% bacitracin, 0.01% soybean trypsin inhibitor]. Dose displacement experiments were performed by incubating broken cells (20–40 μg of protein) for 1 hour at room temperature with 0.8 nM [125I]AngII as tracer and increasing concentrations of AngII. Bound radioactivity was separated from free ligand by filtration through GF/C filters presoaked for at least 3 hours in binding buffer. Receptor-bound radioactivity was evaluated by γ counting. Results are presented as means ± S.D. The Kd values in the displacement studies were determined from the IC50 values using the Cheng-Prusoff equation.

Gq Signaling.

Gq signaling was evaluated by the measurement of inositol 1-phosphate (IP1) production using the IP-One assay (Cisbio Bioassays, Bedford, MA). Necessary dilutions of each analog were prepared in stimulation buffer (10 mM Hepes, 1 mM CaCl2, 0.5 mM MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, 50 mM LiCl, pH 7.4). HEK293 cells stably expressing the AT1 receptor were washed with phosphate-buffered saline (PBS) at room temperature, then trypsinized and distributed at 15,000 cells/well (7 μl) in a white 384-well plate in stimulation buffer. Cells were stimulated at 37°C for 30 minutes with increasing concentrations of AngII or analogs. Cells were then lysed with the lysis buffer containing 3 μl of IP1 coupled to the dye d2. After addition of 3 μl of anti-IP1 cryptate terbium conjugate, cells were incubated for 1 hour at room temperature under agitation. Fluorescence resonance energy transfer signal was measured using an M1000 fluorescence plate reader (TECAN, Salzburg, Austria).

Bioluminescence Resonance Energy Transfer–Based Biosensor Assays.

After 48 hours post-transfection, cells were washed with PBS and resuspended in stimulation buffer. For the βarrestin recruitment assays, the proximity of fusion protein RLuc-βarrestin to the reporter AT1-GFP10 was evaluated. Upon stimulation, RLuc-βarrestin was recruited to the AT1-GFP10 fusion protein, whereby the bioluminescence resonance energy transfer (BRET) signal was increased. For the G12 activation assay, the biosensor measures the proximity of the fusion protein RLuc-Gα12 to GFP10-Gγ. Upon activation, both RLuc-Gα12 and GFP10-Gγ move away from each other, which causes a decrease in the measured BRET. For both the βarrestin recruitment assays and G12 activation assay, cells transfected with the appropriate constructs were stimulated with the indicated ligands in 96-well white plates (50,000 cells/well) for 8 minutes, and then coelenterazine 400A was added at a final concentration of 5 μM. All BRET signals were measured using a TECAN M1000 fluorescence plate reader. The BRET ratio was calculated as the GFP10 emission over luminescence emission. Net BRET ratio was calculated by subtracting the BRET ratio under basal conditions from the BRET ratio upon maximal stimulation. All data were expressed as a percentage of maximal AngII response.

ERK1/2 Activation Assay.

ERK1/2 activation was measured using the ERK1/2 AlphaScreen Surefire kit (PerkinElmer). HEK293 cells stably expressing the AT1 receptor were seeded into 96-well plates at a density of 125,000 cells/well. After 24 hours, cells were starved for at least 16 hours in phenol red–free media before stimulation. Where specified, protein kinase C inhibitor Go6983 [3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione] (1 μM), EGFR tyrosine kinase inhibitor PD168393 [4-[(3-bromophenyl)amino]-6-acrylamidoquinazoline] (250 nM), or a combination of both inhibitors was added 30 minutes before stimulation. For time-course experiments, 100 nM AngII was added for the indicated times. For concentration-response experiments, cells were stimulated with increasing concentrations of indicated ligand. Cells were incubated at 37°C for either 2 minutes (PKC-ERK) or 5 minutes (EGFR-ERK), as determined by the peak responses obtained in the time-course assays. Stimulation of cells was terminated by the addition of lysis buffer to each well. The plate was then agitated at room temperature for 10 minutes, and 4 µl of lysate was transferred to 384-well ProxiPlates (PerkinElmer) and 5 µl of the assay reaction mix was added to each well (reaction buffer:activation buffer:donor beads:acceptor beads = 120:40:1:1). The plate was then incubated in the dark at room temperature for 24 hours under agitation and the signal was measured with an EnSpire Alpha Plate Reader (PerkinElmer) using standard AlphaScreen settings. All data were expressed as a percentage of maximal AngII-induced ERK1/2 phosphorylation.

Western Blotting.

HEK293 cells stably expressing the AT1 receptor that had been transfected with siRNA for 48 hours as described earlier were washed with PBS and then solubilized with lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, pH 7.4) for 30 minutes at 4°C. Insoluble material was precipitated by centrifugation at 15,000g for 20 minutes at 4°C. Cell lysates (30 μg) were separated on an 8% SDS-PAGE gel and were transferred to a 0.2-μm polyvinylidene fluoride membrane in a 96 mM glycine, 10 mM Tris-base, and 20% methanol buffer for 60 minutes at 0.4 A at 4°C. The membranes were blocked for 1 hour at room temperature with Tris-buffered saline/Tween 20 (TBST) buffer (20 mM Tris-base, 150 mM NaCl, and 0.1% Tween 20) containing 5% (w/v) skim milk. They were then incubated for 2 hours at room temperature with rabbit anti-PKCζ (1:1000) or rabbit anti-PKCι (1:250) in TBST supplemented with 5% skim milk. After three washes with TBST, the membranes were incubated with a peroxidase-conjugated donkey anti-rabbit IgG (1:2000) for 30 minutes at room temperature in TBST supplemented with 5% skim milk. They were then washed three times with TBST, and the immune complexes were visualized using Western Lightning Chemiluminescence Reagent Plus.

Data Analysis.

Binding data were analyzed with Prism version 6.0 for Windows (GraphPad Software, San Diego, CA), using a one-site binding hyperbola nonlinear regression analysis. Transduction ratios and bias factors were calculated based on the method of Kenakin et al. (2012), as described in detail by van der Westhuizen et al. (2014). Transduction ratios [log(τ/KA)] were first derived using the operational model equation in GraphPad Prism. The transduction ratio is an assessment of the effect (potency and efficacy) of a compound on receptor conformation and the subsequent ligand-receptor interaction with downstream effectors. To assess true ligand bias, system and observational bias which may be present owing to the different sensitivities of the assays used must be eliminated by comparing ligand activity at a given signaling pathway to that of a reference agonist. AngII, which yielded similar potencies and maximally activated all of the pathways, was the reference compound. By subtracting log(τ/KA) of AngII from the log(τ/KA) value of each analog for a given pathway, a within-pathway comparison was first established, yielding Δlog(τ/KA). Finally, between-pathway comparisons were achieved for a given ligand in the form of ΔΔlog(τ/KA) and the bias factor. ΔΔlog(τ/KA) was calculated by subtracting Δlog(τ/KA) values of one signaling pathway from the Δlog(τ/KA) of the signaling pathway to which it is compared. Bias factor values are the base 10 values of ΔΔlog(τ/KA) and are the actual bias factors.

Results

Binding Properties of AngII Peptide Analogs.

We evaluated the binding properties of eight selected peptide analogs of AngII (Table 1). AngII (Kd = 1.1 nM) and its analogs [Sar1Ile8]AngII (Kd = 1.7 nM), [Sar1]AngII (Kd = 1.8 nM), [Ile8]AngII (Kd = 3.2 nM), AngIII (Kd = 5.2 nM), and [Sar1Ile8]AngIII (Kd = 12 nM) showed high binding affinity in the low nanomolar range, whereas analogs [Sar1Ile4]AngII (Kd = 78 nM) and [Ile4]AngII (Kd = 894 nM) showed lower binding affinities. These analogs modified at positions 1, 4, or 8 were appropriate to compare their relative efficacies in the different signaling pathways.

View this table:
TABLE 1

Binding properties of AT1 receptor ligands

HEK293 cells stably expressing the AT1 receptor were assayed as described in Materials and Methods. Binding affinities (Kd) are expressed as the means ± S.D. of values obtained in n independent experiments performed in duplicate.

Gq Signaling.

To assess AT1 receptor signaling via the heterotrimeric G protein Gq, we measured IP1 production after a 30-minute stimulation with each analog, using AngII as a reference. The AngII dose-response curve shown in Fig. 1A revealed a maximal response of 730.2 nM of IP1 produced (efficacy normalized to 100%) with a half-maximal response (EC50) at a concentration of 3.1 nM. [Sar1]AngII also showed a high affinity with a good efficacy (Fig. 1B), whereas AngIII had a good efficacy but a low affinity (Table 2). [Sar1Ile4]AngII and [Ile4]AngII were partial agonists with low affinities (Fig. 1C; Table 2). [Sar1Ile8]AngII (Fig. 1D), [Ile8]AngII (Fig. 1E), and [Ile8]AngIII (Fig. 1F) did not produce any measurable IP1. All of these data are summarized in Table 2. These results indicate that position 8 of AngII is critical for activating the Gq pathway. These results also indicate that position 4 of AngII affects both the potency and the efficacy of the peptide in the activation of the Gq pathway, whereas modifications at position 1 did not show any impact.

Fig. 1.
Fig. 1.

Inositol 1-phosphate production induced by AngII analogs. HEK293 cells expressing the AT1 receptor were stimulated with increasing concentrations of AngII (A), [Sar1]AngII (B), [Sar1Ile4]AngII (C), [Sar1Ile8]AngII (D), [Ile8]AngII (E), and [Ile8]AngIII (F) for 30 minutes at 37°C. IP1 accumulation was measured with the IP-One assay, as described in Materials and Methods. Data are expressed as a percentage of AngII maximal response. The dotted line represents the AngII dose-response curve. Data are the mean ± S.D. of three to six independent experiments performed in triplicate.

View this table:
TABLE 2

Activation of Gq, βarrestin1, βarrestin2, and G12 by AT1 receptor ligands

HEK293 cells expressing the AT1 receptor were assayed as described in Materials and Methods. EC50 and Emax are expressed as the means ± S.D. of values obtained in at least three independent experiments performed in triplicate.

βArrestin Recruitment.

To evaluate the capacity of the receptor to recruit and engage with either βarrestin1 or βarrestin2 following an 8-minute stimulation with each analog, a BRET-based βarrestin recruitment assay was used. The AngII dose-response curve for βarrestin1 recruitment revealed a maximal net BRET ratio of 0.085 (efficacy normalized to 100%) with an EC50 of 6.1 nM (Fig. 2A), whereas that for βarrestin2 recruitment revealed a maximal net BRET ratio of 0.106 (efficacy normalized to 100%) with an EC50 of 4.6 nM (Fig. 3A). [Sar1]AngII showed a full efficacy and a high affinity for the recruitment of both βarrestins (Figs. 2B and 3B). AngIII showed a high efficacy and a low affinity for the recruitment of both βarrestins (Table 2). [Sar1Ile4]AngII showed a high efficacy but a low affinity for the recruitment of both βarrestins (Figs. 2C and 3C). [Ile4]AngII also showed a good efficacy but a very low affinity for the recruitment of both βarrestins (Table 2).

Fig. 2.
Fig. 2.

βArrestin1 recruitment to the AT1 receptor by AngII analogs. HEK293 cells cotransfected with fusion protein RLuc-βarrestin and the reporter AT1-GFP10 were stimulated with increasing concentrations of AngII (A), [Sar1]AngII (B), [Sar1Ile4]AngII (C), [Sar1Ile8]AngII (D), [Ile8]AngII (E), and [Ile8]AngIII (F) for 8 minutes at 37°C. βArrestin1 recruitment was measured as described in Materials and Methods. Data are expressed as a percentage of AngII maximal response. The dotted line represents the AngII dose-response curve. Data are the mean ± S.D. of three to six independent experiments performed in triplicate.

Fig. 3.
Fig. 3.

βArrestin2 recruitment to the AT1 receptor by AngII analogs. HEK293 cells cotransfected with fusion protein RLuc-βarrestin and the reporter AT1-GFP10 were stimulated with increasing concentrations of AngII (A), [Sar1]AngII (B), [Sar1Ile4]AngII (C), [Sar1Ile8]AngII (D), [Ile8]AngII (E), and [Ile8]AngIII (F) for 8 minutes at 37°C. βArrestin2 recruitment was measured as described in Materials and Methods. Data are expressed as a percentage of AngII maximal response. The dotted line represents the AngII dose-response curve. Data are the mean ± S.D. of three to six independent experiments performed in triplicate.

Dose-response curves were performed with three analogs modified at position 8. [Sar1Ile8]AngII was a partial agonist with a high affinity for the recruitment of both βarrestins (Figs. 2D and 3D). [Ile8]AngII was also a partial agonist with a high affinity for the recruitment of both βarrestins (Figs. 2E and 3E), whereas [Ile8]AngIII was a partial agonist with a low affinity for the recruitment of both βarrestins (Figs. 2F and 3F). All of these results are summarized in Table 2. These results indicate that the analogs modified at position 8 of AngII are partial agonists for the recruitment of βarrestins with relatively high potencies but with efficacies lower (∼50%) than that of AngII. The analogs modified at position 4 are also partial agonists for the recruitment of βarrestins, although with relatively high efficacies (∼80% compared with AngII) and low potencies. Modifications at position 1 of AngII did not show any impact on the recruitment of βarrestins.

G12 Activation.

The ability of the AT1 receptor to engage with and activate the G12 heterotrimer was evaluated using a BRET-based biosensor assay. The AngII dose-response curve shown in Fig. 4A revealed a net BRET ratio of 0.096 (efficacy normalized to 100%) with an EC50 of 4.7 nM. [Sar1]AngII (Fig. 4B) showed a full efficacy and a high affinity, whereas AngIII (Table 2) showed a full efficacy but a low affinity for the activation of G12. [Sar1Ile4]AngII (Fig. 4C) and [Ile4]AngII (Table 2) were partial agonists with low affinities for the activation of G12. [Sar1Ile8]AngII (Fig. 4D) and [Ile8]AngII (Fig. 4E) were partial agonists with high affinities, whereas [Ile8]AngIII (Fig. 4F) was a partial agonist with a low affinity for the activation of G12. All of these results are summarized in Table 2. These results indicate that the analogs modified at position 8 of AngII are partial agonists for G12 activation with relatively high potencies but with efficacies lower (∼40%) than that of AngII. The analogs modified at position 4 are also partial agonists for G12 activation with efficacies lower (∼60%) than that of AngII and with low potencies. Modifications at position 1 of AngII did not show any impact on the activation of G12.

Fig. 4.
Fig. 4.

G12 activation by AngII analogs. HEK293 cells expressing the AT1 receptor and cotransfected with Gα12-RLuc, Gγ1-GFP10, and Gβ1 were stimulated with increasing concentrations of AngII (A), [Sar1]AngII (B), [Sar1Ile4]AngII (C), [Sar1Ile8]AngII (D), [Ile8]AngII (E), and [Ile8]AngIII (F) for 8 minutes at 37°C. G12 activity was measured as described in Materials and Methods. Data are expressed as a percentage of AngII maximal response. The dotted line represents the AngII dose-response curve. Data are the mean ± S.D. of three to six independent experiments performed in triplicate.

ERK Response.

We evaluated the capacity of the selected AngII analogs to activate the ERK1/2 pathway by measuring ERK phosphorylation levels upon stimulation, a hallmark of ERK activity. Figure 5 shows that, under control conditions (circles), the addition of AngII increased ERK phosphorylation, which reached a maximal value at 5 minutes and then slowly declined toward the basal level. In the presence of the EGFR tyrosine kinase inhibitor PD168393 (triangles), the addition of AngII increased ERK phosphorylation, which reached a maximal value at 2 minutes and then slowly declined toward the basal level. In the presence of the PKC inhibitor Go6983 (squares), the addition of AngII increased ERK phosphorylation, which reached a maximal value at 5 minutes and then slowly declined toward the basal level. In the presence of both PD168393 and Go6983 (diamonds), the addition of AngII did not produce any measurable ERK phosphorylation. These results suggest that, in our experimental model, within the limits of our time course, AngII activates the ERK1/2 pathway via PKC following Gq activation and via the transactivation of EGFR. Since the ERK1/2 response is completely abolished in the presence of both inhibitors, we reasoned that the ERK response in the presence of Go6983 was mediated by EGFR (pathway EGFR-ERK), whereas the ERK response in the presence of PD168393 was mediated by PKC (pathway PKC-ERK).

Fig. 5.
Fig. 5.

AngII-induced ERK activation. HEK293 cells expressing the AT1 receptor were pretreated as indicated for 30 minutes and then stimulated with 100 nM AngII for different periods of time. ERK activity was measured as described in Materials and Methods. Data are expressed as a percentage of AngII maximal response. Data are the mean ± S.D. of three to six independent experiments performed in triplicate. DMEM, Dulbecco’s modified Eagle’s medium.

EGFR-Mediated ERK Response.

To evaluate the EGFR-ERK pathway, HEK293 cells stably expressing the AT1 receptor were treated with increasing concentrations of each analog for 5 minutes, in the presence of Go6983 (Fig. 6; Table 3). The AngII dose-response curve shown in Fig. 6A shows a maximal response of 49,677 luminescence arbitrary units (efficacy normalized to 100%) with an EC50 of 2.8 nM. [Sar1]AngII (Fig. 6B) showed a high efficacy and a high affinity, whereas AngIII (Table 3) showed a high efficacy but a low affinity for the activation of the EGFR-ERK pathway. [Sar1Ile4]AngII (Fig. 6C) and [Ile4]AngII (Table 3) showed high efficacies but low affinities for the activation of the EGFR-ERK pathway. [Sar1Ile8]AngII (Fig. 6D), [Ile8]AngII (Fig. 6E), and [Ile8]AngIII (Fig. 6F) were partial agonists with relatively low affinities for the activation of the EGFR-ERK pathway. All of these results are summarized in Table 3. These results indicate that the analogs modified at position 4 are partial agonists, although with very high efficacies (∼94%) for the EGFR-ERK pathway. Furthermore, [Sar1Ile4]AngII showed a higher potency (∼10 times) in the EGFR-ERK pathway than the Gq, G12, and βarrestin pathways. The analogs modified at position 8 of AngII are partial agonists of the EGFR-ERK pathway, with relatively low potencies (∼10 times lower than that of AngII) and with low efficacies (∼30% of that of AngII). Modifications at position 1 of AngII had no major impact on the EGFR-ERK pathway.

Fig. 6.
Fig. 6.

EGFR-dependent ERK activation by AngII analogs. HEK293 cells expressing the AT1 receptor were pretreated with 1 μM Go6983 for 30 minutes and then stimulated with increasing concentrations of AngII (A), [Sar1]AngII (B), [Sar1Ile4]AngII (C), [Sar1Ile8]AngII (D), [Ile8]AngII (E), and [Ile8]AngIII (F) for 5 minutes at 37°C. ERK activity was measured as described in Materials and Methods. Data are expressed as a percentage of AngII maximal response. The dotted line represents the AngII dose-response curve. Data are the mean ± S.D. of three to six independent experiments performed in triplicate.

View this table:
TABLE 3

Activation of Gq, PKC-ERK, and EGFR-ERK by AT1 receptor ligands

HEK293 cells stably expressing the AT1 receptor were assayed as described in Materials and Methods. EC50 and Emax are expressed as the means ± S.D. of values obtained in at least three independent experiments performed in duplicate.

PKC-Mediated ERK Response.

To evaluate the PKC-ERK pathway, HEK293 cells stably expressing the AT1 receptor were treated with increasing concentrations of each analog for 2 minutes, in the presence of PD168393 (Fig. 7; Table 3). The AngII dose-response curve shown in Fig. 7A shows a maximal response of 58,391 luminescence arbitrary units (efficacy normalized to 100%) with an EC50 of 1.2 nM. [Sar1]AngII (Fig. 7B) showed a high efficacy and a high affinity, whereas AngIII (Table 3) also showed a high efficacy but a low affinity for the activation of the PKC-ERK pathway. [Sar1Ile4]AngII (Fig. 7C) and [Ile4]AngII (Table 3) showed high efficacies but low affinities for the activation of the PKC-ERK pathway. [Sar1Ile8]AngII (Fig. 7D), [Ile8]AngII (Fig. 7E), and [Ile8]AngIII (Fig. 7F) were partial agonists with relatively low affinities for the activation of the PKC-ERK pathway. These results indicate that the analogs modified at position 4 are partial agonists, although with high efficacies (∼85%) for the PKC-ERK pathway. Furthermore, the analog [Sar1Ile4]AngII showed a higher potency (∼10 times) in the PKC-ERK pathway than the Gq, G12, and βarrestin pathways. The analogs modified at position 8 of AngII are partial agonists of the PKC-ERK pathway, with relatively low potencies (∼10 times lower than AngII) and low efficacies (∼30% that of AngII). Modifications at position 1 of AngII had no major impact on the PKC-ERK pathway.

Fig. 7.
Fig. 7.

PKC-dependent ERK activation by AngII analogs. HEK293 cells expressing the AT1 receptor were pretreated with 250 nM PD168393 for 30 minutes and then stimulated with increasing concentrations of AngII (A), [Sar1]AngII (B), [Sar1Ile4]AngII (C), [Sar1Ile8]AngII (D), [Ile8]AngII (E), and [Ile8]AngIII (F) for 2 minutes at 37°C. ERK activity was measured as described in Materials and Methods. Data are expressed as a percentage of AngII maximal response. The dotted line represents the AngII dose-response curve. Data are the mean ± S.D. of three to six independent experiments performed in triplicate.

Role of Atypical PKC Isoforms in the PKC-Mediated ERK Response.

It is generally accepted that Gq activation leads to the production of DAG and IP3, leading to the elevation of intracellular Ca2+ concentration. DAG and Ca2+ in turn lead to the activation of most isoforms of PKC. How then can a ligand such as [Sar1Ile8]AngII, which does not engage Gq, activate the PKC-ERK pathway? Actually, PKCs are composed of at least 15 isomers which can be classified into three families: conventional PKCs, including PKCα, PKCβ, and PKCγ, that are activated by Ca2+ and DAG; novel PKCs, including PKCδ, PKCε, PKCη, and PKCθ, that are activated by DAG only; and atypical PKCs, including PKCζ and PKCι, that are not activated by Ca2+ nor by DAG, but by other, less understood mechanisms (Wu-Zhang and Newton, 2013). We therefore hypothesized that [Sar1Ile8]AngII could activate atypical PKC isoform PKCι or PKCζ. To test this hypothesis, we used siRNA directed against either PKCι or PKCζ, and then measured the PKC-dependent ERK response. Figure 8A shows that, upon PKCι knockdown, the PKC-dependent ERK activity promoted by [Sar1Ile8]AngII was reduced by 55%. When PKCζ was knocked down, PKC-dependent ERK activity promoted by [Sar1Ile8]AngII was reduced by 60%. Upon knockdown of both isoforms simultaneously, the PKC-dependent response elicited with [Sar1Ile8]AngII was further diminished by 75%. Western blotting analysis showed that both PKCζ (Fig. 8B) and PKCι (Fig. 8C) were efficiently knocked down. These results support a role for both atypical PKC isoforms PKCι and PKCζ in AT1 receptor signaling that may have previously been underestimated.

Fig. 8.
Fig. 8.

Atypical PKC-dependent ERK activation. HEK293 cells expressing the AT1 receptor were transfected with siRNA against the indicated PKC isoforms as described in Materials and Methods. (A) Cells were stimulated with 100 nM [Sar1Ile8]AngII for 2 minutes at 37°C. ERK activity was measured as described in Materials and Methods. Data are expressed as a percentage of AngII maximal response. Data are the mean ± S.D. of three to six independent experiments performed in triplicate. Western-bot analysis of PKCζ (B) and PKCι (C) following treatment with the indicated siRNA.

Quantification of Ligand Bias.

Large variations in the potencies and efficacies of AngII analogs toward the different signaling pathways were observed (Tables 2 and 3), which suggests the presence of signaling bias. To clearly establish whether an analog was biased toward one pathway over the others, the bias factors were determined for each analog and for all of the signaling pathways. Transduction ratios [log(τ/KA)] were first derived using the operational model (Table 4). The log(τ/KA) of the reference compound AngII was then subtracted from the log(τ/KA) value of each analog for a given pathway, yielding Δlog(τ/KA) as a within-pathway comparison for each signaling pathway. The Δlog(τ/KA) value is indicative of how well a given signaling pathway can be activated by a ligand, where a value of 0 indicates that a given ligand activates a pathway to the same degree as the reference compound, a positive value indicates that the ligand more strongly activates the signaling pathway than the reference compound, and an increasingly negative value indicates that the ligand poorly activates the signaling pathway, if at all. The Δlog(τ/KA) values of each analog calculated for every signaling pathway were represented on a radar plot, adapted from the “web of efficacy” (Evans et al., 2010; Zhou et al., 2013) (Fig. 9). This allowed a graphic representation highlighting to what extent each pathway can be activated (or not) by a given ligand. Ultimately, a between-pathway comparison was achieved for a given ligand in the form of ΔΔlog(τ/KA), which is the actual bias factor (Tables 5 and 6).

View this table:
TABLE 4

Transduction ratios of AT1 receptor ligands

HEK293 cells expressing the AT1 receptor were stimulated with the different analogs and responses were measured for six distinct signaling pathways. Data were analyzed by nonlinear regression using the operational model equation as described in Materials and Methods to determine log(τ/KA). ΔLog(τ/KA) values were calculated from log(τ/KA) using AngII as the reference ligand. Data are the mean ± S.E.M. of three to six independent experiments performed in triplicate.

Fig. 9.
Fig. 9.

Effects of AngII analogs on AT1 signaling pathways. Radar graph representations summarizing the calculated Δlog(τ/KA) values of the different ligand-activated pathways for [Sar1]AngII (A), [Sar1Ile4]AngII (B), [Sar1Ile8]AngII (C), [Ile8]AngII (D), and [Ile8]AngIII (E). The balanced reference analog AngII is represented in blue.

View this table:
TABLE 5

Bias factors of AT1 receptor ligands modified at position 1

ΔΔLog(τ/KA) and BF values were calculated as described in Materials and Methods. Data are the mean ± S.E.M. of three to six independent experiments performed in triplicate.

View this table:
TABLE 6

Bias factors of AT1 receptor ligands modified at position 4

ΔΔLog(τ/KA) and BF values were calculated as described in Materials and Methods. Data are the mean ± S.E.M. of three to six independent experiments performed in triplicate.

The different analogs were partitioned into three groups, based on their functional selectivity profiles. The first group was composed of the analogs modified solely at position 1, [Sar1]AngII and AngIII (Fig. 9A; Table 5). The analog [Sar1]AngII showed Δlog(τ/KA) values ranging from −0.25 to 0.29, indicating that it is balanced relative to AngII (Fig. 9A). These results suggest that position 1 of AngII is likely not an important molecular determinant for functional selectivity toward the AT1 receptor. A second group of analogs included [Sar1Ile4]AngII (Fig. 9B) and [Ile4]AngII, which are modified at position 4 (Table 6). Figure 9B shows that [Sar1Ile4]AngII was a strong activator of both EGFR-dependent and PKC-dependent ERK signaling, with Δlog(τ/KA) values of −1.51 and −1.72, respectively. The rank order of pathways activated were as follows: βarrestin2 recruitment [Δlog(τ/KA) of −2.35], βarrestin1 recruitment [Δlog(τ/KA) of −2.39], the G12 pathway [Δlog(τ/KA) of −2.49], and the Gq pathway [Δlog(τ/KA) of −3.47]. Table 6 shows that the biases of [Ile4]AngII and [Sar1Ile4]AngII toward EGFR-ERK over Gq were 14-fold and 93-fold, respectively. The bias of [Ile4]AngII toward PKC-ERK over Gq was 26-fold, whereas for [Sar1Ile4]AngII it was 57-fold. These represented the strongest biases for AngII analogs modified at position 4. These results suggest that position 4 of AngII is likely an important molecular determinant involved in functional selectivity toward the AT1 receptor. The third group of analogs included [Sar1Ile8]AngII (Fig. 9C), [Ile8]AngII (Fig. 9D), and [Ile8]AngIII (Fig. 9E), which are modified at position 8. Figure 9C shows that, for [Sar1Ile8]AngII, the rank order of pathways activated were as follows: βarrestin2 recruitment [Δlog(τ/KA) of −0.77], βarrestin1 recruitment [Δlog(τ/KA) of −0.79], the G12 pathway [Δlog(τ/KA) of −1.93], EGFR-ERK [Δlog(τ/KA) of −4.03], and PKC-ERK [Δlog(τ/KA) of −4.13]. Analogs [Ile8]AngII and [Ile8]AngIII showed the same rank order of pathway activation as [Sar1Ile8]AngII, but were less proficient at recruiting both βarrestins and activating the G12 pathway. Since none of the analogs in this group activated the Gq pathway, no bias factor could be calculated. However, this in itself represents an obviously strong bias of these ligands for all other pathways over the Gq pathway. Table 7 shows that the bias of [Sar1Ile8]AngII toward βarrestin2 over PKC-ERK was 2252-fold, whereas the bias of βarrestin1 over PKC-ERK was 2147-fold. These represented the strongest biases found for AngII analogs modified at position 8. These results confirm that position 8 of AngII is likely an important molecular determinant involved in functional selectivity toward the AT1 receptor.

View this table:
TABLE 7

Bias factors of AT1 receptor ligands modified at position 8

ΔΔLog(τ/KA) and BF values were calculated as described in the Materials and Methods. Data are the mean ± S.E.M. of three to six independent experiments performed in triplicate.

Discussion

Functional selectivity is a relatively new concept, and its therapeutic potential is becoming more and more acknowledged (Rominger et al., 2014). Ligand bias is the ability of certain ligands to stabilize distinct receptor-transducer pairs at the expense of others, leading to signal pathway selectivity. The goal of our study was to identify biased ligands of the AT1 receptor and to better characterize the molecular determinants of AngII that underlie functional selectivity. A better understanding of the mechanisms leading to the recognition of distinct receptor-effector states by biased ligands should lead to the development of better therapeutics with less off-target effects. For this study, 11 AngII peptide analogs together with the reference ligand AngII were selected to assess their functional selectivity profiles toward six different AT1 receptor signaling outcomes. The pathways investigated were IP1 production (as a reporter of Gq activation), βarrestin1 and βarrestin2 recruitment, G12 activation as assessed by a BRET biosensor assay, and the ERK1/2 activation via either PKC activation or EGFR activation. The ligands were chosen based on the previously characterized importance of positions 1, 4, and 8 for AngII activity, as demonstrated by previous structure-activity relationship studies (Regoli et al., 1974; Holloway et al., 2002).

We showed that the substitution of phenylalanine at position 8 of AngII for isoleucine abolishes Gq signaling, whereas it maintains βarrestin recruitment, G12, and ERK activation. The radar plot shown in Fig. 9 further indicates the strongest bias toward βarrestin recruitment. [Ile8]AngII, [Sar1Ile8]AngII, and [Ile8]AngIII were antagonists for the Gq pathway, and partial agonists with relatively high efficacies for βarrestin recruitment and lower efficacies for the other pathways. It was known for a long time that AngII analogs containing an aliphatic residue at position 8 are antagonists of the Gq pathway (Regoli et al., 1974; Miura et al., 1999). It was only recently shown that these analogs can activate some pathways downstream of the AT1 receptor. For instance, it was shown that [Sar1Ile8]AngII can activate total ERK (Holloway et al., 2002; Ahn et al., 2004a) and recruit βarrestin (Ahn et al., 2004b; Zimmerman et al., 2012). TRV027 ([Sar1d-Ala8]AngII), an analog that contains d-Ala at position 8, is another recently developed biased ligand that favors βarrestin recruitment (Violin et al., 2010). These results suggest that the molecular determinants present at position 8 of AngII contribute to AT1 receptor functional selectivity, whereby Gq activation is abolished, whereas the G12 and βarrestin pathways are maintained and ERK activity is strongly decreased.

Of particular interest, we found that [Sar1Ile8]AngII, despite being unable to generate a Gq-dependent response, was still able to activate ERK in an atypical PKC-dependent manner. It was previously shown that PKCζ plays a role in AT1 receptor ERK activation in vascular smooth muscle cells (Liao et al., 1997; Zhao et al., 2005; Kim et al., 2009). These results support a role for both atypical PKC isoforms PKCι and PKCζ in AT1 receptor signaling that may have previously been underestimated.

We showed that the substitution of aspartate at position 1 for sarcosine ([Sar1]AngII) or its removal (AngIII) had very little impact on the capacity of the AT1 receptor to signal through the different pathways tested (Fig. 9A; Table 5). It has been long known that sarcosine at position 1 of AngII peptides confers resistance to aminopeptidase degradation in vivo (Hall et al., 1974). Substitution at position 1 of AngII was seen to have little or no impact on smooth muscle contraction (an assay dependent on Gq activation) (Hall et al., 1974), inositol phosphates production (Holloway et al., 2002), or total ERK activation (Miura et al., 2004). Here, we further showed that substitution at position 1 of AngII also had no impact on G12 activation, EGFR transactivation, or βarrestin recruitment. Altogether, these results suggest that position 1 of AngII peptides has very little impact on AT1 receptor functional selectivity.

We showed that the substitution of tyrosine at position 4 of AngII for isoleucine caused a bias toward ERK signaling. Both [Ile4]AngII and [Sar1Ile4]AngII are full agonists for PKC-dependent and EGFR-dependent ERK activity, but partial agonists for the G12, βarrestin, and Gq pathways. Although the substitution of Tyr4 for Ile4 decreased the potency for activation of all of the pathways, this decrease was markedly less for the ERK pathway. This modification at position 4 appears to stabilize a receptor conformation that preferentially interacts with the effectors of the ERK pathway. To our knowledge, we are the first to evaluate and compare the impact of position 4 of AngII on different signaling pathways using a single model. We are also the first to evaluate the impact of position 4 on G12 activation. It was previously shown that [Sar1Ile4]AngII is a partial agonist of the Gq pathway in CHO-K1 cells (Miura et al., 2004). Substitution at position 4 was also shown to decrease AngII potency and efficacy in smooth muscle contraction assays (Regoli et al., 1974; Samanen et al., 1989). A previous report indicated that substitutions at position 4 of AngII had very little impact on ERK activation in CHO-K1 cells (Holloway et al., 2002). These results suggest that the molecular determinants present at position 4 of AngII contribute to AT1 receptor functional selectivity, whereby ERK activity is maintained while the G12, Gq, and βarrestin pathways are negatively impacted.

In conclusion, the purpose of our study was to establish the impact of positions 1, 4, and 8 of AngII on different signaling pathways downstream of the AT1 receptor, and thus gain some insight into whether these molecular determinants of AngII were involved in conferring functional selectivity. Our study reveals that position 1 of AngII does not confer functional selectivity, whereas position 4 confers a bias toward ERK signaling over Gq signaling, and that position 8 confers a bias toward βarrestin recruitment over ERK activation and Gq signaling.

Authorship Contributions

Participated in research design: Domazet, Holleran, Lavigne, Escher, Leduc, Guillemette.

Conducted experiments: Domazet, Holleran, Richard, Vandenberghe.

Performed data analysis: Domazet, Holleran, Richard, Vandenberghe, Lavigne, Escher, Leduc, Guillemette.

Wrote or contributed to the writing of the manuscript: Domazet, Holleran, Leduc, Guillemette.

Footnotes

    • Received December 8, 2014.
    • Accepted March 24, 2015.

  • This work was supported by a grant from the Canadian Institutes of Health Research [Grant MOP-136770].

  • This work is part of the Ph.D. thesis of I. Domazet.

  • dx.doi.org/10.1124/mol.114.097337.

Abbreviations

AngII
angiotensin II
AngIII
angiotensin III
BRET
bioluminescence resonance energy transfer
DAG
diacylglycerol
EGFR
epidermal growth factor receptor
ERK
extracellular signal–regulated kinases
Go6983
3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione
GPCR
G protein–coupled receptor
HEK
human embryonic kidney
IP1
inositol 1-phosphate
IP3
inositol 1,4,5-trisphosphate
PBS
phosphate-buffered saline
PD168393
4-[(3-bromophenyl)amino]-6-acrylamidoquinazoline
PEI
polyethyleneimine
PKC
protein kinase C
RLuc
Renilla luciferase
siRNA
small interfering RNA
TBST
Tris-buffered saline/Tween 20
TRV027
[Sar1d-Ala8]AngII

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Functional Selectivity of AngII Analogs

Ivana Domazet, Brian J. Holleran, Alexandra Richard, Camille Vandenberghe, Pierre Lavigne, Emanuel Escher, Richard Leduc and Gaétan Guillemette
Molecular Pharmacology June 1, 2015, 87 (6) 982-995; DOI: https://doi.org/10.1124/mol.114.097337
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