functional selectivity profiling of the angiotensin ii …...1 receptor using pathway-wide bret...

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GPCR S IGNAL ING

1Department of Medicine, Research Institute of the McGill University Health Center(RI-MUHC), McGill University, Montréal, QC H4A 3J1, Canada. 2Department of Bio-chemistry and Molecular Medicine, Institute for Research in Immunology and Can-cer (IRIC), Université de Montréal, Montréal, QC H3T 1J4, Canada. 3Department ofPharmacology and Therapeutics, McGill University, Montréal, QC H3G 1Y6, Canada.4Departamento de Bioquímica e Imunologia, Faculdade de Medicina de RibeirãoPreto, Universidade de São Paulo, Ribeirão Preto, São Paulo 14049-900, Brazil. 5Institutde Pharmacologie de Sherbrooke and Department of Pharmacology-Physiology, Fac-ulty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC J1H5N4, Canada. 6Centre de Recherche de l’Hôpital Sainte-Justine, Montréal, QC H3T1C5, Canada. 7Department of Anatomy and Cell Biology, McGill University, Montréal,QC H3A 0C7, Canada.*These authors contributed equally to this work.†Corresponding author. Email: [email protected] (S.A.L.); [email protected] (M.B.)

Namkung et al., Sci. Signal. 11, eaat1631 (2018) 4 December 2018

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Functional selectivity profiling of the angiotensin II type1 receptor using pathway-wide BRET signaling sensorsYoon Namkung1*, Christian LeGouill2*, Sahil Kumar1, Yubo Cao3, Larissa B. Teixeira2,4,Viktoriya Lukasheva2, Jenna Giubilaro3, Sarah C. Simões4, Jean-Michel Longpré5,Dominic Devost3, Terence E. Hébert3, Graciela Piñeyro6, Richard Leduc5, Claudio M. Costa-Neto4,Michel Bouvier2†, Stéphane A. Laporte1,3,7†

G protein–coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biasedsignaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all thesignaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by givenligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bio-luminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation ofsignaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF,and Rho. Combined with G protein and b-arrestin BRET biosensors, our sensors enabled real-time monitoring ofGPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of theresponses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds intodifferent subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivitybetween Gaq and b-arrestin, there are also biases among G protein subtypes. We also demonstrated that biasesobserved at the receptor and G protein levels propagated to downstream signaling pathways and that these biasescould occur through the engagement of different G proteins to activate a common effector. We also used thesetools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensorsprovides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functionalselectivity at various levels of GPCR signaling.

INTRODUCTIONG protein–coupled receptors (GPCRs) represent the largest class ofmembrane-bound proteins and are involved in diverse biological pro-cesses ranging from hormone action and neurotransmission to cellmigration, proliferation, and differentiation (1, 2). They transmit sig-nals from external stimuli conveyed by natural and synthetic ligands,such as hormones and drugs, by engaging different intracellular cas-cades of signaling effectors and modulators such as the heterotrimericG proteins, GPCR kinases (GRKs), and b-arrestins, as well as down-stream second messenger–generating enzymes and channels. Activa-tion of these receptors by ligands leads to the functional dissociation ofGa and Gbg subunits within the G protein heterotrimer and the ac-tivation of downstream signaling effectors such as adenylyl cyclases(ACs; which can also be inhibited by the action of G proteins), phos-pholipases such as PLC and PLD, second messenger–dependent proteinkinases such as PKA and PKC, and small guanosine triphosphatases(GTPases) such as Rho and Ras. The recruitment of b-arrestin toGRK-phosphorylated, agonist-occupied GPCRs uncouples them from

heterotrimeric G proteins at the plasma membrane (PM), leading toa reduction in G protein–dependent signaling, and targets GPCRs forinternalization (1, 2). GPCRs have been typically categorized by theirability to couple to and activate a preferred subtype of heterotrimericG protein, as defined by the identity of the a subunit (Gas, Gaq, Gai,etc.); hence, they are often qualified as Gs-, Gq-, or Gi-coupled recep-tors with the assumption that signaling is restricted to one of theseG proteins. However, it has become clear that many GPCRs engagemore than one subtype of G protein, albeit with variable efficacies,resulting in the activation of several downstream signaling cascadesthat specify the responses elicited by the receptor in cells and tissues(2–4). Similarly, b-arrestins (b-arrestins 1 and 2) have been found tonot only play a role in receptor desensitization and act as endocyticadaptors for many GPCRs but also to participate in downstreamsignaling, affecting cell responsiveness in distinct ways (1, 5). There-fore, detecting how this multidimensionality of signaling repertoire fora given receptor is faithfully translated into intracellular signals re-mains a challenge.

Observations of distinct ligands acting on the same receptor dif-ferentially activating different subsets of signaling effectors in cellshave led to the idea that receptors can be conformationally stabilizedby ligands in multiple discrete active signaling states (6), a notion thatis supported by receptor structural studies (7, 8). The overall signalingprofile of a particular GPCR-ligand combination can also be modifiedby naturally occurring polymorphisms and engineered mutations, whichalso affect the conformational signaling landscape of the ligand-boundreceptor to preferentially engage one pathway over another (9–12). Sta-bilization of such distinct conformations by different ligands or mutantforms of ligands or receptors thus enables the preferential activation ofspecific subsets of effectors and downstream signaling pathways. Confor-mationally dependent GPCR signaling is now a well-accepted concept

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that has been encapsulated within the pharmacological notion of“biased agonism,” “functional selectivity,” or “pluridimensional efficacy”(13, 14) and has been described for many GPCRs, including the an-giotensin II type 1 receptor (AT1R), which is activated by the octa-peptide angiotensin II (AngII) (15–18). For example, the peptide SII,the first reported ligand with biased activity on AT1R, has been shownto promote b-arrestin engagement independently of Gaq activation(15, 16). Similarly, AT1R signaling elicited by other AngII analogssuch as SI, SVdF, SBpa, DVG, TRV027, and Ang(1–7) have also beenshown to preferentially engage b-arrestin over Gaq, albeit with differ-ent relative efficiencies (18–20), whereas SII was also reported to engageG proteins other than Gaq after its binding to AT1R (17, 21). However,the ability of SII to engage AT1R signaling pathways downstream ofthese other G proteins or other AngII analogs to promote receptor cou-pling to distinct G proteins or to other downstream signaling pathwayshas not been explored carefully.

Exploiting functional selectivity holds great promise for devel-oping more efficient and safer therapeutics (6, 22). Defining thesignaling pathways responsible for desired versus undesired effectswould facilitate the development of novel drugs with better thera-peutic indexes. In principle, this could be achieved by selectivelyactivating the pathways responsible for the desired effects whileavoiding the activation of those involved in tolerance or un-desirable side effects. This notion has been applied to the AT1Rfor which TRV027, a biased peptide ligand favoring b-arrestin overGaq signaling, has been developed to treat heart failure (23). Al-though TRV027 was efficacious in animal models, in a clinical trialtesting the efficacy in human, TRV027 did not improve clinical sta-tus compared to placebo in acute heart failure patients (24). Effortsto develop therapeutically advantageous biased drugs can thus belimited by an incomplete understanding of the mechanisms governingfunctional selectivity, as well as the lack of complete informationabout the entire repertoire of downstream mediators engaged bybiased ligands that could contribute to the desired versus undesiredtherapeutic effects. Studies on GPCR functional selectivity have oftenfocused on a subset of responses or on a narrow range of ligands, orboth, and little information is available about the extent to which theobserved biased engagement of G proteins translates into differencesin downstream signaling outputs. Moreover, how functional selectiv-ity is generally evaluated (for example, comparing biased effects of oneligand or a receptor mutant for a limited subset of responses, as com-pared to a reference agonist or the wild-type receptor) may representa challenge when trying to link biased agonism with therapeutic ef-fects of drugs and of the effects of mutations when those effects arecontingent on an ensemble of signaling outputs engaged at variouslevels and at different times in cells (25). Hence, there is a need for as-sessing functional selectivity in a more global manner.

Here, we describe the development, validation, and use of a suiteof bioluminescence resonance energy transfer (BRET)–based bio-sensors, including G proteins and their downstream effectors as wellas b-arrestin, for studying the functional selectivity of AT1R signaling.Using this platform of pathway-wide BRET-based sensors, we dis-sected AT1R signaling and demonstrated functional selectivity of var-ious AngII analogs and receptor mutants, revealing much greatersignaling profile diversity than previously reported. In addition, theability to probe signaling activities at different levels downstream ofthe receptor, in combination with selective pharmacological inhibi-tors, reveals points of convergence and divergence in the signalingnetworks that are engaged.


RESULTSAssessing G protein and b-arrestin signaling elicitedby AT1RThe AT1R not only couples to the Gaq/11 family of G proteins but alsohas been reported to activate other G proteins, such as Ga12/13 and Gai,as well as b-arrestins (17, 18, 21, 26). Therefore, we first validated theability of AT1R to activate different G proteins and b-arrestins usingBRET-based biosensors (Fig. 1, A and B). These G protein biosensors,which have been previously described, measure separation of the Gaand Gbg subunits after receptor activation using various Ga subunitstagged with the energy donor RlucII and a Gg subunit tagged with theenergy acceptor GFP10, yielding a reduction in BRET signal upon dis-sociation of the heterotrimer (27–31). We also used the b-arrestin2double-brilliance (barr2-DB) BRET sensor that quantifies b-arrestinbinding to AT1R (Fig. 1B), wherein the conformational change inb-arrestin after receptor binding results in a reduction of the BRETsignal (18). In these experiments, human embryonic kidney (HEK)293 cells were transiently cotransfected with complementary DNAs(cDNAs) encodingAT1R and either (i) individualGa-RlucII constructsplus an optimizedGbg pair consisting ofGb1 and aGFP10-taggedGg1/2or (ii) barr2-DB. To compare the efficacy of AT1R coupling to G pro-teins, we measured the BRET signals at the time of the maximal re-sponse after full agonist occupancy. No significant BRET signals overbasal were detected with the sensors upon AngII stimulation whenAT1R was not expressed in cells (fig. S1A). However, significantchanges in maximal BRET signals were observed with the Gaq, Ga12,and Gai1,2,3 sensors after AngII stimulation (Fig. 1C and fig. S1B, re-spectively). The Gas sensor showed no significant BRET change uponAngII stimulation of AT1R (fig. S1B). As expected, the selective GaqinhibitorUBO-QIC (also known as FR900359) (31) completely blockedthe BRET signal from theGaq sensor but not that from theGai2 orGa12sensors (Fig. 1C). AngII concentration dependently promoted barr2 re-cruitment to AT1R, as revealed by a reduction in BRET of the barr2-DBsensor (Fig. 1D).

Characterizing downstream G protein–mediated signalingevents using new pathway BRET sensorsWenext examinedGaq signaling events by engineering newBRET sen-sors that detect the activity of effectors downstream of this G protein:PLC, PKC, the guanine nucleotide exchange factor (GEF) p63RhoGEF,and the GTPase Rho (Fig. 2, A to D). We assessed these responses inHEK293 cells downstream of endogenous G proteins. Because thesecells do not produce endogenous AT1R, we transfected the sensorconstructs into HEK293 cells stably producing a small amount ofAT1R (around 0.5 pmol/mg; hereafter referred to as HEK293/AT1R).This amount of AT1R is comparable to that in vascular smooth musclecells (VSMCs) (32).

To measure the activity of PLC, which cleaves phosphatidylinositol4,5-bisphosphate (PIP2) and releases diacylglycerol (DAG) as one ofthe end products, we devised a sensor that detects DAG generation atthe PM. This sensor was created by introducing the dual acylation(myristoylation and palmitoylation) signal from the kinase Lyn at theN terminus ofGFP10, followed by a 300–amino acid disorganized linker(DIS300), RlucII, and finally the c1b DAG-binding domain of PKC-dat the C terminus (Fig. 2A). UponDAGproduction, the c1b domain isrecruited at the PM, allowingRlucII to be in close proximity toGFP10,thus producing an increased intramolecular BRET signal (Fig. 2A). Asrevealed by microscopy, the DAG BRET sensor localized to the PM,anchored through its fatty-acylated moieties (fig. S2A). Treating cells

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with phorbol 12-myristate 13-acetate (PMA), amimic ofDAG,was suf-ficient to promote the recruitment of the c1b domain to the PM,bringing the RlucII and GFP10moieties into closer proximity and gen-erating a robust BRET signal (Fig. 2, A and E). AngII stimulation ofHEK293/AT1R cells expressing the DAG sensor led to a rapid increaseinBRETwithin 30 s of stimulation (Fig. 2E and fig. S2B), consistentwiththe known time frame of Gaq activation (29). As expected, the BRETsignal then gradually decreased toward baseline over 5 min. AT1R-mediated DAG generation was significantly inhibited by the selectiveGaq inhibitor UBO-QIC but not by the pan-PKC inhibitor Gö6983,which inhibits both conventional and novel PKCs (cPKC and nPKC,respectively), consistent with a Gaq-dependent activation mechanism(Fig. 2E). To test the ability of the sensor to detect the activity of en-dogenous receptors, we took advantage of the fact that HEK293 cellsproduce endogenous muscarinic acetylcholine receptors (mAChRs)(33), including the M3 receptor subtype that couples to Gaq/11. Car-bachol stimulation of these cells containing the DAG sensor led to aconcentration-dependent increase in the BRET response (fig. S2C),confirming that the sensor is sufficiently sensitive to detect signals pro-moted by endogenous amounts of receptors.

To assess the activity of PKC without overexpressing the kinase it-self, which may affect receptor activity, we generated an intramolecularBRET sensor that consists of an N-terminal GFP10 moiety followed bythe FHA1 and FHA2 phosphothreonine binding domains of Rad53,two cassette sequences containing threonine residues in the contextof PKC consensus sites (34), RlucII, and the DAG-binding c1b domainat the C terminus (Fig. 2B). A 50–amino acid DIS linker (DIS50) sep-arates the FHA domains and the phosphothreonine cassettes to allow


the flexibility necessary for the FHA domains to bind the PKC-phosphorylated sites, which would bring the GFP10 and RlucII moietiesinto close proximity, yielding an increased BRET signal (Fig. 2B). Thec1b domain was added to the PKC BRET sensor in the C terminus tobind DAG at the PM once this second messenger signaling lipid isproduced after receptor activation (35). This localized the biosensorin the vicinity of PKC subtypes known to be activated by AT1R(36–39). A Strep-tag II sequence was also inserted in the N terminusin front of the GFP10 to purify the sensor. This sensor is hereafterreferred to as the PKC-c1b sensor. Under basal conditions, the PKC-c1b sensor was found in both the cytosol and nuclei of HEK293 cells(fig. S3A). AngII stimulation of cells led to a rapid translocation of thePKC-c1b sensor from the cytosol to the PM with no obvious changesin the nuclear signal at the time point studied and an increase in BRETsignal (fig. S3, A and B). Activation of the PKC-c1b sensor after AT1Rstimulation resulted in a similar kinetic response as the DAG sensor,with the BRET signal peaking at 1 min and then returning to basalafter 5 min (figs. S2B and S3B). The phosphorylation of the PKC-c1b biosensor upon either AngII or PMA activation was confirmedby Western blot analysis using an antibody recognizing phosphothreo-nine after affinity purification of the biosensor (fig. S3C). No increasein the BRET signal was observed with a mutant form of the sensorlacking the threonine residues in the cassette domains (fig. S3D). DirectPMA activation of endogenous PKCs detected by the PKC-c1b BRETsensor was inhibited by Gö6983, but not by UBO-QIC, whereasforskolin-mediated activation of AC and PKA did not promote anyactivation of the sensor, confirming the selectivity of the sensor forPKCs (Fig. 2F). The AT1R-promoted activation of the PKC-c1b sensor

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Fig. 1. AT1R downstream signaling pathways and BRET sensors for G protein and b-arrestin activation. (A) AT1R stimulates signaling through several differentclasses of heterotrimeric G proteins and activation of barr2. cAMP, adenosine 3′,5′-cyclic monophosphate. (B) Illustration of BRET-based Ga-Gbg and barr2-DB sensors.Upon receptor activation, dissociation of the Ga subunit from Gbg and conformational changes in b-arrestin cause the BRET signal to decrease. L, ligand; GDP, gua-nosine diphosphate. (C) AT1R-induced G protein activation. HEK293 cells were transfected with AT1R along with the indicated Ga-RlucII (Gaq, Ga12, or Gai2) plus GFP10-Gg and Gb. Cells were preincubated in the absence or presence of the selective Gaq inhibitor UBO-QIC or vehicle and then stimulated with AngII for 2 min (Gaq and Gai2) or 10min (Ga12). Data represent means ± SEM from at least three independent experiments. **P < 0.01, unpaired Student’s t test. (D) Concentration-response curve of the barr2-DBsensor upon AngII stimulation of AT1R. Cells were transfected with AT1R along with barr2-DB and stimulated with the indicated concentrations of AngII for 20 min. Datarepresent means ± SEM from nine independent experiments.

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Fig. 2. Generation and validation of BRET-based sensors for activation of PLC, PKC, p63/Gaq, and Rho. (A) Schematic diagram of the DAG BRET sensor, whichmeasures 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 BRETsensor. Phosphorylation of threonine residues in two PKC consensus sequences (TLKI and TLKD) causes a conformational change in the sensor due to interactionsbetween the phosphothreonines and the phosphothreonine-binding domains (FHA1 and FHA2), producing a BRET signal. (C) Schematic diagram of the p63/Gaq BRETsensor. Upon AT1R stimulation, Gaq-RlucII dissociates from Gbg and binds to a minimal PH domain of p63RhoGEF (p63BD) fused to GFP10 (p63BD-GFP10). (D) Illus-tration 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 Rhoactivation increases bystander BRET with the membrane-anchored rGFP-CAAX. (E) Pharmacological validation of the DAG sensor. HEK293/AT1R cells expressing theDAG sensor were pretreated in the absence (DMSO) or presence of either the Gaq inhibitor UBO-QIC or the PKC inhibitor Gö6983 and then stimulated with either AngIIfor 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 representmean ± SEM of at least three independent experiments. (G) Time course of AngII-mediated p63 recruitment to Gaq. HEK293 cells were transfected with AT1R along withp63BD-GFP10 and Gaq-RlucII, preincubated with the vehicle (DMSO) or UBO-QIC before stimulation with AngII (arrow), before BRET measurements. Data representmeans ± SEM of triplicate in a representative experiment that was repeated three times with similar results. (H) Rho activation profiles in cells with compromised Gprotein signaling. Parental HEK293 cells and CRISPR Gq/11 or G12/13 cells (DGq/11 and DG12/13, respectively) were transfected with PKN-RBD-RlucII and rGFP-CAAX alongwith AT1R. Cells were incubated with or without UBO-QIC (UBO) and stimulated with the indicated concentrations of AngII before BRET measurements. Data representmeans ± SEM of three independent experiments.

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depended on activation of Gaq/11, but not on activation of Gai, asillustrated by the inhibition of the BRET response by the Gaq/11 in-hibitor YM-254890, but not by the Gai inhibitor pertussis toxin (PTX)(fig. S3E). The PKC-c1b sensor reported on the activity of all PKCs,with PKCb accounting for half of the response, as revealed by the re-duction in the BRET signal in the presence of the selective PKCbinhibitor LY-333,531 (fig. S3E). Mobilizing intracellular Ca2+ in aGaq/11- and PLC-independent manner using the ionophore A23187activated the PKC-c1b sensor, but to a lesser extent than did theagonist-mediated, DAG- and Ca2+-dependent activation of the sensor(fig. S3, E and F), indicating that the sensor was sufficiently sensitive todetect PKC activated solely by Ca2+ and likely relied on the presenceof DAG in the membrane under basal conditions. To determine therelative role of the two components of the sensor activation (the trans-location to the PM through binding toDAGand the phosphorylation-dependent conformational rearrangement), we designed a PKC sensorconstitutively anchored to the PM through the introduction of the dualacylation domain of Lyn in the N terminus of the sensor, hereafterreferred to as Lyn-PKC (fig. S3, G to I). The agonist-mediated activationand kinetics of the constitutively (Lyn) anchored and DAG-recruited(c1b) PKC sensors were identical (fig. S3, G and H). However, theLyn-PKC sensor generated a larger signal, indicative of a greater sensi-tivity. As was the case for the PKC-c1b, Lyn-PKC was also detected inthe nucleus (fig. S3I). As expected, the Lyn-PKC constitutively presentat the PM and nucleus did not relocate upon AT1R activation. Thecauses for the nuclear localization of both PKC-c1b and Lyn-PKCsensors are unknown, but it did not prevent the detection of receptor-promoted PKC-c1b activity even in response to activation of endoge-nous (not overexpressed) receptors. Similar to the DAG BRET sensor,the PKC-c1b sensor detected the activity of endogenous mAChRs inHEK293 cells (fig. S2C).

The GEF p63RhoGEF, which activates the small G protein RhoA,is also a downstream effector ofGaq (40, 41).We generated a newBRET-based biosensor that detects Gaq activation and engagement of this ef-fector using the minimal PH domain of p63RhoGEF that binds Gaq(40, 42), hereafter referred to as p63 binding domain (p63BD). Pull-down experiments using a glutathione S-transferase (GST)–taggedform of p63BD (GST-p63BD) revealed that p63BD interacted specifi-cally with Gaq but not with Ga12 (fig. S4A). We thus attached thep63BD through its C terminus to GFP10, which when recruited toRlucII-Gaq would generate a BRET signal, and refer to this BRET pairas the p63/Gaq sensor (Fig. 2C). The BRET signal from the p63/Gaqsensor increased over time and reached amaximumafter 60 s of agoniststimulation, and the signal lasted over 5min (Fig. 2G). Its activationwastotally blocked by UBO-QIC. The p63/Gaq sensor was not as robust asthat from the PKC-c1b sensor and required higher amounts of receptorfor activation (compare Fig. 2F to 2G). The greater sensitivity of thePKC-c1b sensor is well illustrated by both its greater maximal BRETresponse and the steeper increase in signal observed upon stimulationwith increasing receptor concentrations (fig. S4B). This is perhaps re-flective of the amplified nature of the PKC response versus that of thep63/Gaq sensor, which requires a stoichiometric recruitment of p63BD-GFP10 to RlucII-tagged Gaq, or differences in the sensitivity betweenthese two sensors due to their specific intrinsic design, or both. The p63/Gaq BRET sensor could be activated by receptors known to couple toGaq/11, such as AT1R and the prostaglandin F2a receptor (FP), but notby receptors known to signal through Gai [the dopamine D4 receptor(D4R)] or Gas [the b2-adrenergic receptor (b2AR)] (fig. S4C). In addi-tion, we detected no BRET signal between p63BD-GFP10 and either


Gai2-RlucII or Gas-RlucII upon activation of the D4R or b2AR, respec-tively, nor between Ga12-RlucII and p63BD-GFP10 upon activation ofAT1R or FP (fig. S4C). Last, consistent with the selectivity shown by thepull-down experiment with GST-p63BD (fig. S4A), YM-254890blocked the activation of the p63/Gaq BRET sensor by both AT1Rand FP, but PTX did not (fig. S4D), also confirming the selectivity ofthe p63/Gaq BRET sensor for detecting the activation of Gaq-coupledreceptors.

To further explore downstream effectors, we generated a sensor toevaluate the activity of the GTPase Rho. We fused the C terminus ofthe RBD of PKN, which is recruited to the PM upon Rho activation,to RlucII (PKN-RBD-RlucII). We monitored the recruitment of PKN-RBD to the PM by coexpressing PKN-RBD-RlucII with the green flu-orescent protein from Renilla reniformis (rGFP) anchored at the PMthrough prenylation of the CAAX domain of kRas (rGFP-CAAX)(Fig. 2D), which generates an enhanced bystander BRET signal (43)upon PKN-RBD-RlucII translocation to the PM. In HEK293 cellsexpressing AT1R and the Rho sensor (PKN-RBD-RlucII plus rGFP-CAAX), AngII increased the BRET ratio within 30 s of agonist addi-tion, and the signal persisted for more than 5 min (fig. S5A). Bothbasal and agonist-mediated BRET responses were significantly re-duced by incubating cells with the Rho inhibitor C3 toxin (fig. S5B)(44), confirming the selectivity of the response. Because Rho can beactivated by both Gaq/11- and Ga12/13-coupled receptors (45, 46), weevaluated the contributions of these two G protein subfamilies to Rhoactivation downstream of AT1R using pharmacological and geneticapproaches. Either inhibiting Gaq/11 (with UBO-QIC or YM-254890)or using CRISPR-Cas9 Gaq/11 knockout cells (31) reduced the AngII-mediated response by 30 to 40%, without affecting receptor abundance(Fig. 2H and fig. S5C). Consistent with the absence of Gaq/11 in theCRISPR-Cas9–generated cells, UBO-QIC or YM-254890 had no fur-ther inhibitory effects on Rho sensor activity in these cells (Fig. 2H).Selective Ga12/13 pharmacological inhibitors are not available, but re-moving both these G proteins in cells through CRISPR-Cas9 [G12/13

knockout cells (47)] reduced the activation of the Rho sensor by 15 to20% (Fig. 2H). Adding UBO-QIC to Ga12/13-depleted cells nearly com-pletely blocked the activation of the Rho sensor by AngII (≈95% inhi-bition; Fig. 2H), demonstrating a role for both Ga12/13 and Gaq/11 inRho activation. Consistent with our BRET data, we found that bothGa12/13 andGaq/11 subtypes of heterotrimeric G proteins were involvedin Rho activation by AT1R in HEK293 cells (fig. S5, D and E), using aclassical GST pull-down assay to monitor Rho activation (48). In addi-tion, to confirm the contribution of the twoGproteins to Rho activationin living cells, these data validate the usefulness of the BRET sensor tomonitor the relative contributions of each G protein to Rho activation.Together, our findings reveal that these BRET sensors are sensitive sur-rogates for detecting signaling of heterologously expressed Gaq/11- andGa12/13-coupled GPCRs in cells. We also tested the PKC-c1b and Rhosensors for endogenous AT1R activity using VSMCs. Similar kineticsand magnitudes of response of PKC-c1b and Rho activation as foundin HEK293 cells were also observed in VSMCs (Fig. 3, A and B). Acti-vation of these sensors by AngII in VSMCs increased dose-dependentlyand was dependent of AT1R and Gaq/11 (Fig. 3, C to F)

Profiling G protein and b-arrestin activation by variousAngII analogsNext, we used the BRET-based sensors to examine the diversity ofG protein and b-arrestin activation profiles by 14 AngII analogs in-cluding AngII itself (table S1). These analogs were selected on the basis

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of their partial agonist activities (49) and, for some, their reported biasfor inducing activation of b-arrestin versus Gaq [such as SI, SVdF,SBpa, SII, DVG, TRV027, Ang(1–7), and AngIII] (18, 20, 50). By se-lecting these ligands, we also sought to assess the impact of modifyingamino acid positions 1 and 8 of AngII on the overall signaling activityof AT1R. Other analogs, including Sarmesin, SII, and SIII, wereselected to also assess the contribution of position 4. We generatedconcentration-response curves for Gaq, Gai2, Gai3, Ga12, and barr2activation in HEK293/AT1R cells and compared the potencies andefficacies of each ligand to AngII (Fig. 4, A to E, and Table 1). TheAngII analogs did not significantly decrease BRET signals over basalin HEK293 cells lacking AT1R (fig. S1A). In cells overexpressingAT1R, however, AngIII and [Val4]-AngIII showed full agonist activityon Gaq, whereas Sarmesin, [Val5]-Sarmesin, SBpa, SVdF, and SI showedpartial activity (Fig. 4A). Saralasin and SIII only marginally activated


Gaq, whereas TRV027, DVG, Ang(1–7), and SII were deemed inactiveon Gaq, because we detected no reliable signal from concentration-response curves (Fig. 4A). We investigated the activity of AngII analogson AT1R-mediated Gai2, Gai3, and Ga12 responses (Fig. 4, B to D, andTable 1). The ligands that did not activate Gaq [TRV027, DVG, Ang(1–7), and SII] had mild-to-strong partial agonist activities on Gai2,Gai3, and Ga12, with Ga12 being the G protein most activated by allfour ligands (Fig. 4, B to D). The rank order of potency for activatingGa12, Gai2, and Gai3 by Gaq-inactive ligands was identical [TRV027≥DVG > SII > Ang(1–7)]. Saralasin acted as a partial agonist for allthree G proteins, with a relatively better efficacy for activating Ga12over Gai2 and Gai3, but had greater potency for engaging Gai2 andGai3 than for engaging Ga12 (Fig. 4, B to D). SIII had similar potenciesfor activating Gai2, Gai3, and Ga12 but better efficacy on Ga12 ascompared to Gai2 and Gai3 (Fig. 4, B to D). The partial Gaq agonistsSarmesin, [Val5]-Sarmesin, SBpa, and SVdF all act as partial agonistson Gai2, Gai3, and Ga12 activation, with similar efficacies and poten-cies (Fig. 4, A to D). SI, which showed weak Gaq activity, was the leastefficacious activator of Gai2, Gai3, and Ga12, whereas the full Gaq ago-nists AngIII and [Val4]-AngIII retained their full agonist properties onthese G proteins. Last, despite their reduced efficacies on Gaq activa-tion, Sarmesin, [Val5]-Sarmesin, SBpa, SVdF, and SI all activatedbarr2 with efficacies and potencies comparable to that of AngII(Fig. 4E and Table 1). The non–Gaq-activating ligands TRV027,DVG, Ang(1–7), and SII also all promoted barr2 recruitment to a sim-ilar extent as did AngII, albeit with different potencies (Fig. 4E, rightpanel, and Table 1). The two full Gaq agonists AngIII and [Val4]-AngIIIwere also full agonists of the barr2 response. Together, these data sug-gest that many AngII analogs preferentially stimulate b-arrestin overGaq signaling downstream of AT1R.

We next evaluated potential pathway-specific effects of the differ-ent AngII analogs. We used the operational model (6) to quantitative-ly determine any bias between two signaling pathways for the testedligands as compared to the reference ligand, AngII (Table 2 and datafile S1). To better visualize ligand rank order, we generated a heat mapof the relative activity of the response to each ligand as compared tothe response to AngII (Fig. 5A). The relative activity [Dlog(t/KA),where KA represents the functional affinity of the ligand for the recep-tor and t is its efficacy in activating a signaling pathway] is deter-mined from the difference in transduction coefficient of ligands, ascompared to that of the reference ligand AngII. Because no reliableEmax or EC50 (median effective concentration) could be determinedfor TRV027-, DVG-, Ang(1–7)-, and SII-mediated activation of Gaq,we arbitrarily assigned them log(t/KA) values equal to the weakest Gaq-activating ligand (SIII) for which such coefficient could be determined(Table 2). Similar clustering and rank orders were observed when theeffectiveness of responses to the ligands was analyzed according to ei-ther the Emax or pEC50 (pEC50, representing the negative log of the con-centration of the ligand that gives EC50; fig. S6, A and B). Analysis ofDlog(t/KA) revealed significant bias responses against Gaq in favor ofGai2, Gai3, Ga12, and b-arrestin for SBpa, SVdF, SI, Saralasin, TRV027,DVG, Ang(1–7), SII, and SIII (Fig. 5A and data file S1). As expectedfrom the heat map clustering of the ligands’ relative activity, we ob-served no strong bias between their abilities to activate Gai2, Gai3,Ga12, and b-arrestin, although a small bias, in favor of Ga12, was de-tected for SBpa, [Val5]-Sarmesin, and SVdF between Gai3 and Ga12.Sarmesin showed functional selectivity in favor of b-arrestin over Gaq,but not between Gai2, Gai3, or Ga12, as compared to Gaq. Ang(1–7)showed a bias against Gaq, and Ga12 while favoring Gai2, Gai3, and

AngIIA B

C D

E F

AngII

PKC-c1b sensor Rho sensor

–12 –11–10 –9 –8 –7 –6 –50

20406080

100120

Log [Ligand] (M)

Res

pons

e(%E

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ngII)

AngIIAngIIISBpaSVdFSarmesinSI 0

20406080

100120

Log [Ligand] (M)

0 60 120 180 240 3000.0

0.10

0.15

0.20

0.25

Time (s)

BRET

ratio

0 60 120 180 240 3000.0

0.20.30.40.50.6

Time (s)

Vehicl

e

PD 1233

19

Losa

rtan YM

0.00

0.02

0.04

0.06

0.08

0.10

AngI

I-ind

uced

BRET

cha

nge

(BR

ET)

Vehicl

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PD 1233

19

Losa

rtan YM

0.00

0.05

0.10

0.15

0.20

0.25

–12 –11–10 –9 –8 –7 –6 –5

Fig. 3. Activation of PKC and Rho sensors in VSMCs. (A and B) Time course ofAngII-mediated activation of virally expressed PKC-c1b (A) and Rho (PKN-RBD-RlucIIplus rGFP-CAAX) (B) BRET sensors in VSMCs. Arrow indicates addition of AngII. Datarepresent means of triplicate in a representative experiment that was repeated threetimes with similar results. (C and D) Concentration-response curves for activation ofthe PKC-c1b (C) and Rho (D) BRET sensors in VSMCs by the indicated concentrationsof various AngII analogs. BRET signals were normalized to that induced by AngII inthe same experiment and expressed as %Emax of AngII and then averaged. Data aremeans ± SEM of at least three independent experiments. (E and F) Validation ofAngII-induced, AT1R-mediated PKC and Rho activation in VSMCs. Cells expressingthe PKC-c1b (E) or Rho (F) BRET sensor were preincubated with vehicle, the AngIItype 2 receptor (AT2R) antagonist PD 123319, the AT1R antagonist losartan, or theGaq inhibitor YM-254890 (YM) and then stimulated with or without AngII. Data rep-resent means ± SEM of AngII-mediated changes in the BRET signal (DBRET) derivedfrom three to five independent experiments.

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b-arrestin, but did not distinguish between Gai2, Gai3, and b-arrestin.These results reveal a diversity of signaling profiles that had not beenpreviously appreciated and demonstrate that such clustering can beuseful to identify pathway-selective ligands with similar biased sig-naling properties.


Assessing G protein–mediated effector signaling bydifferent AngII analogsWe assessed the coupling efficiencies of the 14 different AngII analogson the downstream effectors of Gaq/11 to assess whether their relativeactivities toward this G protein were also propagated to downstream

020406080

100120 AngII

AngIII[Val4]-AngIII

SI

SVdF

[Val5]-SarmesinSarmesin

SBpa

Res

pons

e(%E

max

of A

n gII)

Log [Ligand] (M)

G qA

G i2

G i3

G 12

arr2

B

C

D

E

020406080

100120 AngII

AngIII[Val4]-AngIII

SI

SVdF


SBpa

Log [Ligand] (M)

Res

pons

e(%E

max

of A

ngII)

–200

20406080

100120

AngII

SIISIII

DVGSaralasinTRV

Log [Ligand] (M)

020406080

100120 AngII

AngIII[Val4]-AngIII

SI

SVdF


SBpa

Log [Ligand] (M)

Res

pons

e(%E

max

of A

ngII)

–200

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100120

AngII

SIISIII

DVGSaralasinTRV

Log [Ligand] (M)

020406080

100120 AngII

AngIII[Val4]-AngIII

SI

SVdF


SBpa

Log [Ligand] (M)

Res

pons

e(%E

max

of A

ngII)

–200

20406080

100120

AngII

SIISIII

DVGSaralasinTRV

Log [Ligand] (M)

–12–11–10–9 –8 –7 –6 –5–20

020406080

100120 AngII

AngIII[Val4]-AngIII

SI

SVdF


SBpa

Log [Ligand] (M)

Res

pons

e(%E

max

of A

n gII)

–200

20406080

100120 AngII

SIISIII

DVGSaralasinTRV

Log [Ligand] (M)

Ang(1–7)

–12–11–10–9–8 –7 –6 –5 –40

20406080

100 AngII

SIISIII

DVGSaralasinTRV

Log [Ligand] (M)

Ang(1–7)

Ang(1–7)

Ang(1–7)

Ang(1–7)

–12–11–10–9 –8 –7 –6 –5

–12–11–10–9 –8 –7 –6 –5

–12–11–10–9 –8 –7 –6 –5

–12–11–10–9 –8 –7 –6 –5

–12–11–10–9 –8 –7 –6 –5

–12–11–10–9–8 –7 –6 –5 –4

–12–11–10–9 –8 –7 –6 –5

–12–11–10–9 –8 –7 –6 –5

Fig. 4. Concentration-response curves for G protein and b-arrestin activation by AngII analogs. (A to E) HEK293 cells were transiently transfected with DNAencoding the Gaq (A), Gai2 (B), Gai3 (C), Ga12 (D), or barr2 (E) BRET sensor along with AT1R and stimulated with the indicated concentrations of AngII or various AngIIanalogs. BRET measurements were recorded and normalized to the response of AngII in the same experiment and expressed as %Emax of AngII. Data are means ± SEMof at least three independent experiments.

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effectors. We first generated concentration-response curves withthe DAG, PKC-c1b, and p63/Gaq sensors for the different ligandsin HEK293/AT1R cells (fig. S7). These concentration-response curvesfor many ligands mimicked those observed for Gaq activation (com-pare Fig. 4A and fig. S7, A to D). We also observed a similar rankorder for the Gaq response to partial agonists in VSMCs, although theamplitude of the signal was lower than in HEK293 cells (Fig. 3C). Weperformed the corresponding heat map analysis of the relative activitiesfor each ligand in HEK293 cells (Fig. 5B and tables S2 and S3). Again,we arbitrarily assigned the lowest transduction coefficients for TRV027and Ang(1–7) for the activation of p63/Gaq because we could notextrapolate reliable efficacies or potencies for these ligands. Similarly,we could not determine the transduction coefficient for Saralasin,TRV027, DVG, Ang(1–7), SII, or SIII for the PKC and DAG responsesnor for PKC activation by SI (table S3). The heat map analysis of rel-ative activities or the Emax and pEC50 for activating p63/Gaq, DAG, andPKC mirrored that of Gaq activation, but not that of Gai2, Gai3, andGa12, consistent with Gaq/11 playing a primary role in the activationand generation of these effectors and second messengers (Fig. 5B andfig. S8, A and B). Consistent with these observations, we also found astronger correlation between the transduction coefficients for the differ-ent ligands for activating Gaq versus p63/Gaq (fig. S9A), DAG(fig. S9B), and PKC (fig. S9C) than for other G proteins and b-arretin2with those effectors (fig. S9, A to C, right panels). These findings indi-cate that the bias of signaling flow through downstream of Gaq was


maintained and that the efficiency toward the other pathways had littleimpact on the coupling of Gaq effectors.

We next generated concentration-response curves of AngII analogsinHEK293 cells transfected with bothAT1R and the Rho sensor (Fig. 6,A to D). Because we found that both Gq/11 and G12/13 contributed tothe activation of Rho in HEK293 cells, we also assessed the couplingefficiency of the AngII analogs on the Ga12/13-mediated activation ofRho. We stimulated cells expressing the Rho sensor with the differ-ent AngII analogs and compared the concentration-response curvesand their coupling efficiencies in the presence or absence of the Gaq/11inhibitor UBO-QIC (Fig. 6, A to D, and tables S4 and S5). UBO-QICwas used to isolate the contribution of Ga12/13 in Rho activation. Rhoactivation by AngII, AngIII, Sarmesin, SBpa, SVdF, SI, or SII was signif-icantly reduced in cells treated with UBO-QIC (Fig. 6E), suggesting—although to different degrees—the contribution of both Gaq/11 andGa12/13 in the activation of this effector. Predictably from our analysis,which revealed a stronger bias for the activation of Ga12 over Gaq forSaralasin, TRV027, and DVG among the AngII analogs (data file S1),we observed no significant effects of UBO-QIC on the activation of Rhoby these ligands (Fig. 6, A to E). Despite the undetectable efficacy of SIIfor activating Gaq (Fig. 4A), we nonetheless observed a modest inhib-itory effect of UBO-QIC on Rho activation, suggesting the involvementof both Gaq/11 and Ga12/13. These results illustrate that, notwithstandingAngII analogs activating Rho with varied efficacies in HEK293 cells,they do so by differentially engaging Gaq/11 and Ga12/13. Inhibiting

Table 1. Potency and relative efficacy (Emax) of AngII and AngII analogs for activating G protein and barr2 signaling. HEK293/AT1R cells expressing eachindicated BRET sensor were stimulated with various concentrations of AngII and AngII analogs. BRET signals from each sensor were normalized to the maximalresponse of AngII (%Emax of AngII) and then averaged. pEC50 and Emax were obtained from the nonlinear regression curve of the averaged data. Data representmeans ± SEM of three to eight independent experiments. n.d., not determined due to lack of responses.

Ligand
Gaq Gai2 Gai3 Ga12 barr2
pEC50
Emax pEC50 Emax pEC50 Emax pEC50 Emax pEC50 Emax
%AngII
%AngII %AngII %AngII %AngII
AngII
8.55 ± 0.04 100 8.34 ± 0.06 100 8.56 ± 0.04 100 8.19 ± 0.05 100 8.66 ± 0.08 100
AngIII
8.30 ± 0.08 102.0 ± 3.4 8.10 ± 0.11 86.5 ± 4.1 8.39 ± 0.08 98.5 ± 2.9 7.68 ± 0.07 95.1 ± 2.7 8.22 ± 0.13 101.0 ± 5.2
[Val4]-AngIII
8.31 ± 0.11 93.8 ± 4.2 8.50 ± 0.07 86.7 ± 2.5 8.49 ± 0.11 99.7 ± 4.3 7.78 ± 0.07 89.7 ± 2.8 8.44 ± 0.19 102.4 ± 7.6
hSarmesin
7.53 ± 0.12 39.6 ± 1.9 7.34 ± 0.29 42.0 ± 5.1 7.76 ± 0.23 40.2 ± 3.9 7.38 ± 0.20 53.8 ± 4.4 8.03 ± 0.19 93.2 ± 7.5
[Val5]-Sarmesin
7.46 ± 0.21 49.2 ± 4.2 7.15 ± 0.20 43.2 ± 3.9 7.27 ± 0.20 40.6 ± 3.4 7.56 ± 0.14 54.6 ± 3.1 8.02 ± 0.23 84.3 ± 8.2
SBpa
7.53 ± 0.08 42.5 ± 1.4 7.98 ± 0.08 73.2 ± 2.5 8.06 ± 0.12 55.8 ± 3.0 8.17 ± 0.12 64.0 ± 3.2 8.05 ± 0.15 89.9 ± 5.9
SVdF
7.10 ± 0.09 41.7 ± 1.8 7.83 ± 0.12 59.0 ± 3.0 7.69 ± 0.13 53.7 ± 3.0 7.88 ± 0.09 67.5 ± 2.5 8.16 ± 0.15 82.6 ± 5.1
SI
7.22 ± 0.55 16.5 ± 4.0 8.45 ± 0.35 27.1 ± 3.7 8.59 ± 0.27 25.6 ± 2.7 7.83 ± 0.09 48.4 ± 1.9 8.26 ± 0.18 94.1 ± 6.8
Saralasin
6.75 ± 0.48 11.3 ± 2.6 8.30 ± 0.39 25.6 ± 4.0 8.66 ± 0.34 30.1 ± 4.1 7.90 ± 0.09 60.1 ± 2.3 8.22 ± 0.25 74.8 ± 7.8
TRV
n.d. 2.1 ± 2.0* 8.18 ± 0.15 40.0 ± 2.5 8.23 ± 0.18 29.7 ± 2.1 7.61 ± 0.10 64.0 ± 2.6 8.30 ± 0.28 81.4 ± 9.2
DVG
n.d. 4.6 ± 1.3* 7.84 ± 0.22 36.1 ± 3.3 7.88 ± 0.21 32.0 ± 2.8 7.44 ± 0.10 60.9 ± 2.4 7.62 ± 0.17 82.4 ± 5.6
Ang(1–7)
n.d. 1.5 ± 2.3* 6.36 ± 0.37 31.7 ± 5.8 6.99 ± 0.29 22.9 ± 3.1 5.07 ± 0.25 46.7 ± 6.8 6.54 ± 0.23 53.4 ± 5.7
SII
n.d. 7.3 ± 1.2* 7.06 ± 0.22 27.1 ± 2.7 7.66 ± 0.29 24.4 ± 3.0 6.88 ± 0.17 43.3 ± 3.4 6.69 ± 0.22 73.9 ± 7.4
SIII
5.13 ± 0.39 24.1 ± 7.4 6.44 ± 0.43 23.6 ± 4.9 6.46 ± 0.49 25.9 ± 6.0 6.69 ± 0.21 36.1 ± 3.5 6.21 ± 0.32 72.5 ± 12.3
*Emax was obtained from the response of the ligand at a concentration of 10 mM.

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Gaq/11 differentially affected the relative effectiveness of activation ofRho for some ligands, whereas for others it had no effect, implyingthe contribution of only Ga12/13 familymembers for these biased AngIIligands (fig. S10A). The biased nature of several ligands toward Ga12was well illustrated by the stronger correlation observed betweenGa12 and Rho activation when Gaq/11 was inhibited in these cells(Fig. 6F and fig. S10B, respectively). In VSMCs, however, the activationof Rho by AngII seems to mostly involve the engagement of Gaq/11(Fig. 3, D and F).

Investigating pathway selectivity of AT1R variantsLast, we used the BRET-based platform to study the impact of pre-viously reported naturally occurring AT1R variants (www.uniprot.org/uniprot/P30556) (51–55) on signaling. We focused on nonsyn-onymous variants in the transmembrane (TM) regions of AT1R(Fig. 7A) because we reasoned that those would be the most likely


to affect stabilization of distinct receptor conformations and hencehave specific effects on agonist-mediated signaling. We selected thefollowing five variants: I3.27T, A4.60T, A6.39S, T7.33M, and C7.40W[Ballesteros-Weinstein numbering (56); hereafter abbreviated I103T,A163T, A244S, T282M, and C289W, respectively]. We transfectedconstructs encoding each receptor variant into HEK293 cells alongwith individual BRET sensors and generated concentration-responsecurves for AngII stimulation to determine the EC50 and Emax valuesfor each variant as well as for wild-type AT1R for Gaq, Gai2, Gai3,Ga12, PKC-c1b, Rho, and b-arrestin translocation to the PM and intoendosomes (fig. S11, A to H, and Table 3). Because receptor abun-dance can affect coupling efficacies, we first examined the cell surfaceabundance of all constructs by enzyme-linked immunosorbent assay(ELISA; fig. S12A). T282M was as abundant as wild-type AT1R, whereasthe abundance of A163T was increased by about 50%, and the three var-iants C289W, I103T, and A244S were reduced by 50% or less compared

Table 2. Transduction ratio and relative effectiveness of AngII and AngII analogs for activating AT1R downstream pathways. Concentration-responsedata for each ligand were analyzed by nonlinear regression using the operational model equation in GraphPad Prism with AngII as the reference ligand, asdescribed previously (29). DLog(t/KA)s were calculated by subtracting the log(t/KA) value of AngII in each pathway (Eq. 1). The SEs of Dlog(t/KA) were estimatedby Eq. 3 as described in Materials and Methods. Data represent means ± SEM of three to eight independent experiments. Relative effectiveness (RE) of theligand toward each pathway, relative to AngII, was determined using Eq. 2.

Ligand
Log(t/KA) DLog(t/KA) RE
Gaq
Gai2 Gai3 Ga12 barr2 Gaq Gai2 Gai3 Ga12 barr2 Gaq Gai2 Gai3 Ga12 barr2
AngII
8.55 ±0.03
8.41 ±0.05

8.58 ±0.04

8.22 ±0.04

8.68 ±0.7

0.00 ±0.05

0.00 ±0.07

0.00 ±0.06

0.00 ±0.06

0.00 ±0.11

1
1 1 1 1
AngIII
8.32 ±0.06
8.00 ±0.07

8.36 ±0.07

7.65 ±0.06

8.22 ±0.11

−0.22 ±0.07

−0.41 ±0.09

−0.22 ±0.07

−0.57 ±0.07

−0.47 ±0.13

0.5984
0.3917 0.6039 0.2685 0.3428
[Val4]-AngIII
8.23 ±0.06
7.98 ±0.06

8.50 ±0.07

7.66 ±0.06

8.47 ±0.12

−0.31 ±0.07

−0.43 ±0.08

−0.08 ±0.07

−0.57±0.08

−0.21 ±0.14

0.4853
0.3724 0.8318 0.2723 0.6152
Sarmesin
7.08 ±0.14
7.19 ±0.20

7.49 ±0.16

7.10 ±0.12

8.01 ±0.12

−1.47 ±0.14

−1.22 ±0.21

−1.09 ±0.16

−1.13 ±0.14

−0.68 ±0.14

0.0341
0.0603 0.0813 0.0748 0.2113
[Val5]-Sarmesin

7.12 ±0.13

6.98 ±0.16

7.02 ±0.17

7.26 ±0.13

8.01 ±0.16

−1.43 ±0.13

−1.43 ±0.18

−1.56 ±0.17

−0.95 ±0.14

−0.68 ±0.18

0.0370
0.0372 0.0277 0.1112 0.2109
SBpa
7.13 ±0.13
7.94 ±0.09

7.90 ±0.10

7.95 ±0.10

8.04 ±0.13

−1.42 ±0.14

−0.46 ±0.11

−0.68 ±0.10

−0.27 ±0.11

−0.64 ±0.15

0.0379
0.3459 0.2104 0.537 0.2296
SVdF
6.69 ±0.12
7.75 ±0.13

7.52 ±0.12

7.69 ±0.10

8.14 ±0.15

−1.86 ±0.12

−0.65 ±0.14

−1.06 ±0.12

−0.53 ±0.11

−0.55 ±0.17

0.0137
0.2218 0.0877 0.2985 0.2851
SI
6.74 ±0.33
8.07 ±0.31

8.13 ±0.26

7.48 ±0.14

8.25 ±0.16

−1.81 ±0.33

−0.34 ±0.32

−0.45 ±0.26

−0.74 ±0.15

−0.43 ±0.17

0.0157
0.4581 0.3565 0.1832 0.3715
Saralasin
5.63 ±0.44
7.93 ±0.31

8.33 ±0.23

7.65 ±0.10

8.18 ±0.20

−2.92 ±0.45

−0.48 ±0.32

−0.24 ±0.23

−0.57 ±0.11

−0.51 ±0.21

0.0012
0.3311 0.5702 0.2692 0.3105
TRV
n.d. 7.97 ±0.17
7.85 ±0.20

7.39 ±0.11

8.26 ±0.18

−4.2*
−0.44 ±0.18
−0.73 ±0.20

−0.82 ±0.12

−0.43 ±0.20

0.0000
0.3673 0.1866 0.15 0.3758
DVG
n.d. 7.58 ±0.22
7.52 ±0.22

7.20 ±0.12

7.61 ±0.15

−4.2*
−0.82 ±0.23
−1.05 ±0.22

−1.03 ±0.12

−1.07 ±0.17

0.0000
0.15 0.0883 0.0938 0.0853
Ang(1–7)
n.d. 6.05 ±0.26
6.23 ±0.21

4.93 ±0.15

6.51 ±0.26

−4.2*
−2.36 ±0.26
−2.34 ±0.21

−3.29 ±0.16

−2.18 ±0.27

0.0000
0.0044 0.0045 0.0005 0.0066
SII
n.d. 6.75 ±0.25
7.35 ±0.24

6.44±0.16

6.66 ±0.17

−4.2*
−1.66 ±0.26
−1.22 ±0.24

−1.78 ±0.17

−2.03 ±0.19

0.0000
0.0218 0.0597 0.0168 0.0094
SIII
4.49 ±0.29
6.08 ±0.34

5.76 ±0.21

6.19 ±0.20

6.18 ±0.24

−4.05 ±0.29

−2.32 ±0.35

−2.82 ±0.21

−2.03 ±0.21

−2.51 ±0.26

0.0001
0.0047 0.0015 0.0093 0.0031
*A value of −4.2 for Dlog(t/KA) was arbitrary given for calculation of bias.

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http://www.uniprot.org/uniprot/P30556

http://www.uniprot.org/uniprot/P30556


to wild-type AT1R. The differences in abundance in this relativelynarrow range did not seem to significantly affect the coupling of re-ceptors to Gaq, because all variants exhibited similar efficacy towardthis G protein and because the potency was only marginally affectedfor T282M and C289W (fig. S11A). Consistent with unaffected cou-pling to Gaq, all variants exhibited similar efficacies and potencies toactivate PKC-c1b (fig. S11E). For Gai coupling, only modest effects ofthe variants were observed. The potency or efficacy, or both, wasslightly reduced for C289W, T282M, A244S, and I103T (in the caseof Gai2) and for C289W and T282M (in the case of Gai3) (fig. S11, Band C). For C289W, I103T, and A244S, these small differences mayresult from the reduced receptor abundance having a greater impacton activation of a G protein that is more weakly coupled to the recep-tor. For Ga12, a reduced efficacy or potency, or both, was also observedfor A244S, I103T, and C289W, again consistent with an effect thatcould be a result of reduced receptor abundance (fig. S11D). However,the largest effect was observed for the T282M variant that showed areduced potency of more than 100-fold compared to wild-type AT1R,a difference that cannot be attributed to a reduced amount of the pro-tein because this variant exhibited cell surface abundance similar tothe wild-type receptor (fig. S12A).


We also observed reduced efficacies or potencies for A244S, I103T,C289W, and T282M in promoting barr2 recruitment to the PM. TheT282M variant also reduced the translocation of b-arrestin–receptorcomplexes to the endosome by more than 60% (fig. S11H and Table3), as assessed using a previously described BRET-based traffickingsensor (43). Together, these data suggest that the relatively modesteffects observed for A244S, I103T, and C289W most likely result fromthe modestly reduced cell surface abundance of the variants, whereasthe T282M mutation had a major impact on both Ga12 and b-arrestinpathways (and to a lesser extent on Gai and Gaq pathways) that couldnot be attributed to a reduction in the abundance of the receptor atthe cell surface. The apparent biased effect of this variant was con-firmed by estimating the relative activity, calculated from the ratioof maximal responses over the concentration for half-maximum re-sponses (Emax/EC50) (Table 4). The relative activity of T282M towardGa12 and barr2 was substantially more affected than that of Gaq orGai. This contrasts with the other variants for which the relative ac-tivity for the different pathways was either not affected (A163T, I103T,and A244S) or affected to similar extents (C289W) compared to wild-type AT1R (Fig. 7B and Table 4). Of the panel of variants analyzed,only T282M revealed a biased signaling pattern. Because mutations inthe receptor can also have an effect on ligand binding affinity, we mea-sured the affinity of each of the variants for radiolabeled AngII (fig.S12B and Table 3). The ligand-binding affinities of I103T, A163T,and A244S were similar to that observed for wild-type AT1R; however,although we detected ligand binding for C289W and T282M, a loss inbinding affinity prevented a reliable estimate of the Kd [dissociationconstant (binding affinity)] for these variants. These two variants alsoshowed the greatest impact on downstream signaling. Although the re-duction in affinity can explain the reduced potency, it cannot explainthe reduction in signaling efficacy observed for these variants. Theseresults also suggest that T282M affected both the affinity for AngIIand the transition toward active conformations, which are obviouslyinterrelated.

DISCUSSIONWe report on the development and use of a suite of BRET-based bio-sensors that monitor G proteins, b-arrestin, and signaling effectorsdownstream of G proteins to study the functional selectivity of AT1Rand naturally occurring receptor mutants. This resource is useful for(i) establishing signaling “fingerprints” for different AngII analogs,allowing a global characterization of their biased signaling properties;(ii) uncovering new biased activities for AngII ligands; (iii) showingthe existence of functional selectivity for naturally occurring AT1Rmutants; and (iv) demonstrating that the relative contribution of dis-tinct G protein subtypes to a common downstream effector can varyfrom ligand to ligand, hence revealing a new level of bias.

ThenewBRET-based sensorsmonitor receptor-mediated,Gprotein–dependent engagement of different effectors: PLC (as measured byDAG production), PKC, p63RhoGEF, and Rho. When combined withpreviously describedGprotein sensors (27–31), they allowed us to dem-onstrate that the relative biased activity of ligands for the receptor-mediated engagement of G proteins is conservatively propagated totheir downstream effectors. This was best observed for the Gaq sig-naling pathway. We observed that biased ligands activated the PLC,PKC, andp63/Gaq sensorswith lower efficacies as their ability to engageGaq decreased. This suggests that the biased responses observed are in-trinsic properties of different ligands on AT1R and did not result from

A

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–0.2 –0.5 –1.0 –1.5 –2.0 –2.5 –3.0 –3.5 –4.0 –4.2

AngII AngIII [Val4]-AngIII Sarmesin [Val5]-SarmesinSBpa SVdF SI Saralasin TRV DVG Ang(1–7)SII SIII

AngII AngIII [Val4]-AngIII Sarmesin [Val5]-SarmesinSBpa SVdF SI Saralasin TRV DVG Ang(1–7) SII SIII

Fig. 5. Heat map of AT1R signaling signature of various AngII analogs.(A and B) The transduction coefficients [log(t/KA)] of each AngII analog werecalculated from the concentration-response curves. The relative activity of eachligand [Dlog(t/KA)] represents the difference between the calculated transductioncoefficient [log(t/KA)] for each ligand and the transduction coefficient of referenceligand (AngII). The relative activity [Dlog(t/KA)] of AngII analogs in each signalingpathway was expressed as a heat map for (A) G protein signaling and b-arrestinactivation and (B) Gaq signaling and activation of its downstream effectorsp63RhoGEF (p63/Gaq), PLC (DAG generation), and PKC.

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the expression of the sensors. When the DAG, PKC, and Rho sensorswere used to record the activities of endogenous G proteins (Gaq/11 andGa12/13), we detected similar kinetics and relative efficiency rank orderof the ligands as those observed with the G protein BRET sensors.

The DAG, PKC, and Rho BRET sensors detected amplified re-sponses to receptor-mediated activation of G proteins. They were sen-sitive enough to detect responses from endogenous (not overexpressed)receptors, such as AT1R and mAChRs in VSMCs and HEK293 cells,respectively, and to demonstrate the coupling of overexpressed recep-tors to endogenous G proteins. The use of these BRET biosensors, incombination with pharmacological inhibitors and gene editing tools,also revealed their usefulness for dissecting biases displayed by certainligands among different G protein subtypes in the engagement of thesame effector. We show that some ligands, despite having similar ef-ficiencies for activating Rho, stimulated this effector through the dif-ferential engagement of Gaq/11 versus Ga12/13. This was best illustratedby the stronger correlation observed between the relative effectivenessof the biased ligands for engaging Ga12/13 versus Rho when Gaq/11was inhibited. Because both Gaq/11 and Ga12/13 are known to regulate


vascular tone downstream of many vasoactive GPCRs in VSMCs, in-cluding AT1R (2, 57, 58), similarly biased ligands may have distincteffects on such cellular responses, as well as other functional con-sequences in cells producing different relative amounts of these or otherG protein subtypes. Although Gai is itself inhibitory to AC, the Gbgsubunits of Gai-containing heterotrimeric G proteins can, in somecases, activate specific PLC subtypes, leading to PKC activation (59).However, our analysis of the sensors’ responses reveals a better corre-lation between the effectiveness of the different biased ligands forengaging Gaq/11 versus the PLC or PKC sensor than between Gai2versus these same sensors, again indicating that, in HEK293 cells,AT1R favors signaling through Gaq/11-PLCb-PKC. These observa-tions are consistent with the lack of effects of PTX, which inhibitsGai subtypes, on the activation of the PKC sensor. These results ob-tained with our biosensor set, monitoring the activity at different stepsof the signaling cascade for ligands with diverse biased properties,highlight the granular details of signaling flow that exists in these cells.

The activity of ligands can be influenced by the stoichiometry ofreceptors and G proteins, as well as by the efficacies or kinetics, or

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R2 = 0.9094

–12–11–10 –9 –8 –7 –6 –5

–12–11–10 –9 –8 –7 –6 –5–12–11–10 –9 –8 –7 –6 –5

Fig. 6. Rho activation upon AT1R stimulation by various AngII analogs. (A to D) Concentration-response curves for Rho activation. HEK293 cells expressing the RhoBRET sensor (PKN-RBD-RLucII plus rGFP-CAAX) along with AT1R were pretreated with vehicle or UBO-QIC (UBO) and then stimulated with the indicated concentrationsof 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 tofour independent experiments. (E) The relative activity of each ligand [Dlog(t/KA)] represents the difference between the calculated transduction coefficient [log(t/KA)]for each ligand and pretreatment conditions (vehicle or UBO-QIC) and the transduction coefficient of AngII with vehicle pretreatment. Data represent means ± SEMfrom three to four independent experiments. *P < 0.05 and **P < 0.01, unpaired Student’s t test. (F) Scatterplot of Dlog(t/KA) of Rho activation in the presence of UBO-QICversus Dlog(t/KA) of Ga12 activation by the AngII analogs. R2 analysis was determined from a linear regression.

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both, of G protein activation (25, 60–62). Although we did not per-form a systematic analysis on the sensor responses in relation totheir abundance when expressed in cells, we nonetheless observedthe same activation kinetics of Gaq and Gai by the biased ligands,as well as between Gaq and its downstream effectors, which werealso similar to those promoted by AngII. However, differences be-tween the activation kinetics of Gaq and Ga12 were observed, withmaximal activation occurring after 2 and 10 min of receptor stim-ulation, respectively. Such difference in kinetics has been previouslyobserved, although the underlying cause remains unclear (63). Thisobservation, however, had little impact on our analysis of thesignaling induced by biased ligands, because they induced the ac-tivation of these G proteins similarly to AngII. Nonetheless, the ac-tivation of these G proteins may vary among ligands in othersystems because of variations in the amount or identities of specificintracellular signaling components that are present in the cell, whichmay distinctly influence the signaling signature observed. These con-siderations might be of importance when using the BRET sensors fordrug discovery. To evaluate the effect of drugs, they should be used incell systems containing the appropriate complement of signaling com-ponents, and controls should be performed to assess the impact of thestoichiometric variation between receptors and G proteins on the dif-ferent responses.

Biased agonism has been linked to the affinity or efficacy, orboth, of the ligand-bound receptor for the downstream signalingproteins (64). These parameters were well captured in the transduc-tion coefficients determined for each ligand but may have differen-tially contributed to the biased activity of the ligands and mutant.


When comparing the apparent affinities (as determined in compe-tition binding assays) of the different ligands for AT1R, we gener-ally did not find clear relationships with their bias profiles. Wepreviously reported the following rank affinities order: AngII >>SI = SVdF >> SBpa = DVG >>> SII (1 nM, 7 and 8 nM, 17 and18 nM, and 200 nM, respectively) (18), but found different relativeeffectiveness for Gaq, Gai, and Ga12 activation, as outlined in theheat map. For example, the rank order for Gaq is AngII >> SBpa >SVdF = SI >> DVG = SII, whereas for Ga12 it is AngII = SBpa >SVdF > SI > DVG >> SII (Fig. 5). This implies an efficacy-basedbiased agonism mechanism for many AngII ligands in G proteinsignaling. However, for b-arrestin, the affinities of the ligands forAT1R seem to contribute more to the bias than do their efficacies.

Mechanisms underlying receptor functional selectivity are notfully understood but have been proposed to involve, at least in part,the stabilization of different receptor conformational states by theligands (6–8), which could lead to the binding of different G proteinsubtypes and b-arrestin with different affinities. In that respect, wepreviously showed that AngII-biased ligands promoted different con-formations of the b-arrestin found in complexes with AT1R (18, 65),suggesting a unique stabilization of the receptor-effector complex con-formation by different AngII-biased ligands. Similarly, differences in Gprotein subtype affinities for the agonist-bound muscarinic M3R thatdepended on the identity of the ligand have been reported (66).

Our findings also emphasize the existence of a greater than antici-pated diversity in the biased signaling profiles among AngII analogs.Substitutions or removal of position 8 in the AngII analogs, such as inSBpa, SVdF, SI, TRV027, DVG, SII, and Ang(1–7), has been shown to

MK

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VVGIFGN

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T M L VA K V

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G qG i2G i3G 12

Rhoarr2 at PM

PKC

arr2 in EE

****

N-term

C-term

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 thevariant residues highlighted. (B) Scatterplot of the Dlog(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 fiveindependent 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 (fP < 0.01).

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http://www.gpcrdb.org


bias these ligands toward b-arrestin signaling over Gaq/11 engagement(18–20). SII was also shown to bias AT1R against Gaq but in favor ofother G proteins, such as Gai1, Gai2, Gai3, and Ga12/13 (17, 21). Ourdata reveal previously undetected biased activities for known biasedligands [SBpa, SVdF, SI, TRV027, DVG, and Ang(1–7)] as well asfor other AngII ligands for which bias had not previously been re-ported (Sarmesin, Saralasin, and SIII). SBpa, SVdF, SI, TRV027,DVG, and Saralasin have high efficacies, compared to AngII, for acti-vating Gai2, Gai3, and Ga12 relative to Gaq. The ability of the ligandsto better engage b-arrestin rather than Gaq inversely correlated withthe bulkiness of the residue in position 8 of AngII. Our findings withthe Sarmesin peptides (where a methyl-tyrosine replaces the tyrosine atposition 4 in AngII) are consistent with the importance of the fourthresidue of AngII, as recently shown by the biased response to othersignaling pathways of the analog [Sar1, Ile4]AngII at the expense of areduction in its agonist activity on Ga12 as compared to AngII (21).They also demonstrate the importance of this position for the receptor-mediated activation of Gai2 and Gai3. Altogether, our data suggestthat the presence of Phe at position 8 of AngII is required for stabiliz-ing a conformation in AT1R compatible with its efficient binding andactivation of Gaq, whereas the Tyr at position 4 is important for sta-bilizing a conformation for the activation of Gaq, Gai, and Ga12 pro-teins. Notwithstanding that further structure-activity relationshipstudies on the AngII peptide would be needed to better understand


the contribution of each amino acid residue in the biased responsesof AT1R, our initial analysis suggests the possibility of developingnew classes of AngII ligands with specific biases.

Our findings using BRET sensors and different AngII ligands arecompatible with the ability of AT1R to couple to different G proteinsin vivo. Although AngII is known to activate Gaq/11 in different tissues,it has been shown to promote AT1R coupling to PTX-sensitive Gai/oproteins in the rat adrenal glomerulosa, liver, kidney, and pituitaryglands and to Ga12/13 proteins in rat portal vein myocytes (26, 57, 58).These observations suggest that some of the AngII-biased ligandsdescribed heremay have different actions on target tissues, depend-ing on their ability to engage different G proteins and downstreameffectors as well as on the relative abundance of these proteins inthese tissues.

The octapeptide TRV027, which has been developed for thera-peutic use (23) and has been described as a b-arrestin–biased lig-and, much like SII (15, 16), also promoted the activation of otherG proteins (for example, Gai2, Gai3, and Ga12) with somewhat bet-ter efficacies than did SII. To what extent the activation of theseG proteins contributes to the cardioprotective properties of TRV027in preclinical models remains an open question. Ang(1–7) is an activecirculating metabolite of AngI and AngII that has been proposed toact as a physiological antagonist of AngII, but its main endogenoustarget(s) still remains open to debate (67). Evidence suggests that

Table 3. Binding affinity, potency, and relative efficacy of wild-type and variant AT1Rs for activating Gaq, Gai2, Gai3, Ga12, PKC, Rho, and barr2.HEK293 cells were transfected with wild-type (WT) or variant AT1R or along with the indicated BRET sensor (Gaq, Gai2, Gai3, Ga12, PKC-c1b, Rho, barr2-PM, orbarr2-EE) and stimulated with various concentrations of AngII. BRET signals for each pathway were normalized to the maximal response of wild-type (%Emax ofWT) and then averaged. pEC50 and Emax were obtained from the nonlinear regression curve of the averaged data. Data represent means ± SEM of three to fiveindependent experiments. AngII binding affinities were obtained from [125I]-AngII saturation binding assays. Data represent means ± SD of two to threeindependent experiments performed in duplicate.

Receptor
Kd (nM) Gaq Gai2 Gai3 Ga12
pEC50
Emax pEC50 Emax pEC50 Emax pEC50 Emax
%Emax of WT
%Emax of WT %Emax of WT %Emax of WT
WT
0.53 ± 0.14 8.13 ± 0.05 100 7.71 ± 0.05 100 7.97 ± 0.08 100 7.97 ± 0.07 100
A163T
1.03 ± 0.52 8.19 ± 0.08 102.3 ± 3.3 7.63 ± 0.08 102.9 ± 3.3 7.89 ± 0.13 92.4 ± 4.9 8.00 ± 0.14 108.9 ± 5.9
T282M
n.d. 7.48 ± 0.09 98.7 ± 3.5 6.78 ± 0.09 97.0 ± 3.9 7.44 ± 0.15 73.9 ± 4.7 6.34 ± 0.10 87.4 ± 4.3
C289W
n.d. 7.86 ± 0.07 98.5 ± 3.1 6.98 ± 0.11 84.4 ± 4.4 7.30 ± 0.13 73.4 ± 4.0 7.37 ± 0.10 103.3 ± 4.4
I103T
0.24 ± 0.03 8.18 ± 0.08 94.2 ± 3.7 7.58 ± 0.19 73.0 ± 7.2 8.04 ± 0.26 85.5 ± 10.7 8.37 ± 0.24 65.9 ± 6.4
A244S
0.29 ± 0.09 8.17 ± 0.08 97.7 ± 3.7 7.66 ± 0.24 67.9 ± 8.4 8.20 ± 0.24 94.4 ± 10.9 8.48 ± 0.15 75.5 ± 4.4
Receptor
PKC Rho barr2-PM barr2-EE
pEC50
Emax pEC50 Emax pEC50 Emax pEC50 Emax
%Emax of WT
%Emax of WT %Emax of WT %Emax of WT
WT
9.02 ± 0.07 100 8.60 ± 0.07 100 8.16 ± 0.07 100 8.59 ± 0.09 100
A163T
8.85 ± 0.07 104.1 ± 3.0 8.46 ± 0.10 109.1 ± 4.4 7.97 ± 0.11 100.7 ± 4.9 8.54 ± 0.10 96.2 ± 3.7
T282M
8.31 ± 0.12 94.9 ± 4.7 7.91 ± 0.14 100.8 ± 5.8 6.94 ± 0.05 86.0 ± 2.0 7.47 ± 0.09 34.6 ± 1.3
C289W
8.49 ± 0.10 104.6 ± 4.2 7.96 ± 0.11 101.5 ± 4.8 7.18 ± 0.08 85.4 ± 3.2 7.90 ± 0.10 93.1 ± 4.0
I103T
8.98 ± 0.09 99.3 ± 4.4 8.57 ± 0.07 89.3 ± 3.0 8.25 ± 0.05 74.8 ± 1.7 8.55 ± 0.08 62.6 ± 2.4
A244S
9.03 ± 0.07 95.2 ± 3.4 8.60 ± 0.05 86.7 ± 2.1 8.26 ± 0.07 73.9 ± 2.6 8.52 ± 0.05 82.5 ± 1.9
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Ang(1–7) operates in vivo through the Mas oncogene and Mas-relatedreceptors (Mas-R and MrgD, respectively), but recent findings by usand others demonstrate that it also acts as a b-arrestin–biased agonistfor AT1R (20, 50). Consistent with these findings, we show here thatAng(1–7) not only acts as a b-arrestin–biased ligand but also biasesAT1R signaling to G proteins other than Gaq with a somewhat similareffectiveness and functional selectivity profile as SII. However, as forSII and TRV027, the physiological implications of such signaling bi-ases by Ang(1–7) are yet to be determined.

Our findings on the functional selectivity of AT1R variants are alsocompatible with a mechanism whereby biased signaling is conferredby the stabilization of distinct receptor conformations. In that respect,substituting a Thr for a Met residue in AT1R at position 282 generateda biased receptor that most likely was stabilized in a conformation thatreduces its efficacy toward Ga12 and b-arrestin activation more thanfor the other G proteins. Cocrystallization studies using nonpeptider-gic antagonists of AT1R have revealed that this amino acid in theN-terminal region of TM domain 7 (TM7) is located close to the bind-ing domain of these antagonists (54). No crystal structure of AT1Rwith AngII exists, but molecular dynamic and docking studies suggestthat position 282 is located near the AngII orthosteric binding site(68, 69). This is consistent with the observed loss of affinity theT282M variant has for AngII. Because TM6 and, to a lesser extent,TM7 reorientation upon agonist binding to GPCRs has been shownto be important for the functional coupling of receptors to G proteins(70, 71), it is tempting to speculate that mutating the Thr at position282 to Met affects transitional changes in the TM7 conformation. Thegreater loss of AngII potency to activate Ga12 and b-arrestin than to


activate Gaq observed in the T282M variant suggests that this positionplays a particular role in driving the active conformation preferred forthe engagement or activation of both Ga12 and b-arrestin. These find-ings are also consistent with the recent suggestion that the binding ofGa12 and b-arrestin to the receptor is functionally linked (63).

This new suite of BRET sensors presented here helped define thesignaling pathways downstream of AT1R and should be useful instudying the signaling of other receptors for which coupling to G pro-teins remains incompletely characterized. These tools have been usedto analyze Gaq/11-PLC-PKC signaling downstream of the GPCRFrizzled 5 (FZD5) in response to the ligand WNT-5A (72). The useof these biosensors in combination with the pharmacological analysisof biased agonism for AT1R has also led to the generation of specific“fingerprints” for different ligands and naturally occurring receptormutants. They allowed us to cluster signaling behaviors of GPCR li-gands and mutants, which, when compared to responses in relevanttissues, should also help better understand the relationship betweendifferent signaling pathways and the physiological or pathophysio-logical responses downstream of receptors. Genomic studies havenow started to reveal the preponderance of polymorphisms in GPCRs(11, 73) and, in some cases, their impact on drug efficacies. However,such efforts are limited by the lack of resources needed to mechanis-tically understand the impact of these genetic variations on signaling.Our suite of BRET biosensors should not only aid in reducing this gapbut also provide insight on the impact thesemutations have on receptorsignaling and drug efficacy and hence facilitate the decision-makingabout which drugs to be used or developed for a more personalizedmedicine.

Table 4. Relative activity of AT1R and mutant receptors for activating each signaling pathway. Top: Relative activity [log(Emax/EC50)] of each AT1R variantobtained from the concentration-response curves from each individual experiment. Bottom: DLog(RA)s were calculated by subtracting the log(RA) value of thewild-type receptor for activating the same pathway (Eq. (5)). Data represent means ± SEM of three to five independent experiments.

Receptor
Log(Emax/EC50)
Gaq
Gai2 Gai3 Ga12 PKC Rho barr2-PM barr2-EE
WT
8.13 ± 0.05 7.71 ± 0.03 7.96 ± 0.06 8.00 ± 0.10 9.03 ± 0.10 8.60 ± 0.11 8.16 ± 0.08 8.60 ± 0.14
A163T
8.20 ± 0.09 7.64 ± 0.09 7.82 ± 0.16 8.10 ± 0.12 8.87 ± 0.06 8.52 ± 0.20 7.97 ± 0.07 8.57 ± 0.15
T282M
7.44 ± 0.09 6.72 ± 0.15 7.31 ± 0.27 6.30 ± 0.11 8.26 ± 0.12 7.86 ± 0.26 6.88 ± 0.03 7.00 ± 0.12
C289W
7.82 ± 0.10 6.84 ± 0.14 7.19 ± 0.17 7.35 ± 0.10 8.49 ± 0.07 7.96 ± 0.17 7.11 ± 0.09 7.87 ± 0.16
I103T
8.18 ± 0.07 7.84 ± 0.02 7.93 ± 0.16 8.20 ± 0.06 8.95 ± 0.13 8.49 ± 0.10 8.09 ± 0.04 8.22 ± 0.10
A244S
8.17 ± 0.07 7.89 ± 0.07 8.02 ± 0.11 8.35 ± 0.06 9.09 ± 0.11 8.42 ± 0.06 8.09 ± 0.04 8.39 ± 0.04
Receptor
DLog(Emax/EC50)
Gaq
Gai2 Gai3 Ga12 PKC Rho barr2-PM barr2-EE
WT
0 0 0 0 0 0 0 0
A163T
0.068 ± 0.093 −0.078 ± 0.067 −0.141 ± 0.100 0.099 ± 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
I103T
0.034 ± 0.079 −0.051 ± 0.080 −0.073 ± 0.090 −0.0234 ± 0.112 −0.189 ± 0.094 0.172 ± 0.16 −0.205 ± 0.034 −0.348 ± 0.077
A244S
0.025 ± 0.049 −0.007 ± 0.127 0.015 ± 0.072 0.131 ± 0.127 −0.049 ± 0.088 −0.402 ± 0.228 −0.202 ± 0.022 −0.187 ± 0.022
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MATERIALS AND METHODSReagentsSome of the angiotensin ligands used here were described elsewhere(18). The sequences of all ligands used in this study are listed in tableS1. AngII (Sigma), DVG (NeoBioLab), and Ang(1–7) (Bachem) werepurchased from commercial suppliers. All other AngII analogs weresynthesized at the Université de Sherbrooke (Quebec, Canada). Iodine-125 was obtained from PerkinElmer. Dulbecco’s modified Eagle’smedium (DMEM), minimal essential medium (MEM), fetal bovine ser-um (FBS), and other cell culture reagents were purchased from Gibco,Life Technologies. Coelenterazine 400a was purchased from NanoLightTechnology and Gold Biotechnology. UBO-QIC (31) [l-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3- idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4-methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone(9CI)] was purchased fromInstitute for Pharmaceutical Biology of the University of Bonn (Bonn,Germany). YM-254890 was purchased from FUJIFILMWako ChemicalsU.S.A. Phusion DNA polymerase was from Thermo Scientific. Restrictionenzymes, T4 DNA ligase, and Gibson assembly mix were obtained fromNew England Biolabs. Oligonucleotides were synthesized at IntegratedDNA Technologies. Strep-Tactin sepharose and Strep-Tactin conjugatedto horseradish peroxidase (Strep-Tactin HRP) were purchased from IBAGmbH. The anti-phosphothreonine (#9381) and anti-RhoA (#2117) anti-bodies were purchased from Cell Signaling Technology. Anti–Renillaluciferase antibody (MAB4400) was obtained from EMD Millipore.Bradford protein assay was from Bio-Rad. Gö6983 and Gö6976 werefrom Calbiochem, and LY333531 was from Tocris Bioscience. PD 123319,PTX, and A23187 were purchased from Sigma. Glutathione Sepharose4B was from GE Healthcare.

BRET biosensor constructsThe polycistronic Gaq BRET sensor (29), Ga12(136)-RlucII, Gai1(91)-RlucII (30), Gai2-RlucII (30), Gai3-RlucII (30), GFP10-Gg1 (74),GFP10-Gg2 (74), Flag-Gb1 (29), GFP10-barr2-RlucII (29), barr2-RlucII,rGFP-FYVE and rGFP-CAAX (43), and signal peptide–Flag–taggedhuman AT1R (sp-Flag-AT1R) were described previously (32). DNAencoding the c1b domain of PKCd, the FHA1 and FHA2 domainsof yeast Rad53, and the DIS300 linker were codon-optimized andsynthesized by GenScript. The DIS300 linker and DIS50 are artificiallydesigned linkers 300 and 50 residues in length, respectively. The DIS300linker was created by generating a random sequence respecting thecomposition of natural disordered sequences. The random sequencewas of a few thousand residues, and the sequence was analyzed forthe absence of any nuclear localization sequence motif, for phosphoryl-ation and known protein-protein interaction sites, and for its predictedglobularity. Structure prediction was used to identify the most dis-ordered stretches of the sequence, and the best 300- and 50-residue-long sequences were selected. The DIS300 sequence is as follows:KEGEKQKGAMQPSEQQRGKEAQKEKNGKEPNPRPEQPK-PAKVEQQEDEPEERPKREPMQLEPAESAKQGRNLPQKVEQ-GEERPQEADMPGQAQSAMRPQLSNSEEGPARGKPAPEEP-DEQLGEPEEAQGEHADEPAPSKPSEKHMVPQMAEPEK-G E E A R E P Q G A E D K P A P V H K P K K E E P Q R P N E E -KAPKPKGRHVGRQENDDSAGKPEPGRPDRKGKEKEPEEE-PAQGHSLPQEPEPMPRPKPEVRKKPHPGASPHQVSDVE-DAKGPERKVNPMEGEESAKQAQQEGPAENDEAERPERP. TheDIS50 sequence is as follows: EPGRPDRKGKEKEPEEEPAQGHSLP-QEPEPMPRPKPEVRKKPHPGASPHQ.


To construct the PKC sensor, Strep-tag II (stII)–GFP10–FHA2–RFRRFQTLKI [a PKC substrate (34)] was synthesized by GenScriptand cloned into theN terminus of RlucII in pcDNA3.1 (pPKC2). DNAsencoding FHA1 and c1b were polymerase chain reaction (PCR)–amplified and subcloned into the pPKC2. A DNA oligo encodingthe second PKC substrate (RFRRFQTLKD) was inserted by linkerligation. Last, the DIS50 linker was inserted between FHA2 and thesubstrate cassette. Two threonine residues found in PKC substrateswere mutated to alanine residues by linker ligation. Lyn-PKC wasgenerated by inserting the Bsr GI–Xho I fragment from the PKCsensor (FHA1-FHA2-linker-PKC substrates) into a vector containingthe Lyn-GFP10 andRlucII-stII by ligation. This generated a PKC sensorlacking the c1b domain and with a Lyn sequence in its N terminus. Toconstruct the DAG sensor, a DIS300 linker and RlucII-c1b were sub-cloned into the Lyn-GFP10 (43) in pcDNA3.1 by using in-fusion tech-nology. To generate PKN-RBD-RlucII, the coding sequence of the first93 residues of the human PKN1 (GenBank accession no. AAH94766.1)was synthesized by GeneArt (Thermo Fisher) as a DNA string andsubcloned to pcDNA3.1 hygro (+) GFP10-RlucII, using Nhe I andAge I sites, replacing GFP10 by the RBD of PKN1 (PKN-RBD). Alinker sequence (IDTGGRAIDIKLPAT) is present between PKNand RlucII. For the pIRES hygro p63-GFP10-stII construct, GFP10was PCR-amplified from the EPAC db sensor construct (75) using thefollowing primers: 5′-GCTAGCGGATCCGCCGGTACCATGGT-GAGCAAGGGCGAGGAG-3′ and 5′-ATCGGATCCTTAT-TTTTCGAACTGCGGGTGGCTCCACTTGTACAGCTCGTC-CATGCC-3′. PCRproductswere subcloned inpIRESHygro3 (Clontech)using Nhe I and Eco RV sites. The construct encoding the Gaq bindingdomain of the human p63RhoGEF (residues 295 to 502, p63BD) taggedwith GFP10 was done by PCR amplification from an IMAGE clone(OpenBiosystems) encoding the p63RhoGEF and subcloned byGibsonassembly in pIRES Hygro3 GFP10-stII digested with Nhe I. A peptidelinker (PASGSAGT) is present between the p63BD andGFP10. For theGST-tagged p63BD, the Gaq binding domain was PCR-amplified frompIRES Hygro p63BD-GFP10-stII using the following primers: 5′-CTGATCGAAGGTCGTGGGATCCCCGAATTCATGATTAT-GAAGTACCAGTTGCTC-3′ and 5′-GCGGCCGCTCGAGTC-GACCCGGGAATTCAACTTCCAACTCCGGGTCCTCTGGG-3′,and subcloned by Gibson assembly in pGEX 5X2 digested with Eco RI.

Site-directed mutagenesis of AT1RThe AT1R variants (A4.60T, T7.33M, and C7.40W; also referred to asA163T, T282M, and C289W) were generated by PCR with overlappingends and Gibson assembly. Two complementary oligonucleotidescontaining the desired mutations were used as 5′ and 3′ primers withits matching Xba I reverse primer (5′-GTGACACTATAGAATAG-GGCCCTCTAGA-3′) and Eco RI forward primer (5′-CTACTGAA-GATGGCATCAAAAGAATTCAAGATGA-3′) from the sp-Flag-AT1R in pcDNA3.1, respectively. Two PCR amplicons, overlappingat the location of the altered bases, were assembled with Eco RI–XbaI–cleaved sp-Flag-AT1R in pcDNA3.1 in one step using the NEBuilderHiFi DNA Assembly Cloning Kit (New England Biolabs). The 5′ prim-ers were as follows, with the mutated nucleotides underlined: A163T,GCCAGTTTGCCAACTATAATCCATCGA; T282M, AATTGCA-GATATTGTGGACATGGCCATGCC; C289W, GCCATGCCTAT-CACCATTTGGATAGCT. The I3.27T and A6.39S variants (I103T andA244S) were generated by the whole-plasmid PCR technique. Briefly,primers containing the desired mutations were designed complement-ing opposite strands of the plasmid and extended using DNA polymerase

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(Phusion Flash High-Fidelity PCR Master Mix, Thermo Scientific).Dpn I endonuclease (Invitrogen, Thermo Fisher Scientific) was used todigest the DNA template and to select the synthesized DNA containingmutations. The 5′ primers were as follows, with the mutated nucleotidesunderlined: I103T, GGCAATTACCTATGTAAGACTGCTTCAGC-CAGCGT (forward) and ACGCTGGCTGAAGCAGTCTTACA-TAGGTAATTG (reverse); A244S, TTTAAGATAATTA-TGTCAATTGTGCTTTTCTTT (forward) and AAGCACAATT-GACATAATTATCTTAAAAATATCATCATT (reverse). Allconstructs were validated by sequencing.

Cell culture and transfectionWe used a HEK293 clonal cell line (HEK293SL cells) previously de-scribed in (43, 76), hereafter referred to as HEK293 cells. HEK293and HEK293/AT1R cells (HEK293SL cells, which stably express thesp-Flag-AT1R) (32) were cultured in MEM and DMEM, respectively,supplemented with 10% FBS and gentamicin (20 mg/ml). Cells weregrown at 37°C in 5% CO2 and 90% humidity. Cells were seeded at adensity of 7.5 × 105 cells per 100-mm dish and were transiently trans-fected the next daywith sp-Flag-AT1R (3 mg) alongwith either theGaq-polycistronic BRET sensor (4.5 mg), or with the Gai2-RlucII (60 ng), orthe Gai3-RlucII (0.24 mg) and GFP10-Gg2 (0.6 mg) and Gb1 (0.6 mg)sensor, or with the Ga12(136)-RlucII (0.24 mg) and GFP10-Gg1 (0.6 mg)andGb1 (0.6 mg) sensor, orwith theGFP10-barr2-RlucII (0.075mg) sen-sor using the calciumphosphate precipitationmethod, as previously de-scribed (77). For the DAG and PKC sensors, 0.15 and 0.18 mg of sensorDNA were transfected in HEK293/AT1R cells, respectively, using thecalcium phosphate precipitationmethods. For the Rho sensor, 0.67 mgof sp-Flag AT1R, along with 0.12 mg of PKN-RBD-RlucII and 0.48 mgof rGFP-CAAX, was transfected. After 18 hours of transfection, themediumwas replaced and cells were divided for subsequent experiments.For the p63/Gaq sensor, cells were seeded onto polyornithine-coatedwhite 96-well plate at a density of 1 × 104 cells per well. The next day,cells were transfected by Lipofectamine 2000 (Invitrogen) with 34 ngof AT1R, 51 ng of p63BD-GFP10, and 3.4 ng of Gaq(118)-RlucII perwell. For receptor titration experiments, HEK293 cells were trans-fected in suspension using polyethyleneimine [PEI; 1 mg/ml inphosphate-buffered saline (PBS), linear 25 kDa, Polysciences]. Briefly,20 ng of PKC sensorDNA, alongwith different amounts of AT1RDNA[adjusted total DNA amount to 1 mg by single-stranded salmon spermDNA (ssDNA, Sigma-Aldrich)] in 100 ml of PBS, wasmixedwith 100 mlof PBS containing 3 ml of PEI. After 20min of incubation, theDNA-PEIcomplexes (200 ml) weremixed with 3.5 × 105 cells in 1ml ofmedia andthen distributed into 12 wells in a poly-D-lysine–coated white 96-wellplate (100 ml per well). For the p63/Gaq sensor, 50 ng of Gaq(118)-RlucIIand 1 mg of p63-GFP10, along with different amounts of AT1R (0 to300 ng) and ssDNA (for adjustment of a total of 1.35 mg ofDNA),wereused. For testing the specificity of p63BD-GFP10 for different receptorsand Ga-RlucII, 1 mg of p63-GFP10, along with either Gaq(118)-RlucII(50 ng), Ga12(136)-RlucII (50 ng), Gas-RlucII (40 ng), or Gai2-RlucII(50 ng) with different receptorDNAs [b2AR (250 ng), D4R (100 ng), FP(300 ng), or AT1R (300 ng)] and ssDNA (for adjustment of a total of1.35 mg of DNA), was transfected in cells. All assays were performed48 hours after transfection. CRISPR Gq/11 and G12/13 knockout celllines were a gift from A. Inoue (Tohoku University, Sendai, Miyagi,Japan) and derived as previously described (31, 47). Briefly, eitherGNAQ andGNA11 orGNA12 andGNA13 genes in HEK293 cells weresimultaneously targeted using a CRISPR-Cas9 system to generate theGq/11 and G12/13 knockout cell lines, respectively. For experiments with


AT1R mutants, HEK293 cells were transfected with 3 mg of receptorDNA along with the G protein sensors or 1 mg of receptor DNA alongwith the PKC-c1b or Rho sensor using the calcium phosphate method.For the barr2 BRET experiments, HEK293 cells were transfected with120 ng of barr2-RlucII along with either 480 ng of rGFP-CAAX (PMtranslocation) or rGFP-FYVE (endosomal translocation) along with1 mg of the receptor DNA.

BRET measurementsOne day after transfection, HEK293 cells were detached and seededonto polyornithine-coated white 96-well plates at a density of 2.5 ×104 cells per well in media. The next day, cells were washed oncewith Tyrode’s buffer [140 mMNaCl, 2.7 mM KCl, 1 mM CaCl2, 12 mMNaHCO3, 5.6 mM D-glucose, 0.5 mM MgCl2, 0.37 mM NaH2PO4,25 mM Hepes (pH 7.4)] and left in Tyrode’s buffer. For kinetic mea-surements, BRET signals were monitored every 2 s after addition ofthe cell-permeable coelenterazine 400a at a final concentration of5 mM, using a Synergy2 (BioTek) microplate reader. The filter set was410/80 nm and 515/30 nm for detecting the RlucII (Renilla luciferase)(donor) and GFP10 (acceptor) light emissions, respectively. AngII (at afinal concentration of 100 nM) was injected after 11 measurements (20 s),and BRET was recorded over a period of 5 min. For concentration-response curves of the Gaq-poly, Gai2 and Gai3, p63/Gaq, and Rhosensors, BRET signals were measured after the cells were stimulatedwith various concentrations of ligand in Tyrode’s buffer for 2 min atroom temperature (21°C). For detecting DAG, PKC-c1b, and Lyn-PKCsensors, cells were stimulated with ligands for 1 min before BRETmeasurement. For the Ga12 sensor, cells were stimulated for 10 min at37°C. For detecting barr2 at the PM or in endosomes, cells were stimu-lated with either various concentrations of AngII for 4 min at roomtemperature or for 30 min at 37°C, before BRET measurements. Coe-lenterazine 400a (final concentrations of 5 mM) was added 3 to 5 minbefore BRET measurements. For the barr2 sensor, BRET signals weremeasured 20 min after addition of various concentrations of ligands,and coelenterazine 400a was added 10 min before BRET measure-ments. The Gaq/11 inhibitors [UBO-QIC (100 nM) and YM-254890(200 nM)] and PKC inhibitors [Gö6983 (2 mM), Gö6976 (3 mM),and LY333531 (3 mM)] were added for 30 min before ligand stimula-tion. PMA and forskolin were used at a concentration of 1 and 10 mM,respectively, for 10 min. A23187 was used at a concentration of 1 mMfor 1 min. To inhibit Gai, cells were incubated overnight with PTX(100 ng/ml) before ligand stimulation. PD 123319 and losartan wereadded at a concentration of 10 mM for 30 min, before adding 1 mMAngII. For single-concentration stimulations of the p63/Gaq sensor,cells were stimulated for 1 min with either AngII (100 nM), PGF2a(100 nM), dopamine (1 mM), or isoproterenol (1 mM). The BRET ratiowas determined by calculating the ratio of the light emitted by GFP10over the light emitted by the RlucII. All BRET experiments were per-formed in triplicate.

Radioligand binding experimentsAffinities and receptor abundance were assessed by ligand bindingassays using [125I]-AngII in either dose-displacement or saturationexperiments. [125I]-AngII was prepared using the Iodogen method,as previously described (18). For binding experiments, HEK293 cellsfrom a 10-cm culture dish transiently transfected with 3 mg of wild-type AT1R or mutant receptors or HEK293/AT1R cells were seeded ata density of ~1.5 × 105 cells per well in polyornithine-coated 24-wellplates, 1 day before binding experiments. The following day, cells were

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washed once with cold PBS and then incubated in the absence or pres-ence of increasing concentrations of AngII with a fixed concentrationof [125I]-AngII (0.18 nM; ~150,000 cpm at 2200 Ci/mmol) in bindingbuffer [25 mM tris-HCl (pH 7.4), 100 mM NaCl, 5 mM MgCl2, with0.2% (w/v) bovine serum albumin (BSA)] at 4°C overnight. For satu-ration binding experiments, cells were incubated at room temperaturefor 1 hour in the same binding buffer containing increasing concen-trations of [125I]-AngII (at 180 Ci/mmol). Nonspecific binding wasdetermined in the presence of 1 mM AngII. Cells were then washedthree times with ice-cold PBS and solubilized in 0.5 ml of 0.2 MNaOH/0.05% SDS, and bound radioactivity was counted using aPerkinElmer Wizard 1470 automatic g-counter. Protein amounts weredetermined using a Bradford assay after harvesting cells using 10 mMEDTA. AT1R, in transient transfection experiments, was expressed atlevels between 1 and 3 pmol/mg of total proteins, whereas in HEK293/AT1R cells it was expressed at 0.5 pmol/mg. AngII affinity (Kd) forAT1R was determined to be between 0.1 and 0.5 nM for both con-ditions. Mutant receptors were expressed between 1 and 5 pmol/mgfor A163T and between 0.5 and 1.5 pmol/mg for both I103T andA244S with affinities between 0.5 and 0.9 nM. The C289W mutantaffinity was between 2 and 5 nM with an expression level between0.5 and 1.5 pmol/mg. The expression of the T282M mutant couldnot be determined from the saturation binding isotherm because ofthe lack of apparent saturation and was rather extrapolated for ELISAdata (see below) by comparing it to that of the binding and ELISAdata obtained from the wild type expressed at different levels. It wasestimated to be between 1 and 3 pmol/mg. The affinity was estimatedto be at least greater than 100 nM.

Intact cell ELISAThe cell surface abundance of AT1R mutants was assessed by cellsurface ELISA. Cells were transfected with 3 mg of wild-type or mutantreceptors. The next day, cells were plated onto polyornithine-coatedtransparent 96-well plates at a density of 4.5 × 104 cells per well. After24 hours, cells were washed once with PBS and fixed with 3% para-formaldehyde for 10 min at room temperature. Cells were washedtwice with PBS and incubated for 1 hour with 0.05% BSA in PBSfor blocking. Then, cells were incubated with M2 anti-FLAG antibody(1:1000 in PBS/BSA) for 1 hour at room temperature, washed twicewith PBS/BSA, and reblocked with PBS/BSA for 10 min. Cells wereincubated with HRP-conjugated secondary antibody (donkey anti-mouse antibody, 1:1000 in PBS/BSA) for 1 hour and washed fourtimes with PBS, and then, colorimetric HRP substrate (SIGMAFASTOPD) was added. The reaction was stopped after 10 min by adding3 M HCl, and the plate was read at an absorbance of 492 nm with amicroplate reader (Synergy2, Biotek). To obtain specific signal, non-specific signal from mock DNA (pcDNA)–transfected cells was sub-tracted. Protein amounts were determined using a Bradford assay aftercell lysis using 0.01% SDS.

Strep-Tactin pull-down experimentsBriefly, HEK293 cells were seeded at a density of 1 × 105 cells perwell in polyornithine-coated 6-well plates. The next day, the cells weretransfected with the PKC-c1b sensor (200 ng per well) along withAT1R (500 ng per well) using the calcium phosphate precipitationmethod. Forty-eight hours after transfection, the cells were serum-starved with DMEM containing 20 mM Hepes and stimulated with1 mM of either AngII or PMA for 1 or 10 min, respectively. Cells werethen transferred to ice, washed twice with ice-cold PBS, and solubi-


lized for 1 hour at 4°C in 0.4 ml of solubilization buffer [50 mMHepes, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100(pH 7.4)] supplemented with protease inhibitors [leupeptin (20 mg/ml),aprotinin (10 mg/ml), pepstatin A (2 mg/ml), and 10 mM phenyl-methylsulfonyl fluoride (PMSF)] and phosphatase inhibitors (40 mMsodium pyrophosphate and 10 mM NaF). The samples were clearedby centrifugation and then transferred to fresh tubeswith 20 ml of Strep-Tactin sepharose (IBA) and incubated for 1 to 2 hours withmixing at4°C. The samples were then washed three times with solubilizationbuffer and then incubated in Laemmli buffer for 10 min at 65°C, fol-lowed by SDS–polyacrylamide gel electrophoresis (PAGE), transferredto nitrocellulose membranes, and immunoblotted to detect threoninephosphorylation of the PKC sensor. The total amount of pull-down sen-sor was determined by blotting with Strep-Tactin–HRP after stripping.

GST pull-down assaysGST-tagged Rhotekin-RBD (GST-Rhotekin-RBD) and p63BD (GST-p63BD) were expressed in Escherichia coli BL21 cells by inductionwith 0.6 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at 30°Cfor 3 hours and 0.12 mM IPTG at 15°C for 16 hours, respectively.The GST fusion proteins were purified using Glutathione Sepharose4B as previously described (78). For the Rho activation assay usingGST-Rhotekin-RBD, the parental HEK293 cells and the CRISPRGaq/11 knockout (DGaq/11) and Ga12/13 knockout (DGa12/13) cells weretransiently transfected in 100-mm dishes with 3 mg of sp-Flag-AT1R bycalcium phosphate methods. The next day, the cells were reseeded ontopolyornithine-coated six-well plates (~8×105 cells perwell). Forty-eighthours after transfection, cells were serum-starved for ~3 hours withDMEM containing 20 mM Hepes and incubated in the absence(DMSO, vehicle) or presence of YM-254890 (200nM) for 30minbeforebeing either left unstimulated or stimulated with 1 mMAngII for 5 minat 37°C. For the GST-p63BD pull-down experiments, HEK293 cellswere transfected in 100-mm dishes with AT1R (3 mg) along with eitherGaq(118)-RlucII (500 ng) or Ga12(136)-RlucII (500 ng). The next day,cells were reseeded in a six-well plate. Transfected cells were washedonce with ice-cold PBS and lysed for 30 min at 4°C in 300 ml of lysisbuffer [50 mM tris-HCl (pH 7.4), 137 mM NaCl, 5 mM MgCl2, 10%glycerol, 1% NP-40, 5 mM dithiothreitol, 20 mM NaF] supplementedwith protease inhibitors [1 mM PMSF, leupeptin (10 mg/ml), aprotinin(5mg/ml), andpepstatinA (1mg/ml)] andphosphatase inhibitors (20mMNaF and 0.025 mM sodium pervanadate). Samples were cleared bycentrifugation, and 30 ml was kept as total cell lysate. The remaininglysate was transferred to fresh tubes; incubated with 30 mg of eitherGST,GST-Rhotekin-RBD, orGST-p63BD coupled to glutathione resin;and rotated for at least 1 hour at 4°C. Beadswere thenwashed twicewithlysis buffer, and proteinswere eluted in 25ml of sample buffer by heatingat 65°C for 10 min. Proteins were resolved on SDS-PAGE, transferredto nitrocellulose membranes, and immunoblotted for detecting RhoAor RlucII.

Lentiviral vector production and transduction of rat VSMCsThe coding regions of PKC-c1b sensor, PKN-RBD-RlucII, andrGFP-CAAX were PCR-amplified and subcloned into Asc I/BamHI, Spe I/Mfe I, and Spe I/Not I sties of pLVXi2H (29), respectively.Sensor coding viruses were generated as described previously (79).Briefly, Lenti-X 293T (Clontech) cells were seeded at a density of3 × 106 cells onto polyornithine-coated 100-mm dishes. The nextday, cells were transfected with either 6 mg of pLVXi2H-PKC or 4 mgof rGFP-CAAX/2 mg of PKN-RBD-RLucII in pLVXi2H along with

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4.5 mg of psPAX2 and 1.5 mg of pMD2.G packaging DNA by a PEImethod. The medium was changed 16 hours after transfection.Forty-eight to 60 hours after transfection, the virus-containing me-dium was harvested by centrifugation at 300g for 10 min and filteredthrough 0.45-mm pore size filters. Virus was stored either at 4°C up to2 days or at −80°C. Rat VSMCs (passages 13 to 15) were seeded at adensity of 20,000 cells per well in polyornithine-coated white 96-wellplates 1 day before viral vector transduction. The cells were transducedwith the sensor-encoding virus (60 to 100 ml of viral soup for PKC-c1bsensor or 15 to 25 ml for Rho sensor) in the presence of polybrene(8 mg/ml), and the next day, the medium was changed. Forty-eight hoursafter viral transduction, the cells were washed once with Tyrode’s buf-fer, and BRET assays were performed as described above.

Confocal microscopyOne day before transfection, cells were seeded in 35-mm glass-bottom dishes at a density of 1 × 105 cells per dish. HEK293 cells weretransfected with 67 ng of AT1R along with either 33 ng of DAG sensoror 33 ng of PKC-c1b or Lyn-PKC sensor per dish using the calciumphosphate method. Forty-eight hours after transfection, cells wereimaged before and after stimulation with 1 mM AngII with a ZeissLSM-510 laser scanningmicroscope. To detect GFP10, an ultravioletlaser was used with 405-nm excitation and 505- to 550-nm bandpassemission filter. Images (1024 × 1024) were collected using a 63× oilimmersion lens.

Data analysisData were first normalized as percentage of the Emax of AngII (ref-erence ligand) in each experiment. The normalized data were analyzedusing “Operational Model” in GraphPad Prism 6 to determine the trans-duction ratio, log(t/KA), of each ligand on each signaling pathway, aspreviously described (29, 80). A user-defined equation AngII, AngIII,and [Val4]-AngIII were treated as full agonists, and others were trea-ted as partial agonists. The relative effectiveness (RE) of the ligandscompared to the reference ligand for a specific pathway is then deter-mined from the difference between their log(t/KA) values usingEqs. 1 and 2

DlogtKA

� �¼ log

tKA

� �ligand

� logtKA

� �AngII

ð1Þ

Relative effectivenessðREÞ ¼ 10Dlog t

KA

� �ð2Þ

SEs on the Dlog(t/KA) are calculated using Eq. 3

SEDlog t

KA

� �� ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðSEligandÞ2 þ ðSEAngIIÞ2

qð3Þ

To quantify biased agonism of the mutant AT1R receptors rel-ative to the wild-type receptor, we used a “relative activity (RA)” scale(6, 81). This scale uses a ratio of the maximal response to the EC50

value for an agonist. Data were normalized to the Emax of AngII inwild-type receptor in a specific signal pathway, and EC50 and Emax

were estimated from the nonlinear regression curve fitting equationsin GraphPad Prism. Relative activity of the mutant receptors com-pared to the reference receptor (wild-type receptor) for a specific


pathway is then determined from the difference between their log(Emax/EC50) values using Eqs. 4 and 5

Relative activityðRAÞ ¼ EmaxEC50′

EC50Emax′¼ Emax

EC50

� �Emax′EC50′

� �ð4Þ

DlogEmaxEC50

� �¼ log

EmaxEC50

� �mutant

� logEmaxEC50

� �WT

ð5Þ

Statistical analyses were performed using GraphPad Prism 6 software(GraphPad Software Inc.) using Student’s t tests, two-way analyses of var-iance (ANOVAs), or Tukey’s post hoc multiple comparisons tests, whenappropriate. Normality from a typical BRET experiment (AngII-mediated Gaq activation, n = 13) was analyzed using a D’Agostino-Pearson test, and data were found to follow a normal probabilitydistribution. Because all BRET experiments were performed in a sim-ilar manner, we infer normal distribution of data for all the other BRETexperiments. Curves presented throughout this study represent the bestfits and were generated using GraphPad Prism software. P values ≤0.05were considered significant.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/11/559/eaat1631/DC1Fig. S1. Responses of G protein and barr2 BRET sensors in naïve and AT1R-expressing HEK293cells.Fig. S2. Characterization of the DAG BRET sensor.Fig. S3. Characterization of the PKC BRET sensors.Fig. S4. Characterization of the p63RhoGEF (p63/Gaq) 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/Gaq), PKC,and PLC (DAG) BRET sensors by AngII analogs.Fig. S8. Heat map signaling signatures of Gaq and downstream effectors by AngII analogs.Fig. S9. Correlation plot analysis of Dlog(t/KA) for G proteins and b-arrestin sensors againstdownstream signaling effectors.Fig. S10. Heat map and correlation plot of Dlog(t/KA) for Rho and Ga12 activation by AngIIanalogs.Fig. S11. Concentration-response curves for G proteins, PKC, Rho, and b-arrestin activation bywild-type and mutant AT1Rs.Fig. S12. Assessment of cell surface abundance and AngII affinities for wild-type and mutantAT1Rs.Table S1. Sequences of AngII and AngII analogs.Table S2. Potency and relative efficacy of AngII and AngII analogs for activating thep63RhoGEF (p63/Gaq), PKC, and PLC (DAG) sensors.Table S3. Transduction ratio and relative effectiveness of AngII and AngII analogs for activatingthe p63RhoGEF (p63/Gaq), PKC, and PLC (DAG) sensors.Table S4. Potency and relative efficacy of AngII and AngII analogs for activating the Rho sensorin the absence or presence of Gaq/11 inhibition.Table S5. Transduction ratio and relative effectiveness of AngII and AngII analogs for activatingthe Rho sensor.Data file S1. Statistical analysis of Dlog(t/KA) of AngII analogs between signaling pathways.

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Acknowledgments: We thank the RI-MUHC for the use of their imaging platform and themembers of Laporte laboratory for helpful discussion. Funding: This work was supported bygrants from the Canadian Institutes of Health Research (CIHR) (MOP-74603 to S.A.L. andFDN-14843 to M.B.), the Sao Paulo Research Foundation (FAPESP 2012/20148-0 to C.M.C.-N.),and the CQDM (Consortium Québécois sur la Découverte du Médicament; to G.P., T.E.H.,R.L., S.A.L., and M.B.). M.B. holds a Canada Research Chair in Signal Transduction and MolecularPharmacology. Author contributions: Y.N., S.K., Y.C., J.G., L.B.T., and S.C.S. performedexperiments and produced data for presentation. Y.N., C.L., V.L., J.-M.L., D.D., T.E.H., G.P., R.L.,M.B., and S.A.L. contributed to the development and the validation of sensors. Y.N.,C.M.C.-N., M.B., and S.A.L. designed experiments and interpreted the data. Y.N., Y.C., R.L., T.E.H.,M.B., and S.A.L. contributed to writing the manuscript. Competing interests: The BRETbiosensors presented here have been licensed to Domain Therapeutics for commercialization.M.B. is the president of the Scientific Advisory Board of Domain Therapeutics, a companyinvolved in the discovery of drugs targeting GPCRs. Data and materials availability: TheBRET sensors can be obtained upon request to S.A.L. or M.B. and used for academic researchwith a standard academic material transfer agreement (MTA).

Submitted 30 January 2018Accepted 13 November 2018Published 4 December 201810.1126/scisignal.aat1631

Citation: Y. Namkung, C. LeGouill, S. Kumar, Y. Cao, L. B. Teixeira, V. Lukasheva, J. Giubilaro,S. C. Simões, J.-M. Longpré, D. Devost, T. E. Hébert, G. Piñeyro, R. Leduc, C. M. Costa-Neto,M. Bouvier, S. A. Laporte, Functional selectivity profiling of the angiotensin II type1 receptor using pathway-wide BRET signaling sensors. Sci. Signal. 11, eaat1631 (2018).

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