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* Cite This: https://doi.org/10.1021/acs.jproteome.1c00080 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information 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 trans- membrane 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.