Mapping Angiotensin II Type 1 Receptor-Biased Signaling Using Proximity Labeling and Proteomics Identifies Diverse Actions of Bi

pubs.acs.org/jpr Article

Mapping Angiotensin II Type 1 Receptor-Biased Signaling Using Proximity Labeling and Proteomics Identiﬁes Diverse Actions of Biased Agonists Conrad T. Pfeiﬀer, Jialu Wang, Joao A. Paulo, Xue Jiang, Steven P. Gygi, and Howard A. Rockman*

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ABSTRACT: Angiotensin II type 1 receptors (AT1Rs) are one of the most widely studied G-protein-coupled receptors. To fully appreciate the diversity in cellular signaling profiles activated by AT1R transducer-biased ligands, we utilized peroxidase-catalyzed proximity labeling to capture proteins in close proximity to AT1Rs in response to six different ligands: angiotensin II (full agonist), S1I8 (partial agonist), TRV055 and TRV056 (G-protein-biased agonists), and TRV026 and TRV027 (β-arrestin-biased agonists) at 90 s, 10 min, and 60 min after stimulation (ProteomeXchange Identifier PXD023814). We systematically analyzed the kinetics of AT1R trafficking and determined that distinct ligands lead AT1R to different cellular compartments for downstream signaling activation and receptor degradation/recycling. Distinct proximity labeling of proteins from a number of functional classes, including GTPases, adaptor proteins, and kinases, was activated by different ligands suggesting unique signaling and physiological roles of the AT1R. Ligands within the same class, that is, either G-protein- biased or β-arrestin-biased, shared high similarity in their labeling profiles. A comparison between ligand classes revealed distinct signaling activation such as greater labeling by G-protein-biased ligands on ESCRT-0 complex proteins that act as the sorting machinery for ubiquitinated proteins. Our study provides a comprehensive analysis of AT1R receptor-trafficking kinetics and signaling activation profiles induced by distinct classes of ligands. KEYWORDS: AT1R, biased agonism, APEX, proximity labeling

Downloaded via DUKE UNIV on May 6, 2021 at 12:58:58 (UTC). ■ INTRODUCTION the extracellular face subsequently leads to activation of − G-protein-coupled receptors (GPCRs) constitute the largest heterotrimeric G protein by guanosine diphosphate guanosine class of cell surface receptors in mammals. The biological role of triphosphate exchange on the intracellular side of the receptor. G these receptors is to detect various external stimuli and initiate or protein activation results in the dissociation of the G protein terminate internal signals that regulate a diverse range of complex into α and β/γ subunits, which initiate various See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. physiological processes from immune activity and behavioral intracellular signaling cascades. Additionally, GPCR activation and mood regulation to cardiovascular and blood pressure leads to recruitment of G-protein-coupled receptor kinases homeostasis. Due to their critical roles in physiological (GRKs)4,5 that phosphorylate serine and threonine residues on processes, GPCRs are one of the most important subgroups of the c-terminal tail of the receptor. This phosphorylation protein targets for therapeutics with estimates that a third of all increases the aﬃnity of the receptor for the multifunctional 1,2 FDA approved drugs target these receptors. protein and transducer, β-arrestin, which plays a role in the Also known as seven-transmembrane receptors, GPCRs share termination of G protein signaling, internalization/translocation similar structural features such that a seven-helix bundle passes of the receptor, and activation of β-arrestin-dependent signaling through the cellular membrane with the ends of the helices pathways.6 The ratio between G-protein-dependent and β- forming both extracellular and intercellular domains. Ligands of GPCRs range from small molecules to proteins and typically bind to the transmembrane domain of the extracellular face of Received: January 27, 2021 the receptor. Ligands that act as blockers of receptor activity are known as antagonists. Conversely, agonists, or activators of receptor activity, result in conformation changes in the receptor most notably including a large outward movement of transmembrane helix (TM) 6.3 GPCR activation by ligand binding at

© XXXX American Chemical Society https://doi.org/10.1021/acs.jproteome.1c00080 A J. Proteome Res. XXXX, XXX, XXX−XXX Journal of Proteome Research pubs.acs.org/jpr Article arrestin-dependent signaling is often defined by the endogenous confluency, the medium was replaced with a fresh growth ligand of the GPCR, which is considered a “balanced” agonist medium containing 1 μg/mL doxycycline hydrochloride to because it activates both pathways to a quantifiably defined induce receptor overexpression. Approximately 43 h following extent.7 Ligands that bind a GPCR and drive signaling efficacies induction, the medium was replaced with a starvation medium more toward G-protein-dependent pathways are termed G- (DMEM, +4.5 g/L D-glucose, +L-glutamine, and sodium biased ligands, whereas those ligands that preferentially promote pyruvate supplemented with 0.1% bovine serum albumin β-arrestin-dependent pathways are defined as β-arrestin (BSA), 10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesul- biased.7,8 Importantly, this concept of biased agonism, the fonic acid, and 10 μg/mL gentamicin) and incubated for at balance of signaling between these two distinct but overlapping least 4 h. During the course of the experiment, biotin tyramide pathways, is hypothesized to account, in part, for the difference was added to a final concentration of 500 μM, ligands were in cellular responses in response to different ligands.9 added to a final concentration of 10 μM except for angiotensin The angiotensin II type 1 receptor (AT1R) is a GPCR that II, which was added to a final concentration of 1 μM, and plays a key role in the renin-angiotensin system that regulates hydrogen peroxide was added to a final concentration of 1 mM blood pressure, cardiac contractility, vasoconstriction, and [having been freshly diluted to 1 M in Dulbecco’s phosphate- cardiac hypertrophy in response to the peptide hormone buffered saline (DPBS) from 30%]. Biotin incubation was 10 angiotensin II (Ang II). For this reason, AT1Rs have been exactly 1 h, hydrogen peroxide incubation was exactly 1 min, and targeted by antagonists for the treatment of heart failure, ligand incubation was either 90 s, 10 min, or 1 h. Following 11 diabetic kidney disease, and hypertension. However, in recent peroxide treatment, the medium was quickly decanted and the years, there has been evidence to suggest that targeting AT1R cells were washed three times with quench buffer (DPBS with a β-arrestin-biased ligand rather than a neutral antagonist containing 10 mM sodium ascorbate, 10 mM sodium azide, and 12,13 may provide additional benefit. This has led to the 5 mM trolox). Cells were then harvested in quench buffer with 5 development of a diverse set of G-protein- and β-arrestin-biased mM EDTA with scraping and collected by centrifugation. The AT1R ligands that have been characterized with respect to supernatant was discarded and the cell pellet was flash-frozen in transducer coupling, cellular signaling, and, more recently, liquid nitrogen then stored at −80 °C. Four independent 7,14,15 receptor conformation. A detailed mapping of direct AT1R experiments were performed for each ligand incubation time. interactors and nearby proteins following biased ligand The raw data include separate experiments that used osmotic stimulation would provide greater insights into the diversity of stretch to stimulate cells. For technical reasons due to poor cell cellular pathways being activated in response to biased agonism. viability and low protein recovery, these experiments were not To obtain a detailed intracellular signaling map downstream included in the final data analysis. of the AT1R, one approach is to use peroxidase-catalyzed Streptavidin Enrichment and Digestion proximity labeling with an engineered soybean ascorbate peroxidase (APEX2).16 Expressing AT1R fused to APEX2 in All buffers were prepared in Milli-Q filtered water the day of use mammalian cells allows labeling of proteins in close proximity and filtered through 0.22 μm filters. Cell pellets were thawed and and provides a snapshot in time of the local protein environment resuspended in alkaline lysis buffer (2 M NaOH, 7.5% 2- in a manner similar to the widely used BioID technique.17 An mercaptoethanol) and incubated on ice for 20 min. TCA was advantage of APEX2 over traditional protein-labeling techni- added to reach 25% and the samples were vortexed thoroughly ques is the rapid labeling kinetics of the enzyme that allow before being incubated on ice for 1.5 h. Protein pellets were labeling on a time scale similar to that of GPCR activation collected by centrifugation at 4 °C and 16,100g for 20 min. resulting in high temporal resolution.18,19 In this study, we Pellets were washed by adding ice cold acetone, vortexing, systematically investigated the profiles of proximal proteins to collected by centrifugation for 5 min at the speed above, and the AT1R upon stimulation with a comprehensive panel of ligands: supernatant was discarded. This washing procedure was the full agonist Ang II, the partial agonist S1I8, the G-protein- repeated a total of four times and the pellet was air-dried for biased agonists TRV055 and TRV056, and the β-arrestin-biased approximately 5 min. Resuspension buffer [8 M urea, 100 mM agonists TRV026 and TRV027. We performed time courses of NH4HCO3, 100 mM sodium phosphate pH 8, 1% sodium 90 s, 10 min, and 60 min for each ligand to precisely determine dodecyl sulfate (SDS), and 10 mM TCEP] was added to each the kinetics of receptor trafficking and signaling activation and sample and samples were sonicated for four 30 s cycles in a bath demonstrate both distinct and overlapping signaling patterns sonicator then incubated at 37 °C with vortexing for 1 h. between the AT1R ligands. Samples were then sonicated again as before and incubated for another 1 h. Samples were centrifuged at 16,100g for 10 min and ■ EXPERIMENTAL PROCEDURES transferred to new tubes. Freshly prepared iodoacetamide in 50 fi Cell Culture mM NH4HCO3 was added to a nal concentration of 20 mM and samples were incubated for 30 min in the dark with Flp-In T-REx 293 cells stably expressing AT1R-APEX2 were a vortexing. Alkylation was quenched by adding freshly prepared generous gift of Dr. Andrew C. Kruse (Harvard). Cells were fi ° dithiothreitol in 50 mM NH4HCO3 to a nal concentration of cultured at 37 C and 5% CO2 in 15 cm culture plates with the ’ fi ’ 50 mM and vortexing well. Each sample was then diluted by growth medium [Dulbecco smodied Eagle smedium doubling the sample volume using Milli-Q water. An aliquot of (DMEM), +4.5 g/L D-glucose, +L-glutamine, and sodium each sample was saved for control western blotting. Magnetic pyruvate supplemented with 10% fetal bovine serum, 10 μg/ streptavidin beads were washed three times in wash buffer (4 M mL gentamicin, 10 μg/mL blasticidin, and 100 μg/mL urea, 100 mM sodium phosphate buffer pH 8, and 0.5% SDS) hygromycin B]. and before being added to each sample. Samples were then Proximity Labeling Experiments rocked at 4 °C for approximately 18 h. Protein-loaded beads All buffers were prepared in Milli-Q filtered water the day of use were then washed three times with wash buffer using a magnetic and filtered through 0.22 μm filters. When cells reached 40−50% rack to collect the beads and discard the supernatant. Samples

B https://doi.org/10.1021/acs.jproteome.1c00080 J. Proteome Res. XXXX, XXX, XXX−XXX Journal of Proteome Research pubs.acs.org/jpr Article were then washed once with 200 mM EPPS pH 8.5 and then Consortium via the PRIDE24 partner repository with the dataset transferred to new tubes before being washed twice more with identifier PXD023814. EPPS. Beads were resuspended in 200 mM EPPS pH 8.5 with Mass Spectrometry Data Analysis 8% high-performance liquid chromatography-grade MeCN and ° μ Mass spectra were processed using a SEQUEST-based pipe- digested at 37 C with vortexing for 3 h with 2 g of LysC. An 20 equal volume of 200 mM EPPS pH 8.5 was added and the line. Spectra were converted to mzXML using MSConvert. samples were digested further overnight with 2 μg of trypsin. Database searching included all entries from the human UniProt The next day, a magnetic rack was used to transfer the database (April 20, 2016). This database was concatenated with supernatant to new tubes and then samples were flash-frozen one composed of all protein sequences in the reversed order. using liquid nitrogen and stored at −80 °C. Searches were performed using a 50 ppm precursor ion tolerance for total protein level profiling and fully tryptic Tandem Mass Tag Labeling specificity was required. The product ion tolerance was set to 0.9 Samples were processed using the streamlined-tandem mass tag Da. These wide mass tolerance windows were chosen to (SL-TMT) protocol.20 In brief, a final acetonitrile concentration maximize sensitivity in conjunction with Sequest searches and of ∼30% (v/v) was added along with 10 μL of TMT reagent (20 linear discriminant analysis.25,26 TMT tags on lysine residues ng/μL) to 100 μg of peptides. Following incubation at room and peptide N termini (+229.163 Da) and carbamidomethyla- temperature for 1.5 h, the reaction was quenched with tion of cysteine residues (+57.021 Da) were set as static hydroxylamine to a final concentration of 0.3% (v/v) for 15 modifications, while oxidation of methionine residues (+15.995 min. The TMT-labeled samples were pooled at a 1:1 ratio across Da) was set as a variable modification. Peptide-spectrum all samples. The combined sample was vacuum centrifuged to matches (PSMs) were adjusted to a 1% false discovery rate near dryness and subjected to C18 solid-phase extraction via (FDR).27,28 PSM filtering was performed using a linear Sep-Pak (Waters, Milford, MA). discriminant analysis, as described previously,26 while consid- Δ Off-Line Basic pH Reversed-Phase Fractionation ering the following parameters: XCorr, Cn, missed cleavages, peptide length, charge state, and precursor mass accuracy. For We fractionated the pooled TMT-labeled peptide sample using TMT-based reporter ion quantitation, we extracted the summed the Pierce high pH reversed-phase peptide fractionation kit (cat. signal-to-noise (S/N) ratio for each TMT channel and found the # 84868). 12 fractions were collected using: 7.5, 10, 12.5, 15, closest matching centroid to the expected mass of the TMT 17.5, 20, 22.5, 25, 27.5, 30, 35, and 60% acetonitrile and reporter ion. PSMs were identified, quantified, and collapsed to a concatenated into six, by pooling every sixth fraction. Samples 1% peptide FDR and then collapsed further to a final protein- fi were subsequently acidi ed with 1% formic acid and vacuum level FDR of 1%. Moreover, protein assembly was guided by centrifuged to near dryness. Each fraction was desalted via principles of parsimony to produce the smallest set of proteins StageTip, dried again via vacuum centrifugation, and recon- necessary to account for all observed peptides. Proteins were stituted in 5% acetonitrile and 5% formic acid for liquid quantified by summing reporter ion counts across all matching − chromatography (LC) tandem mass spectrometry processing. PSMs, as described previously.26 PSMs with no MS3 spectra Liquid Chromatography and Tandem Mass Spectrometry were excluded from quantification.29 Protein identification and quantitation values were exported for further analysis in Mass spectrometry data were collected using an Orbitrap fusion Microsoft Excel. mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to a Proxeon EASY-nLC 1200 LC pump (Thermo Post-mass Spectrometry Data Analysis fi Fisher Scienti c, San Jose, CA). Peptides were separated on a Using Excel, columns not needed for further analysis were μ ∼ 100 m inner diameter microcapillary column packed with 40 removed, data were separated into individual experiments, and μ cm of Accucore150 resin (2.6 m, 150 Å, Thermo Fisher Excel-based gene symbol errors30 were corrected before being fi ∼ μ Scienti c, San Jose, CA). For each analysis, we loaded 2 g exported as .csv files. Custom python scripting was then used for onto the column and separation was achieved using a 2.5 h the analysis outlined below. These scripts removed protein fl gradient of 7 to 27% acetonitrile in 0.125% formic acid at a ow contaminants and false positives from FDR filtering as well as rate of ∼550 nL/min. Each analysis used an SPS-MS3-based 21,22 any protein with a TMT reporter sum across all channels less TMT method, which has been shown to reduce ion than 100. Samples within each experimental round were then fi 23 interference compared to MS2-based quanti cation. The scan median normalized before being compared to each round sequence began with an MS1 spectrum (Orbitrap; resolution, unstimulated sample. Only proteins reported in all four − 120,000; mass range, 400 1400 m/z; automatic gain control experiments underwent advanced analysis. Individual proteins × 5 (AGC) target, 5 10 ; and maximum injection time, 100 ms). were classified using the PANTHER database31 (searched Precursors for MS2/MS3 analysis were selected using a Top10 March 26, 2020). Pathway enrichment analysis was performed method. MS2 analysis consisted of collision-induced dissocia- by submitting full protein lists with log 2-fold change values to × 4 tion (quadrupole ion trap; AGC 2 10 ; normalized collision String database version 11.032 (searched April 10, 2020). energy (NCE), 35; and maximum injection time, 150 ms). Volcano plots were made by first making 1v1 comparisons Following acquisition of each MS2 spectrum, we collected an between ligands in log 2 space and p-values were calculated with MS3 spectrum using our recently described method in which an independent two-tailed T-test. multiple MS2 fragment ions were captured in the MS3 precursor population using isolation waveforms with multiple frequency Western Blotting notches.21 MS3 precursors were fragmented by high-energy Aliquots saved from the experiment were quantified using the collision-induced dissociation and analyzed using the Orbitrap Bio-Rad protein assay reagent, and an equal quantity of protein (NCE 65; AGC 1 × 105; maximum injection time, 150 ms; and was loaded onto 10% SDS-polyacrylamide gel electrophoresis resolution was 50,000 at 200 Th). The mass spectrometry gel and separated by electrophoresis. Proteins were transferred proteomics data have been deposited to the ProteomeXchange to a poly(vinylidene difluoride membrane (Bio-Rad) and

C https://doi.org/10.1021/acs.jproteome.1c00080 J. Proteome Res. XXXX, XXX, XXX−XXX Journal of Proteome Research pubs.acs.org/jpr Article

Figure 1. (A) GPCR−APEX technique and workﬂow. Cells expressing GPCR−APEX fusion protein are incubated with biotin tyramide then peroxide is added to promote biotinylation of nearby proteins. After labeling is quenched, cells are harvested and biotinylated proteins are enriched by aﬃnity chromatography before enzymatic digestion and mass spectrometric analysis. (B) Experimental setup used for the AT1R ligand panel. No peroxide no ligand and no hydrogen peroxide were added; no stimulationno ligand was added but hydrogen peroxide was added; losartana small molecule clinically used as an angiotensin receptor blocker; Ang IIangiotensin II peptide; S1I8sarcosine1, isoleucine8-AngII; TRV055synthetic peptide TRV120055; TRV056synthetic peptide TRV120056; TRV026synthetic peptide TRV120026; and TRV027synthetic peptide TRV120027; and (C) timeline of the experiment. Biotin tyramide and hydrogen peroxide (except for the “no peroxide” sample) incubation was held constant at 60 and 1 min, respectively. Ligands were added at 60 min, 10 min, or 90 s before quenching.

Figure 2. Overlap of proteins detected in each replicate for each time point. Proteins were required to have at least two unique peptides and a minimum TMT reporter ion-summed signal-to-noise ratio of 100 for identification. The number of proteins seen in all four replicates at each time point is highlighted in bold and indicates good reproducibility between independent replicates. confirmed with Ponceau S staining (Sigma) following ■ RESULTS manufacturer’s instructions. Blots were blocked with 5% dry milk in TBST (0.1% Tween-20 in Tris-buffered saline) AT1R-APEX2 Proximity Labeling Reveals Time-Dependent Clustering of Ligands Based on G Protein or β-Arrestin Bias overnight at 4 °C, washed extensively with TBST, then incubated with 1:5000 streptavidin-HRP (Thermo Fisher To test the hypothesis that biased AT1R ligands activate distinct Scientific) in 5% BSA in TBST for 1 h at room temperature. signaling patterns, we utilized APEX218,19 to directly compare Immunoblots were washed again with TBST then detected AT1R signaling profiles over time following stimulation by a using enhanced chemiluminescence reagents (Thermo Fisher panel of diverse AT1R ligands. We chose ligands with a wide Scientific) and imaged using a Syngene G:BOX Chemi system. array of activities that include a balanced agonist (Ang II), a

D https://doi.org/10.1021/acs.jproteome.1c00080 J. Proteome Res. XXXX, XXX, XXX−XXX Journal of Proteome Research pubs.acs.org/jpr Article partial agonist (S1I8), G-protein-biased agonists (TRV055, trafficking of the receptor, we speculated that differences TRV056), β-arrestin-biased agonists (TRV026, TRV027), and between agonists at 90 s come from differences in the rate of an antagonist (Losartan). To determine the cellular proteins in internalization, whereas events at 60 min would be due to close proximity to AT1R following receptor activation, we stably cellular localization. 16,18 overexpressed AT1R fused to APEX2 in HEK293 cells Rate of Receptor Internalization Is Slower for preincubated with biotin tyramide prior to the addition of β-Arrestin-Biased AT1R Agonists hydrogen peroxide to promote robust biotinylation of nearby proteins (Figure S1). Labeled proteins were enriched by affinity Statistical analysis was used to determine which proteins are chromatography, digested to peptides, and quantified by liquid confidently undergoing changes in labeling due to the presence chromatography−mass spectrometry (Figure 1A). of each ligand. We considered proteins with a log 2-fold change Ligand stimulation occurred at saturating concentrations (1 in proximity labeling greater than 1 or less than −1 and a p-value μM for Ang II and 10 μM for all other ligands) at three different <0.05 as meaningfully changing in response to ligand and plot time points (90 s, 10 min, and 60 min) and each experiment was these data meeting this threshold as increasing (red) or performed four independent times (Figure 1B,C). Two or more decreasing (blue) at each time point in volcano plots (Figure peptides were required to confidently identify each protein and a 4). As expected, the antagonist losartan did not produce TMT reporter sum greater than 100 was needed for meaningful differences relative to the unstimulated sample at fi quanti cation to ensure a good signal-to-noise ratio. This any stimulation time. The remaining ligands produced robust fi ∼ fi fi noise ltering resulted in 2500 proteins con dently quanti ed changes in protein labeling that continued to increase at later per experiment (Figure 2). The majority of these proteins did ff fi stimulation times. The most noticeable observation between not show di erences in labeling pro les in the presence of volcano plots in Figure 4 is the fewer amount of red and blue ligands (Figure S2) allowing us to median normalize our data for data points for TRV026 and TRV027 at the earliest time point each round to the entire dataset of unchanged proteins. Pearson indicating that these β-arrestin-biased ligands promoted fewer correlation coefficients between independent replicates of the same ligand ranged from 0.75 to 0.92 with a median value of 0.85 changes in the proximal environment of AT1R at this early time. (Figure S3) and approximately 75% of the proteins reported at Many of the proteins that undergo a meaningful increase in each time point were detected in all four biological replicates labeling at 90 s after treatment are associated with receptor further indicating high reproducibility (Figure 2). To maintain internalization. To examine this more closely, PCA was the highest data rigor, all subsequent analyses used only proteins performed while limiting the analysis to proteins associated ffi fi that were identified in all four experiments at any of the time with receptor internalization and tra cking as de ned by the 31 points. PANTHER database. This analysis shows primary grouping Principle component analysis (PCA) of our dataset shows by time of agonist treatment, but also secondary agonist grouping of ligands relative to time of treatment mostly in the clustering is observed at 90 s that separates the β-arrestin-biased PC1 axis, which accounts for 76.7% of data variance (Figure 3). and G-protein-biased ligands (Figure 5A). Consistent with the hypothesis that differences in the rate of internalization occur, the individual proteins that show the largest increase in the level of proximity labeling at 90 s are found following Ang II, S1I8, TRV055, or TRV056 treatment and are associated with internalization and trafficking (Figure 5B). The proteins that undergo the largest increases in proximity labeling are PICALM, ITSN2, EPS15L1, CLTC, and CLTA (Figure 5B), which are all involved in clathrin-mediated AT1R internalization.33 At 90 s, substantially less labeling of proteins involved in endocytosis is found in the samples treated with the β-arrestin-biased ligands, TRV026 and TRV027. However, the level of proximity labeling of these trafficking proteins is similar across all agonists at 10 min suggesting that the difference in internalization rates between Figure 3. PCA of the full dataset. The shape of data points indicates the ligands early after stimulation was lost at later time points time point and the color indicates each ligand. The percent of variance explained by each component is included in the axis label. Data points (Figure 5B). of agonists cluster first by time along the PC1 axis (highlighted in Interestingly, the samples treated with Ang II, TRV055, and shaded circles) then form two secondary clusters containing Ang, TRV056 show higher levels of proximity labeling of proteins TRV055, and TRV056 in one and then S1I8, TRV026, and TRV027 in associated with late endosomes (STX7, STX12, PTPN23, and the other. This subclustering is the most apparent at 90 s and 60 min VPS35) at 60 min than samples treated with TRV026 and and occurs in the PC2 and PC3 directions. Losartan samples cluster TRV027. In contrast, at 60 min treatment with the partial together regardless of when the ligand was added. agonist S1I8 induced a labeling profile more similar to that of the β-arrestin-biased ligands, whereas at 90 s, S1I8 more closely Secondary clustering is observed at each time point between matched the responses to Ang II, TRV055, and TRV056. This Ang II- and G-protein-biased agonists (TRV055 and TRV056) divergent behavior for S1I8 is also observed in the PCA compared to β-arrestin-biased agonists (TRV026 and TRV027) clustering (Figure 5A), where at 90 s S1I8 promoted robust and the partial agonist S1I8, especially apparent at 90 s and 60 AT1R endocytosis similar to Ang II, TRV055, and TRV056, min. This implies that the major differences between agonists while at 60 min S1I8 directed AT1Rs to shuttle to late- occur at the earliest and latest time points of the experiment. endosomesa finding more similar to that of the β-arrestin- Because early events after AT1R stimulation often involve biased ligands, TRV026 and TRV027.

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Figure 4. Volcano plots of each ligand-treated sample compared to the unstimulated sample. Raw p-values were calculated using an independent two- tailed T-test. The fold changes on the x-axis are the mean of the four replicates. Proteins with a log 2-fold change greater than 1 or less than −1 and a p- value less than 0.05 are colored red and blue, respectively. A less robust change in proximity labeling is seen for TRV026 and TRV027 at 90 s compared to the other ligands as indicated by the fewer number of red and blue data points. A similar number of large changes are observed for all agonists at 10 and 60 min. Losartan shows no meaningful changes at any time point.

Figure 5. PCA of proteins within selected functional classes and heatmaps that highlight the proteins that underwent the largest changes in proximity labeling after ligand treatment (a log 2-fold change greater than 2 or less than −2). (A,B) PCA and heatmap of proteins involved in receptor internalization/transportation. Primary clustering by time of ligand stimulation is seen for all agonists. At 90 s stimulation, the β-arrestin-biased agonists cluster more closely with losartan-treated samples, suggesting that the internalization of AT1R activated by β-arrestin-biased ligands occurs at a slower initial rate. (C,D) Proteins classified as G proteins or involved in G protein signaling. In addition to the primary clustering by treatment time, there is notable secondary clustering at 90 s and 60 min, where the β-arrestin-biased agonists and S1I8 form one cluster and the G-protein-biased agonists and AngII form the other cluster. (E,F) Proteins classified as scaffolds or adaptors. PCA shows primary grouping by the treatment time, as well as notable secondary agonist clustering at all time points. Interestingly, S1I8 appears to cluster more closely to the G-protein-biased agonists at 90 s but then cluster with the β-arrestin-biased agonists at later time points. Note the proximity-labeling pattern of ARRB1 and ARRB2 shown in the heatmap is consistent with the secondary clustering of the PCA, specifically with regard to the level of proximity labeling induced by S1I8. (G,H) Proteins classified as kinases or signaling molecules. Primary clustering by time of ligand stimulation is seen for all agonists. There is notable secondary clustering at 90 s and 60 min, where the β-arrestin-biased agonists and S1I8 form one cluster and the G-protein-biased agonists and AngII form the other cluster. The proximity labeling of PRKCA at 90 s shown in the heatmap mirrors the clustering seen for the PCA analysis and suggests that S1I8 and the β- arrestin-biased ligands do not activate PKC-mediated signaling.

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Figure 6. (A) Volcano plots that compare ligands of the same class. Proteins with a log 2-fold change greater than 1 or less than −1 and a p-value less than 0.05 are colored red and blue, respectively. These comparisons show no substantial differences between ligands of the same class with an exception between the β-arrestin-biased ligands at 10 min. The proteins that are differentially labeled between TRV026 and TRV027 at 10 min suggest a difference in mRNA catabolism and metabolism. (B) Volcano plots of each β-arrestin-biased treated sample compared to G-protein-biased ligands. Proteins with a log 2-fold change greater than 1 or less than −1 and a p-value less than 0.05 are colored red and blue, respectively. These comparisons show that there are substantial differences between ligands of different classes at each time point.

GTPase and Adaptor Proteins are Labeled to Different differences in receptor localization at 60 min.35 Together this Levels by Biased Agonists suggests that S1I8 induces receptor internalization at a rate β To investigate the processes that drive receptor localization similar to Ang II, TRV055, and TRV056 but promotes -arrestin following activation by biased agonists, we performed an analysis interaction at a duration similar to TRV026 and TRV027. This fi 31 might be explained by examining the levels of C-terminal of proteins classi ed by the PANTHER database as being ff associated with GTPase signaling or having scaffold/adaptor phosphorylation of AT1R induced by each ligand and the e ect that the amount of phosphorylation has on the duration of roles. Proteins involved in GTPase signaling show primary β 36 clustering based on the stimulation time and secondary AT1R/ -arrestin interaction. Also of note, Ang II and the G- clustering at 90 s and 60 min (Figure 5C). AT1R is regarded biased agonists showed a slight increase in labeling of the α scaffold/adaptor proteins ABI1 and ABI2 at 90 s that was not as primarily a G q-coupled receptor that has also been shown to α 10,34 seen for S1I8, TRV026, and TRV027 followed by a distinct require recruitment of G i for some signaling pathways. Interestingly, the addition of all agonists resulted in a similar decrease for all agonists at 10 and 60 min (Figure 5F). ABI1 and reduction over time in labeling of all heterotrimeric G proteins, ABI2 are linked to actin cytoskeleton dynamics and are including Gα ,Gα , and Gα (Figure 5D), indicating that prior regulators of nonreceptor tyrosine kinases ABL1 and ABL2 q s i involved in processes of cell growth and differentiation. to ligand stimulation all Gα subunits are all held in close proximity to the AT1R at the cellular membrane regardless of Kinases and Signaling Proteins Are Labeled to Different specificity for coupling or activation. Levels by Biased Agonists PCA of proteins defined as scaffolds/adaptors shows clear To examine the link between AT1R and various signaling secondary clustering of β-arrestin-biased agonists compared to pathways, we performed PCA analysis of proteins classified as G-protein-biased agonists/Ang II at each time point indicating nucleotide kinases, amino acid kinases, carbohydrate kinases, large class differences (Figure 5E). Interestingly, in this analysis tyrosine protein kinases, kinases, nonreceptor serine/threonine of scaffold/adaptor proteins, S1I8 clusters more closely with protein kinases, kinase activators, kinase inhibitors, kinase Ang II, TRV055, and TRV056 at 90 s but then clusters with β- modulators, transmembrane signal receptors, intercellular signal arrestin-biased agonists at 10 and 60 min following a similar molecules, and membrane-bound signaling molecules by the pattern observed for internalization. Proximity labeling of the PANTHER database.31 While primary clustering is based on the scaffold/adaptor proteins ARRB1 and ARRB2 (Figure 5F) stimulation time, this category showed clear secondary underwent a marked increase after agonist treatment, with the clustering at 90 s and 60 min (Figure 5G). The largest increase highest and most sustained labeling observed for Ang II, in labeling for this category was seen at 90 s for a protein kinase, TRV055, and TRV056. Proximity labeling of ARRB1 and BMP2K, that plays a role in clathrin-mediated internalization,33 ARRB2 following treatment with S1I8 is more similar to Ang II, especially for Ang II, TRV055, TRV056, and S1I8 (Figure 5H). TRV055, and TRV056 at 90 s and more similar to TRV026 and Interestingly, labeling of PKC and PKC-like kinases PRKCA, TRV027 at 10 and 60 min. This is consistent with the concept PRKCB, and PKN2 occurred primarily by Ang and G-biased that the duration of receptor interaction with β-arrestin plays a agonists (Figure 5H) consistent with PKC activation down- ffi α 37 role in tra cking and, therefore, may be the primary driver of the stream of G q signaling. A unique feature was the large

G https://doi.org/10.1021/acs.jproteome.1c00080 J. Proteome Res. XXXX, XXX, XXX−XXX Journal of Proteome Research pubs.acs.org/jpr Article decrease in labeling of kinases MARK1, MARK2, and MARK3 In addition, β-arrestin-biased ligands resulted in higher levels of observed at 90 s for Ang II, TRV055, and TRV056, and, to a proximity labeling of PODXL, found in apical cell membrane lesser extent, S1I8 (Figure 5H). MARK family kinases are regions, and PCM1, a centrosome protein found at the nuclear associated with PKD signaling downstream of PKC and surface of myocytes, indicating potential AT1R localization at collectively this signaling is associated with regulation of gene these compartments following β-arrestin-biased agonist treat- expression and, in the context of AT1R, has been shown to ment (Figure 7C). promote pathological heart remodeling.38 Taken together, our data suggest that G-protein-biased Biased AT1R Activation Results in Differential Trafficking ligands may shuttle a larger population of AT1R to endosome, and Degradation lysosome, and clathrin-coated vesicles, whereas β-arrestin- biased ligands likely direct the receptor to the cytoskeleton, We next applied binary comparisons of labeled proteins over ruffle, and lamellipodium. These distinct patterns of receptor time between ligands of the same functional class: TRV026 trafficking may lead to the different level of AT1R ubiquitination versus TRV027 and TRV055 versus TRV056. No meaningful and degradation. differences were identified within each ligand class (Figure 6A). β When comparing the labeled proteins between -arrestin-biased ■ DISCUSSION and G-protein-biased ligands, we noted across all time points, 60 fi In this study, we used mass spectrometry-based proteomics to proteins were labeled signi cantly higher in the G-protein- fi biased agonist samples, while 16 proteins were higher in the β- quantitatively pro le proteins in close proximity to AT1R arrestin-biased stimulation (Figure 6B, Table 1). following stimulation by a panel of diverse ligands: the balanced agonist Ang II, the partial agonist S1I8, the G-protein-biased β Table 1. Proteins Distinctly Labeled by Biased Agonists agonists TRV055 and TRV056, and the -arrestin-biased fi agonists TRV026 and TRV027. The rapid labeling kinetics of Identi ed in the Binary Comparison between G-Protein- fi Biased and β-Arrestin-Biased Ligands in Figure 6B the engineered peroxidase APEX2 facilitates the quanti cation of AT1R-proximal proteins with high spatial and temporal resolution. This allows the precise characterization of the kinetics and destinations of AT1R trafficking by quantifying the proteins proximal to the receptor at different time points after ligand stimulation. In addition, the ligand-induced proximity of AT1R with other endogenous proteins infers the potential formation of signaling complexes, providing novel perspectives for studying AT1R signaling regulation and cellular function. Ligand-activated AT1Rs undergo rapid desensitization by a process that involves receptor internalization, thereby limiting potential detrimental effects of prolonged receptor activation.10 Gene ontology (GO) analysis of these proteins revealed their Internalized receptors are then targeted to different organelles association with distinct subsets of cellular components (Figure such as mitochondria and the nucleus to regulate cell physiology, 7A,B). Labeled proteins in the G-protein-biased agonist group recycled back to the plasma membrane for future activation, or are associated with 20 cellular component terms (Figure 7A), transported to lysosomes for degradation. The process of AT1R with the majority of them located in the cytoplasm. Endosome, internalization is regulated by GRK-mediated phosphorylation lysosome, and clathrin coat machinery are also the major target on the carboxyl-terminal tail of the receptor followed by the areas of labeled proteins in the G-protein-biased group. In recruitment of β-arrestin. Previous studies used fluorescence- or contrast, the latter two are not identified in the β-arrestin-biased bioluminescence resonance energy transfer-based assays to group (Figure 7B). A more detailed analysis of the labeled determine the localization of AT1Rs or its association with proteins reveals a number of interesting differences between the trafficking proteins, such as members of the Rab protein two classes of biased agonists. For example, the proximity family.43,44 Here, we provide a systematic analysis of the kinetics labeling of HGS is considerably greater following receptor and fate of AT1R trafficking induced by distinct ligands with stimulation with Ang II, TRV055, or TRV056 compared to the diverse signaling properties. Our data suggest that AT1R β-arrestin-biased ligands (Figure 7C). STAM and STAM2 internalization induced by the β-arrestin-biased agonists follow a similar labeling pattern to HGS (Figure 7C). TRV026 and TRV027 is slower than that induced by the Collectively, these three proteins are associated with the balanced agonist Ang II, the partial agonist S1I8, or the G- ESCRT-0 complex that recognizes ubiquitinated proteins and protein-biased agonists TRV055 and TRV056, despite high generates multivesicular bodies.39,40 These data suggest that G- affinity for the AT1R. Moreover, Ang II- and G-biased agonist- protein-biased agonists may result in differences in ubiquitina- stimulated AT1Rs show a higher level of proximity to late tion levels and subsequent degradation of AT1R. endosomes after a prolonged stimulation of 60 min, as well as a Ten of the 16 proteins in the β-arrestin-biased group are higher labeling level of ARF1 (ADP-ribosylation factor 1), which cytosolic proteins (Figure 7B). Six are involved in the is involved in recycling GPCR from the Golgi complex to the cytoskeleton, consistent with previous evidence of a role for plasma membrane,45 supporting the notion that distinct ligands AT1Rs in F-actin cytoskeletal remodeling.41 Notably, PAC- target the receptor to different cellular compartments for SIN2, PODXL, MYO10, and SRGAP2 identified in the β- downstream signaling activation or receptor recycling/degrada- arrestin-biased group are known to be involved in ruffle and tion. lamellipodium. While a previous study has demonstrated that It is well established that AT1R can engage distinct signaling the AT1R can regulate membrane ruffling and cell migration transducers including multiple G protein subtypes or β-arrestins through ARF6 and Rac1,42 we show here that the AT1R- to activate pleiotropic signaling pathways.8,10 The underlying activated through β-arrestin signaling is involved in this pathway. mechanism for the selectivity of transducer coupling appears to

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Figure 7. (A,B) GO analysis identifying cellular components associated with proteins differentially labeled by biased agonists, as listed in Table 1. (C) Fold difference of proximity labeling compared to the unstimulated sample over time for selected proteins. involve a ligand-induced conformational change of the receptor−proximal protein profiles suggests that the two G- receptor.15,46 The full agonist Ang II induces an outward biased agonists resemble each other with regard to the structural movement of AT1R transmembrane domain 6 (TM6), an and functional changes they induce. Taken together, our data inward movement of TM7, and a conformational rearrangement support the concept that the ligand-induced receptor function- of residue N111 on TM3.15,46 These conformational changes ality is highly correlated with their specific conformational together disrupt the receptor core and induce an active “open” features. conformation allowing for G protein and β-arrestin coupling.15 Our study also provides the possibility to explore new In contrast, while the partial agonist S1I8 and the β-arrestin- directions in studying AT1R signaling and physiological biased agonists also induce shifts in TM6 and TM7, the functions. For instance, previous studies suggest that AT1R signature conformational change for GPCR activation, they lack blockers suppress cell proliferation in various cancers such as effect on N111, therefore leave the receptor core to a similar breast cancer,47 endometrial cancer,48 and esophageal cancer,49 conformation of the antagonist-bound inactive receptor.46 The but their mechanism of action remains to be elucidated. Our similar effects of S1I8 and β-arrestin-biased agonists on receptor study identified the proximity of β-arrestin-biased agonist- conformation are consistent with the protein-labeling profiles induced AT1R to centrosome proteins such as PCM1, we observed in our study showing similar proximal protein suggesting the potential involvement of AT1R in cell cycle profiles at 60 min between S1I8, TRV026, and TRV027. regulation. MYO10, an actin-based motor protein associated In addition to the structural difference of the receptor bound with filopodia, was also highly labeled in the β-arrestin-biased to ligands from different functional classes, ligands within one ligand samples. Both AT1R and MYO10 are targeted by the class, like TRV026 and TRV027, can also induce the receptor to microRNA miR-155, which is upregulated in pancreatic distinct conformational heterogeneity.15 This leads to the cancer.50 In addition, we also identified that β-arrestin-biased question of whether the two β-arrestin-biased agonists also ligands resulted in higher levels of proximity labeling of the LPS- have distinct functional consequences. In this study, our data do responsive beige-like anchor protein (LRBA). A previous study not demonstrate differences in the proximal protein profiles of suggested that both β-arrestin and LRBA mediate the S1P1 TRV026- and TRV027-induced AT1R. Although a number of receptor signaling that regulates human B cell circulation, which proteins, such as VDAC1 and HSPD1, showed higher levels of is involved in chronic lymphocytic leukemia.51 Future studies labeling after TRV027 stimulation compared to TRV026, the examining the interaction of AT1Rs with cell cycle proteins and overall profile was quite similar for these two ligands of the same their regulation of cell proliferation and cancer-related proteins class. On the other hand, the high similarity of the effects of may shed light whether AT1Rs are a potential target for cancer TRV055 and TRV056 on the receptor structure as well as prevention or treatment.

I https://doi.org/10.1021/acs.jproteome.1c00080 J. Proteome Res. XXXX, XXX, XXX−XXX Journal of Proteome Research pubs.acs.org/jpr Article GPCR−APEX2 proximity labeling offers the ability to enrich ■ ACKNOWLEDGMENTS and identify nearby proteins to compare the effects of differential We thank Dr. Andrew C. Kruse (Harvard University, USA) for ligand stimulation; however, the exogenous overexpression of providing the AT1R-APEX2 stable cell line. We thank Dr. Paul the receptor in HEK293 cells and treatment with saturating A. Grimsrud, Dr. Laura M. Wingler, and Dr. Dean P. Staus doses of ligand may imply protein interactions that are not seen (Duke University, USA) for technical consultations and helpful in a more endogenous setting. To more precisely determine the role of AT1Rs in a specific signaling pathway, such as those discussions. This work was supported by National Institutes of discussed above, further experimentation is needed to validate Health grants HL056687 and HL075443 to H.A.R., GM132129 the protein−protein interactions suggested by these data. to J.A.P., GM67945 to S.P.G., and an Institutional NIH training Finally, while this study does not identify the specific grant T32HL007101 to C.T.P. biotinylation sites of the AT1R-proximal proteins due to the sensitivity and resolution of experimental methods, the use of ■ REFERENCES similar approaches has the potential to add conformational (1) Sriram, K.; Insel, P. A. G Protein-Coupled Receptors as Targets for information of nearby proteins by analyzing the changes in Approved Drugs: How Many Targets and How Many Drugs? Mol. 52 accessibility of specific biotinylation sites. Pharmacol. 2018, 93, 251−258. In conclusion, our study profiled proteins proximal to the (2) Hauser, A. S.; Attwood, M. M.; Rask-Andersen, M.; Schiöth, H. 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