Cell culture, antibodies, and reagents<\/p>\n
HEK-293 cells (ATCC), including transient and stable lines, as well as CRISPR\u2013Cas9 \u03b2ARR1\/2-knockout and parental lines61<\/a>, were maintained in Eagle\u2019s Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37\u2009\u00b0C and 5% CO2. U2OS cells (DiscoveRx PathHunter) were cultured in MEM containing 2\u2009mM l-glutamine, 10% FBS, and 1% penicillin-streptomycin under similar conditions. U2OS-based \u03b2-arrestin recruitment and internalization assays were performed per the manufacturer\u2019s protocol (DiscoveRx). For chemokine-induced migration assays, leukocytes were isolated from wild-type mice as described previously18<\/a>,37<\/a>,61<\/a>. Escherichia coli strains DH5\u03b1 and BL21 (DE3) (New England Biolabs) were cultured in LB or Terrific Broth (Fisher Scientific) at 37\u2009\u00b0C. DH5\u03b1 was used for plasmid amplification, and BL21 was used for recombinant protein expression. Sf9 insect cells (Spodoptera frugiperda, Expression Systems, 94-001F) were cultured in ESF 921 medium (Expression Systems) at 27\u2009\u00b0C. Baculoviruses were generated and amplified according to the manufacturer\u2019s instructions. For serum starvation experiments, cells were cultured in serum-free medium supplemented with 0.1% BSA, 10\u2009mM HEPES, and 1% penicillin-streptomycin. Transfections were performed using FuGene 6 (Promega) or Lipofectamine 3000 (Invitrogen), according to manufacturers\u2019 protocols. All cell lines were confirmed mycoplasma-free by routine testing through the Duke University cell culture facility. Monoclonal anti-Flag M2\u2013horseradish peroxidase (HRP) (A8592) and anti-ERK1\/2 (ABS44) antibodies were obtained from Sigma-EMD Millipore. HRP-conjugated secondary antibodies (NA9340-1ML and NA9310-1ML) were purchased from Cytiva. Protease and phosphatase inhibitor tablets (cOmplete, PhosSTOP) were obtained from Roche. Anti\u2013phospho-p44\/42 MAPK (Thr202\/Tyr204) antibody (9101L) was sourced from Cell Signaling Technology.<\/p>\n Recombinant protein expression and purification<\/p>\n The expression and purification of \u03b2-arrestins have been described previously44<\/a>,62<\/a>,63<\/a>,64<\/a>. In brief, E. coli BL21 (DE3) pLysS cells (New England Biolabs: C2527I) carrying pGEX4T1-Rattus norvegicus \u03b2ARR1 or \u03b2ARR2 construct, with their C\u00a0terminus truncated (at amino acid 393 or 394, respectively), were cultured in Terrific Broth (Teknova) medium at 37\u2009\u00b0C. After OD600 reached 0.6\u20130.8, the cells were induced with 0.1\u2009mM isopropyl-\u03b2-d-thiogalactopyranoside (IPTG) at 18\u2009\u00b0C overnight. Bacteria were collected by centrifugation (4,000\u2009rpm), and cell pellets were resuspended in lysis buffer (20\u2009mM HEPES, pH 8, 150\u2009mM NaCl, 10% glycerol, 1\u2009mM EDTA, 0.2\u2009mM dithiothreitol (DTT), 1\u2009mM phenylmethylsulfonyl fluoride, and 1\u2009mM benzamidine) at 4\u2009\u00b0C for 1\u2009h. Cell suspensions were sonicated, centrifuged (14,000\u2009rpm, 30\u2009min, 4\u2009\u00b0C), and the supernatant was loaded onto pre-equilibrated glutathione\u2013agarose resin (GoldBio). After 3\u2009h of binding at 4\u2009\u00b0C, the beads were washed, and thrombin was added for overnight cleavage. The proteins were further purified by anion exchange chromatography followed by size-exclusion chromatography using a Superdex 200 Increase 10\/300 GL column on an \u00c4KTA FPLC system (GE Healthcare). Eluted fractions were analysed by SDS\u2013PAGE, pooled, concentrated using 30\u2009kDa MWCO Amicon Ultra-15 Centrifugal Filter devices, flash-frozen in liquid nitrogen, and stored in aliquots at \u221280\u2009\u00b0C. The same rat \u03b2ARR1 constructs with mutations (L129A, L129S, L129G, P131A, P133A, E134A, D135A, K138A, C140A, Y249A, C251A, E283A, K284A and R285A) were generated and purified in a similar manner. Recombinant G\u03b1 subunits (G\u03b1s, G\u03b1q and G\u03b1i) were purified as GST fusion proteins using glutathione\u2013agarose affinity chromatography as previously described65<\/a>. The heterotrimeric Gs protein complex, composed of G\u03b1s, G\u03b21 and G\u03b32 subunits, was expressed in Sf9 cells using the baculovirus expression system and purified as previously described66<\/a>. M2R expression, purification and reconstitution into high-density lipoprotein (HDL) particles were performed as previously described58<\/a>. ERK2, SRC, p38\u03b1 and JNK3 kinases were expressed and purified using previously established protocols6<\/a>,16<\/a>. Protein concentrations for each protein were determined by ultraviolet absorption at 280\u2009nm and extinction coefficients estimated using the ExPASy ProtParam tool67<\/a>.<\/p>\n Expression of \u03b22AR constructs in Sf9 cells using the baculoviral system<\/p>\n The N-terminal Flag-tagged T4 lysozyme fusion-\u03b22V2R construct (\u03b22AR residues 1\u2013341 fused to V2R residues 328\u2013372), bearing a TEV cleavage site, was co-expressed with GRK2\u2013CAAX (membrane-anchored GRK2) or wild-type \u03b22AR in Sf9 insect cells using the Baculovirus Expression System, as described previously44<\/a>,62<\/a>,63<\/a>,68<\/a>,69<\/a>. At 66\u2009h after\u00a0infection, cells expressing T4L\u2013\u03b22V2R were stimulated with 20\u2009\u03bcM isoproterenol for 20\u2009min at 37\u2009\u00b0C to induce receptor phosphorylation (p\u03b22V2R), while \u03b22AR-expressing cells remained untreated. All cells were washed extensively to remove residual agonist. For membrane preparation from these cells, briefly, cells were resuspended in cold homogenization buffer (75\u2009mM Tris-HCl, pH 7.4, 2\u2009mM EDTA, and cOmplete protease inhibitor) and collected by centrifugation at 500g for 5\u2009min at 4\u2009\u00b0C. After 2 additional rounds of centrifugation at 500g for 5\u2009min at 4\u2009\u00b0C, the supernatant was centrifuged at 21,000g for 30\u2009min at 4\u2009\u00b0C to collect crude membrane fractions. Pellets were then washed with resuspension buffer (75\u2009mM Tris-HCl, pH 7.4, 2\u2009mM EDTA, 12.5\u2009mM MgCl2, cOmplete protease inhibitor, and PhosSTOP phosphatase inhibitor), passed through a 0.4\u2009mm gauge needle 30 times using a syringe on ice, aliquoted, flash-frozen in liquid nitrogen, and stored at \u221280\u2009\u00b0C.<\/p>\n Small-molecule library and reagents<\/p>\n A collection of structurally diverse, drug-like small-molecule libraries used in this work was obtained from the NCI\/DTP Open Chemical Repository. The compound library comprised approximately 3,500 compounds, representing the structural diversity of over 250,000 unique small molecules. This DTP library included the NCI Diversity Set, natural products, and FDA-approved oncogenic drugs. Most compounds were certified as >95% pure by the supplier (NCI DTP Discovery Services). Powdered compounds were dissolved in 100% DMSO and stored at \u221220\u2009\u00b0C. Isoproterenol (Sigma, I2760-1G), ICI-118,551, carvedilol, angiotensin II (ANGII; Sigma, A9525), and human epidermal growth factor (EGF) were purchased from Sigma-Aldrich, and AVP was obtained from GenScript. All small-molecule compounds, including BI-167107\u00a0(ref. 70<\/a>), were dissolved in DMSO and stored at \u221220\u2009\u00b0C as 100\u2009mM stock solutions. The C-terminal peptide of the GPCR vasopressin-2 receptor (V2R), known as V2Rpp, was synthesized by the Tufts University Analytical Core Facility. Cellular agonist stimulations were performed at 37\u2009\u00b0C, as described in the figure legends.<\/p>\n Differential scanning fluorimetry<\/p>\n DSF assay was performed to identify \u03b2-arrestin-binding small molecules from the drug-like compound library (DDLC) described above. The screen was conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems) with the fluorescent reporter probe SYPRO Orange (Thermo Fisher Scientific) in a 96-well format. Proteins were buffered in 20\u2009mM HEPES (pH 7.5) with 100\u2009mM NaCl. Small molecules (100\u2009\u03bcM) were screened with either \u03b2ARR1 or \u03b2ARR2 (5\u2009\u03bcM). DMSO was used as a vehicle control, and V2Rpp (50\u2009\u03bcM) served as a positive control.<\/p>\n For DSF experiments involving the three positive control ligands (V2Rpp, IP6 and heparin, each at 50\u2009\u03bcM) binding to \u03b2-arrestins, HEPES buffer was used as the vehicle control. Excitation and emission filters for SYPRO Orange were set to 475\u2009nm and 580\u2009nm, respectively. The temperature was increased by 0.5\u2009\u00b0C every 30\u2009s, from 25\u2009\u00b0C to 99\u2009\u00b0C, with fluorescence readings taken at each interval. Raw DSF data were analysed using Applied Biosystems Protein Thermal Shift Software. Fluorescence intensities were plotted as a function of temperature, and the midpoint of transition, or melting temperature (Tm), was calculated by plotting the first derivative of fluorescence emission as a function of temperature (dF\/dT). The difference between the Tm of the protein\u2013ligand complex and that of the protein alone represents the thermal shift (\u0394Tm), which indicates ligand binding to the protein of interest. For statistical analysis, experiments were conducted with at least three independent replicates per condition. The final selection of compounds for this study was guided by their efficacy in inhibiting \u03b2ARR1\/2 activity, along with favourable chemical properties, such as aqueous solubility and permeability, as described in the Results section. Three compounds were selected as candidate inhibitors: Cmpd-5 (NSC 250682), (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6,14-tetrahydroxy\u22124,4-dimethyl-8-methylenedecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-7(8H)-one; Cmpd-46 (NSC 302979), (Z)-3-ethoxy-6-hydroxy-4,4,13a-trimethyl-9-methylene-1,2,3,4,4a,5,6,9,10,11,12,13a-dodecahydro-7,10-(metheno)benzo[11]annulene-8,13-dione; and Cmpd-64 (NSC 22070), (Z)-4,10a-dimethyl-7-methylene-8-oxo-1a,2,3,6,6a,7,8,9a,10,10a decahydrooxireno[2\u2019,3\u2019:8,9]cyclodeca[1,2-b]furan-6-yl acetate.<\/p>\n Measurement of \u03b2-arrestin recruitment using the PathHunter assay<\/p>\n \u03b2-Arrestin recruitment to the agonist-activated receptor (\u03b22V2R) was measured using the DiscoveRx PathHunter \u03b2-arrestin assay28<\/a>, which uses enzyme fragment complementation. In this assay, the \u03b22V2R is fused to an inactive portion of \u03b2-galactosidase (ProLink tag), and \u03b2ARR2 is fused to the complementary enzyme acceptor (EA) portion, each stably expressed in U2OS cells. Upon agonist-induced recruitment of \u03b2ARR2 to \u03b22V2R, the \u03b2-gal fragments complement to form a functional enzyme, generating a measurable chemiluminescent signal. The signal intensity correlates directly with the extent of \u03b2ARR2 recruitment. U2OS cells co-expressing \u03b22V2R and \u03b2ARR2 were plated at a density of 25,000 cells per well in white, clear-bottom 96-well plates 24\u2009h before treatment. On the day of the experiment, cells were treated with either a single dose (50\u2009\u03bcM) or varying concentrations of a specific \u03b2-arrestin modulator or vehicle control in Hanks\u2019 balanced salt solution\u00a0(HBSS) (Sigma-Aldrich) with 20\u2009mM HEPES (pH 7.4) and 0.05% BSA. Cells were incubated at 37\u2009\u00b0C, 5% CO2, and about\u00a0100% relative humidity for about\u00a030\u2009min, followed by stimulation with either 10\u2009nM Iso or a serial dilution of Iso for 60\u2009min at 37\u2009\u00b0C. After agonist stimulation, PathHunter reagents were added, and cells were incubated for another 60\u2009min at ambient temperature. Luminescence signals were measured using a CLARIOstar microplate reader (BMG Labtech).<\/p>\n NanoBiT luciferase complementation assays<\/p>\n For each NanoBiT assay, specific components are described below. For NanoBiT-based \u03b2-arrestin recruitment assays, CRISPR\u2013Cas9 \u03b2ARR1\/2-knockout HEK293 cells were maintained in MEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37\u2009\u00b0C and 5% CO2. Cells were seeded in poly-d-lysine-coated white 96-well plates at 100,000 cells per well. \u03b2-Arrestin recruitment was monitored using the NanoBiT complementation system, which uses split NanoLuc luciferase fragments (LgBiT and SmBiT). Two assay formats were used. In the first, the LgBiT\u2013CAAX (plasma membrane) and SmBiT\u2013\u03b2ARR1 format, cells were transfected with 125\u2009ng Flag-tagged GPCR, 25\u2009ng LgBiT\u2013CAAX, 125\u2009ng SmBiT\u2013\u03b2ARR1, and pcDNA filler using polyethylenimine (PEI) at a 3:1 PEI:DNA ratio. After 24\u2009h, cells were lifted and replated in MEM with 1% FBS. The next day, cells were incubated with 2.5\u2009\u03bcM fluorofurimazine and either modulator (50\u2009\u03bcM) or DMSO for 20\u2009min at 37\u2009\u00b0C. Luminescence was recorded before and after agonist or vehicle addition using a Berthold Mithras LB 940 with a 480\u2009nm filter (NanoLuc). For each condition, data were normalized to the mean baseline and vehicle-stimulated cells. Modulator responses were then normalized to the maximal signal observed in DMSO-pretreated cells stimulated with the highest agonist dose. In the second, the V2R\u2013LgBiT and \u03b2ARR1\u2013 or \u03b2ARR2\u2013SmBiT format, cells were transfected with 25\u2009ng V2R\u2013LgBiT and 125\u2009ng \u03b2ARR1\u2013 or \u03b2ARR2\u2013SmBiT using Lipofectamine 3000 and incubated for 48\u2009h. Cells were washed and treated with coelenterazine-h and modulator (50\u2009\u03bcM) or DMSO for 30\u2009min at 37\u2009\u00b0C. Baseline luminescence was recorded for 2\u2009min followed by stimulation with 100\u2009nM AVP or vehicle. NanoBiT luminescence was measured for 20\u2009min using a PHERAstar FSX plate reader (BMG LabTech). AUC was used to quantify \u03b2-arrestin recruitment normalized to baseline and vehicle. NanoBiT assays between \u03b22AR\u2013LgBiT or CCR7\u2013LgBiT and SmBiT\u2013mini-Gi71<\/a> were performed similarly as above. HEK293 \u03b2ARR1\/2-knockout cells were seeded into white poly-D-lysine-coated 96-well flat-bottom plates (Corning; 353296). Coelenterazine-h (NanoLight; 301) was added at a final concentration of 10 \u03bcM before measurement. After establishing a 3-min baseline,\u00a0cells were stimulated with 10\u2009\u03bcM Iso or 250\u2009nM CCL19 in HBSS to measure mini-Gi recruitment in the presence of 50\u2009\u03bcM modulator in HEK293 \u03b2ARR1\/2-knockout cells. Luminescence was recorded for 27\u2009min using a CLARIOstar plate reader (BMG LabTech).<\/p>\n FRET-based cAMP accumulation measurement<\/p>\n To measure cellular cAMP production in live cells mediated by stimulatory G protein, G\u03b1s-coupled \u03b22AR activation, FRET-based Epac sensors were used as described previously30<\/a>. The Epac2 (ICUE2) sensor contains a CFP and YFP FRET pair. HEK293 cells stably expressing ICUE2 were plated in poly-d-lysine-coated, black, clear-bottom 96-well plates (Corning) at a density of 50,000 cells per well. At least 16\u2009h after plating, cells were washed with PBS and incubated in HEPES-buffered saline solution (10\u2009mM HEPES, 150\u2009mM NaCl, 5\u2009mM KCl, 1.5\u2009mM MgCl2, 1.5\u2009mM CaCl2, 10\u2009mM glucose, 0.2% BSA, pH 7.4) for 1\u2009h at 37\u2009\u00b0C. Cells were then treated with either \u03b2-arrestin small-molecule modulators (40\u2009\u03bcM) or DMSO for 5\u2009min, and baseline fluorescence was monitored. Real-time cAMP measurement was initiated by stimulating cells with 10\u2009\u03bcM Iso at 37\u2009\u00b0C. FRET changes corresponding to cAMP accumulation were measured as changes in the background-subtracted 480\u2009nm\/535\u2009nm fluorescence emission ratio (CFP\/YFP), reflecting changes in cAMP levels. The entire cAMP accumulation profile was quantified by calculating the AUC for the time course. To assess the effect of modulators on the agonist-induced cAMP response, AUC values were expressed as a percentage of the response to Iso in the presence of DMSO (set as 100%), enabling comparison of the effect of each modulator relative to this agonist-alone control.<\/p>\n Intracellular calcium measurement<\/p>\n Intracellular (Ca2+) release was measured using the FLIPR Calcium 6 assay kit with a FlexStation 3 microplate reader, following the manufacturer\u2019s instructions (Molecular Devices) and as described previously31<\/a>. In brief, HEK293 cells stably expressing human angiotensin II type 1 receptor (AT1R), parental HEK293 cells transiently expressing AT1R, or HEK293 CRISPR\u2013Cas9 \u03b2ARR1\/2-knockout cells transiently expressing AT1R were seeded in poly-d-lysine-coated, black 96-well assay plates at a density of 40,000 cells per well and incubated for 24\u2009h. On the day of the experiment, cell plates were loaded with FLIPR Calcium 6 reagents and treated with \u03b2-arrestin small-molecule modulators (10\u2009\u03bcM) or vehicle (DMSO) for 30\u2009min. After establishing basal fluorescence (F0), the cells were treated with the agonist ANGII (30\u2009pM for stably expressing AT1R or 120\u2009pM for transiently expressing AT1R) while fluorescence intensity (F) was monitored in real time. These sub-maximal ANGII concentrations were empirically optimized to elicit moderate, temporally resolved Ca2+ transients, allowing sufficient time for \u03b2-arrestin-mediated desensitization to develop prior to the peak response. The complete Ca2+ transient profile was quantified by calculating the AUC over time. AUC values were normalized to the response to ANGII alone (set as 100%) to compare the effects of modulators relative to the agonist-alone control.<\/p>\n Measurement of receptor internalization by PathHunter assay<\/p>\n \u03b2-Arrestin-mediated receptor internalization was measured using the DiscoveRx PathHunter active receptor endocytosis assay according to the manufacturer\u2019s protocol (DiscoveRx) and as previously described28<\/a>. In brief, \u03b22V2R was transiently transfected into U2OS cells stably expressing an EA-tagged \u03b2ARR2 and an endosome-localized ProLink-tagged protein. The next day, cells were seeded at 25,000 cells per well in white, clear-bottom 96-well assay plates and incubated for 24\u2009h before the experiment. On the day of the experiment, cells were treated with varying concentrations of a specific \u03b2-arrestin modulator or vehicle control in HBSS (Sigma-Aldrich) with 20\u2009mM HEPES (pH 7.4) and 0.05% BSA. Cells were incubated at 37\u2009\u00b0C, 5% CO2, and ~100% relative humidity for ~30\u2009min, followed by stimulation with a series of concentrations of agonist (Iso) for 60\u2009min at 37\u2009\u00b0C. After agonist stimulation, PathHunter reagents were added, and cells were incubated for another 60\u2009min at ambient temperature. Receptor\u2013\u03b2-arrestin complex internalization was detected as luminescence resulting from the complementation of \u03b2-gal fragments (enzyme acceptor and ProLink) within endosomes. Luminescence signals were measured using a CLARIOstar microplate reader (BMG Labtech).<\/p>\n BRET-based receptor internalization assay<\/p>\n BRET-based assays were conducted to measure receptor internalization using a bystander BRET format as previously described33<\/a>. In brief, HEK293 cells transiently expressing V2R\u2013RLucII (BRET donor) and the early endosome marker 2\u00d7FYVE-mVenus (BRET acceptor) were pretreated with vehicle or \u03b2-arrestin modulator (40\u2009\u03bcM) for 30\u2009min. Receptor association with the endosome marker was measured as a BRET signal after stimulation with a range of AVP concentrations. BRET measurements were performed using the Synergy2 (BioTek) microplate reader with filter sets of 410\/80\u2009nm and 515\/30\u2009nm to detect RLucII (donor) and mVenus (acceptor) emissions, respectively. The BRET signal was calculated as the ratio of light intensity emitted by the acceptor over the donor, and the \u2018net BRET\u2019 ratio was determined by subtracting the vehicle control ratio from the corresponding AVP-treated ratio.<\/p>\n Gi activity assay<\/p>\n Gi-mediated inhibition of adenylyl cyclase was assessed using the GloSensor cAMP bioluminescence biosensor (Promega). HEK293 cells were transiently transfected with D2R or M2R together with the GloSensor cAMP reporter construct, using FuGENE 6 per the manufacturer\u2019s protocol. Cells were seeded into poly-d-lysine-coated white clear-bottom 96-well plates at 60,000 cells per well and cultured for 24\u2009h. GloSensor reagent was added per the manufacturer\u2019s instructions and cells were equilibrated for 1\u2009h at room temperature. Where indicated, pertussis toxin (PTX; 100\u2009ng\u2009ml\u22121) was applied overnight as a positive control for Gi blockade. Compounds (Cmpd-5, Cmpd-46 or Cmpd-64; 50\u2009\u03bcM) or vehicle (DMSO) in HBSS supplemented with 20\u2009mM HEPES (pH 7.4) and 0.05% BSA were applied for 15\u2009min at room temperature. Cells were stimulated with forskolin (2\u2009\u03bcM, 5\u2009min), followed by concentration-response stimulation with quinpirole (D2R) or acetylcholine (M2R). Luminescence was recorded every 5\u2009min over 30\u2009min using a CLARIOstar microplate reader (BMG Labtech). Forskolin-stimulated cAMP, quantified at 10\u2009min after\u00a0agonist addition, was normalized to the maximal forskolin response per condition. Gi activity was derived from the inhibitory plateau and expressed as a percentage of the DMSO plus agonist control.<\/p>\n Intramolecular FlAsH-BRET assay<\/p>\n Intramolecular FlAsH-BRET assays72<\/a> were performed in HEK293 \u03b2ARR1\/2-knockout cells transiently transfected using FuGENE4K with 2\u2009\u03bcg total DNA per 15\u2009cm dish. Cells were transfected with 1.5\u2009\u03bcg of pcDNA3.1 encoding human AT1R and 0.4\u2009\u03bcg of a plasmid encoding the indicated RLuc\u2013\u03b2-arrestin2\u2013FlAsH biosensors. Twenty-four hours after\u00a0transfection, cells were replated into poly-d-lysine-coated 96-well white, clear-bottom plates at 100,000 cells per well in MEM supplemented with 10% FBS. Forty-eight hours after transfection, cells were labelled with FlAsH-EDT2 (FlAsH II In-Cell Tetracysteine Detection Kit, Thermo Fisher) at a final concentration of 2.5\u2009\u03bcM for 30\u2009min at room temperature. Following labelling, cells were washed twice with BAL wash buffer (250\u2009\u03bcM 2,3-dimercapto-1-propanol in HBSS), then equilibrated in HBSS supplemented with 20\u2009mM HEPES (pH 7.4). Modulators were applied at 50\u2009\u03bcM for 15\u2009min, followed by addition of Prolume Purple substrate (Nanolight Technologies). Cells were then stimulated with 10\u2009\u03bcM angiotensin II. BRET signals were recorded using a CLARIOstar Plus plate reader in kinetic mode (480\u2009nm donor, 530\u2009nm acceptor) for 30\u2009min, and peak values were used for quantification. \u0394Net BRET was calculated by subtracting baseline values from from each condition, including agonist alone, modulator alone, or agonist plus modulator.<\/p>\n TRUPATH BRET assay<\/p>\n TRUPATH assays73<\/a> were performed in HEK293 \u03b2ARR1\/2-knockout cells transiently transfected using FuGENE4K with a total of 4\u2009\u03bcg per 15\u2009cm dish plasmid DNA, including pcDNA3.1 constructs encoding human GPCRs (\u03b22AR, AT1R, M2R or NTSR1), G\u03b1\u2013RLuc8, G\u03b23 and G\u03b39\u2013GFP2 at a 1:1:1:1 ratio. TRUPATH components were obtained from B. Roth via Addgene (1000000163). Twenty-four hours after\u00a0transfection, cells were replated into poly-d-lysine-coated 96-well white, clear-bottom plates at 20,000\u201330,000 cells per well in phenol red-free DMEM supplemented with 2% FBS. BRET signals were acquired 48\u2009h after transfection. On the day of the assay, cells were washed and incubated in HBSS containing 20\u2009mM HEPES (pH 7.4). Modulators were applied at 50\u2009\u03bcM for 15\u2009min, followed by addition of Prolume Purple substrate (Nanolight Technologies). Cells were then stimulated with increasing concentrations of agonist (angiotensin II, neurotensin, isoproterenol or acetylcholine). BRET2 was recorded in kinetic mode for 30\u2009min using a CLARIOstar Plus plate reader (480\u2009nm\/510\u2009nm), and peak signals were used for quantification. \u0394Net BRET ratios were calculated by subtracting the BRET ratio of the vehicle-treated control from each condition, agonist alone or agonist plus modulator. Data represent the mean of three technical replicates across 3\u20135 independent experiments and were fitted using a three-parameter logistic model in GraphPad Prism.<\/p>\n Chemotaxis assays<\/p>\n Chemotaxis assays were performed similarly to previously described protocols37<\/a>. In brief, T cells were isolated from the spleens of wild-type mice, subjected to erythrocyte lysis, and filtered through a 70-\u03bcm filter. The cells were then suspended in RPMI 1640 medium containing 0.5% BSA and treated for 30\u2009min with \u03b2-arrestin modulators or vehicle. A total of 1\u2009\u00d7\u2009106 cells in 100\u2009\u03bcl of medium were added to the upper chamber of 6.5-mm diameter, 5-\u03bcm pore polycarbonate Transwell filters (Corning Costar), and cells migrated towards 100\u2009nM CCL19 in the lower chamber for 2\u2009h at 37\u2009\u00b0C. T cells that migrated to the lower chamber were collected, resuspended, washed, and stained for flow cytometry analysis using a Live\/Dead marker (Aqua Dead, Thermo Fisher) and antibodies for cell surface markers (CD45+, CD3+, CD4+ and CD8+) prior to paraformaldehyde fixation. The total live T cell population (CD45+ and CD3+) and subset populations (CD45+, CD3+ and CD4+, or CD45+, CD3+ and CD8+) were measured using a BD LSR Fortessa machine from the Flow Cytometry Shared Resource (FCSR) at the Duke Cancer Institute (Durham, NC). CountBright beads (Thermo Fisher) were added immediately after resuspension of the lower chamber contents to correct for volume differences and any cell loss during wash steps. Per\u00a0cent migration was calculated by determining the percentage of migrated cells treated with compounds relative to control wells, with migration in CCL19-treated wells alone set as 100%. The use of mouse splenocytes ex vivo for this T cell migration was conducted under institutional guidelines for the care and use of laboratory animals. No live animal procedures were performed. A representative gating tree is shown in Extended Data Fig. 5<\/a>.<\/p>\n Adult ventricular cardiomyocyte isolation and contractility analysis<\/p>\n All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Duke University Medical Center and performed in accordance with relevant guidelines and regulations. Ventricular cardiomyocytes were freshly isolated from 12- to 16-week-old C57BL\/6J wild-type mice using a standard Langendorff perfusion system39<\/a>,74<\/a>. The heart was initially cannulated through the aorta and then perfused with an oxygenated buffer at 37\u2009\u00b0C (120\u2009mM NaCl, 14.8\u2009mM KCl, 0.6\u2009mM KH2PO4, 0.6\u2009mM Na2HPO4, 1.2\u2009mM MgSO4\u00b77H20, 10\u2009mM HEPES, 4.6\u2009mM NaHCO3, 30\u2009mM taurine, and 5.6\u2009mM glucose, pH 7.3). After 2\u2009min, the buffer was switched to a digestion solution containing 2.4\u2009mg\u2009ml\u22121 collagenase (Worthington). Following 8\u2009min of digestion, ventricles were transferred to a stopping buffer that included 10% calf serum. Myocytes were then manually dissociated and gradually brought to a physiological Ca2+ concentration (1.2\u2009mM). Isolated cardiomyocyte was first pretreated with DMSO (0.1%), C5, C46 or C64 (25\u2009\u00b5M) for 20\u2009min followed by treatment with HBSS\u00a0(basal), angiotensin II (positive control, 10\u2009\u00b5M) or TRV027 (10\u2009\u00b5M). Cells were plated in a chamber (Ionoptix) on a Nikon Eclipse TE300 inverted microscope (40\u00d7 0.9 NA objective, MRF00400, Nikon). Cardiomyocytes were paced at 1\u2009Hz and 20\u2009V (MyoPacer, Ionoptix), and sarcomere length was recorded by a MyoCam-S camera (Ionoptix). Ten consecutive contractions per cell (7\u201312 cells per condition) were averaged to quantify contractility and kinetic parameters (IonWizard 7.2). Only those cells exhibiting proper single cardiomyocyte morphology and responsiveness to electrical stimulation were included. Sample size represents the number of hearts at each condition derived from an average of 7\u201312 cells per heart for each condition. Statistical comparisons between conditions were performed using one-way ANOVA followed by Sidak post hoc test.<\/p>\n Isothermal titration calorimetry<\/p>\n ITC measurements were performed on a MicroCal iTC200 system at 25\u2009\u00b0C. Prior to ITC experiments, all proteins (\u03b2ARR1, \u03b2ARR2 or \u03b2ARR1 mutants) were extensively dialysed against 20\u2009mM HEPES (pH 7.4), 100\u2009mM NaCl, and 3\u2009\u03bcM TCEP. Protein concentrations were determined by spectrophotometry at 280\u2009nm, using extinction coefficients calculated from each protein sequence via the ProtParam program67<\/a>. The dialysis buffer was used to dilute DMSO stock solutions of \u03b2-arrestin small-molecule modulators to their final concentrations for measurements. For each ITC experiment, \u03b2-arrestin modulators (Cmpd-5 at 350\u2009\u03bcM, Cmpd-46 at 450\u2009\u03bcM, and Cmpd-64 at 450\u2009\u03bcM) were loaded into the syringe and titrated into the calorimetric cell containing \u03b2ARR1, \u03b2ARR1 mutants or \u03b2ARR2 (at about\u00a030\u201340\u2009\u03bcM, depending on the protein and experiment). Additional ITC experiments were also performed using purified GST-fused G\u03b1 subunits (G\u03b1s, G\u03b1q and G\u03b1i; about\u00a030\u2009\u03bcM) and G\u03b1i\u03b2\u03b3 heterotrimer (about\u00a030\u2009\u03bcM). G\u03b1 subunits were prepared in the same buffer (20\u2009mM HEPES, pH 7.4, 100\u2009mM NaCl, 3\u2009\u03bcM TCEP), whereas the G\u03b1i\u03b2\u03b3 heterotrimer was maintained in buffer supplemented with 0.05% DDM and 10\u2009\u03bcM GDP, with matched DMSO carrier conditions. Modulators (300\u2009\u03bcM) or corresponding DMSO controls were loaded and titrated as described above. The reference cell was filled with distilled water. In all experiments, the titration sequence typically consisted of an initial 0.4\u2009\u03bcl injection, followed by 19 injections of 2\u2009\u03bcl each, with a 180-s interval between injections to allow thermal power to return to baseline. During the experiment, the reference power was set to 7\u2009\u03bccal\u2009s\u22121, and the sample cell was stirred continuously at 750\u2009rpm. Raw data, excluding the first injection, were baseline-corrected, integrated, and normalized. Data were analysed and fit using a one-site independent binding model to obtain the equilibrium dissociation constant, Kd (from the association constant Ka\u2009=\u20091\/Kd), stoichiometry (N), and thermodynamic parameters including enthalpy (\u0394H) and entropy (\u2212T\u0394S) of binding. Data were analysed using MicroCal PEAQ-ITC analysis software (v1.1.0.1262).<\/p>\n Radioligand-binding experiments<\/p>\n To assess the effects of \u03b2-arrestin modulators on \u03b2-arrestin- or G\u03b1s\u2013\u03b2\u03b3-promoted high-affinity agonist binding, [3H]-Fen binding assays were performed using membranes from Sf9 cells expressing phosphorylated \u03b22V2R (p\u03b22V2R) or \u03b22AR28<\/a>. Membranes were prepared from Sf9 cells co-expressing Flag-tagged T4L\u2013\u03b22V2R and GRK2\u2013CAAX under agonist stimulation, or unstimulated \u03b22AR alone as described above28<\/a>. Reactions (150\u2009\u03bcl) contained 6\u2009nM [3H]-Fen (12.6\u2009Ci\u2009mmol\u22121), p\u03b22V2R membranes, \u03b2ARR1\/2 or \u03b2ARR1 mutants (2\u2009\u03bcM), and \u03b2-arrestin modulators (100\u2009\u03bcM) or vehicle in HN100 buffer (20\u2009mM HEPES, pH 7.4, 100\u2009mM NaCl, 12.5\u2009mM MgCl2). For G\u03b1s\u2013\u03b2\u03b3 assays, \u03b22AR membranes were incubated with 4.3\u2009nM [3H]-Fen and G\u03b1s\u2013\u03b2\u03b3 heterotrimer (200\u2009nM) in G protein assay buffer with or without modulators. Nonspecific binding was defined using propranolol (25\u2009\u03bcM). Following 90\u2009min incubation at room temperature, reactions were filtered onto PEI-soaked GF\/B filters and washed with cold buffer. Bound [3H]-Fen was extracted overnight in scintillation fluid and quantified by scintillation counting. Specific binding was calculated by subtracting nonspecific from total binding.<\/p>\n Live-cell ERK1\/2 activation assay<\/p>\n To evaluate the effect of small-molecule modulators on \u03b2-arrestin-dependent ERK1\/2 signalling, HEK293 cells stably expressing \u03b22AR were serum-starved for 6\u2009h (refs. 34<\/a>,75<\/a>), then pretreated with \u03b2-arrestin modulators (30\u2009\u03bcM) or vehicle for 20\u2009min at 37\u2009\u00b0C. Cells were subsequently stimulated with carvedilol (10\u2009\u03bcM, 5\u2009min), a \u03b2-arrestin-biased agonist known to promote ERK1\/2 phosphorylation via \u03b2-arrestin scaffolding of RAF\u2013MEK\u2013ERK signalling components. Cells were lysed, sonicated (15\u2009s), and centrifuged at 14,000g for 15\u2009min at 4\u2009\u00b0C. Equal amounts of total protein were resolved by SDS\u2013PAGE on 4\u201320% Tris-Glycine gels (Thermo Fisher Scientific), transferred to nitrocellulose or PVDF membranes, and immunoblotted with anti\u2013phospho-ERK1\/2 (1:2,000; Cell Signaling) and total ERK1\/2 (1:10,000; Millipore Sigma). HRP-conjugated secondary antibodies were used for detection. Protein bands were visualized using Pierce SuperSignal West Pico ECL substrate (Thermo Fisher Scientific), imaged on a ChemiDoc XRS system (Bio-Rad) and quantified by densitometry using ImageLab (Bio-Rad). Data were analysed using GraphPad Prism.<\/p>\n Pulldown analysis of \u03b2-arrestin\u2013effector interactions<\/p>\n To assess the effects of the compounds on \u03b2ARR1 interactions with its effector proteins SRC and JNK3, \u03b2ARR1\u2013Flag (5\u2009\u03bcM) was incubated with a 20-fold molar excess of Cmpd-5, Cmpd-46 or Cmpd-64 for 30\u2009min at room temperature. Subsequently, 15\u2009\u03bcM of SRC or JNK3 was added. To evaluate compound effects on \u03b2ARR1\u2013ERK2 binding, \u03b2ARR1 (15\u2009\u03bcM) was pre-incubated with a 20-fold molar excess of Cmpd-5, Cmpd-46 or Cmpd-64 for 30\u2009min at room temperature, followed by the addition of 5\u2009\u03bcM ERK2\u2013Flag. Afterward, 50\u2009\u03bcl of anti-Flag M2 affinity gel (Millipore Sigma) was added, and the mixtures were incubated for 1\u2009h at room temperature with rotation. The resin was then collected by centrifugation and washed 3 times with 1\u2009ml of 20\u2009mM HEPES (pH 7.5), 100\u2009mM NaCl. Bound proteins were eluted using 0.2\u2009mg\u2009ml\u22121 Flag peptide in 20\u2009mM HEPES (pH 7.5), 100\u2009mM NaCl, mixed with Laemmli sample buffer (Bio-Rad), and analysed by SDS\u2013PAGE followed by western blotting. Detection was performed using monoclonal anti-Flag M2-peroxidase (HRP) antibody (1:2,000, Sigma-Aldrich A8592, RRID:AB_439702) for \u03b2ARR1\u2013Flag and ERK2\u2013Flag; polyclonal A1CT antibody76<\/a> (1:5,000) for \u03b2ARR1; anti-SRC monoclonal antibody (1:10,000, EMD Millipore 05-184, RRID:AB_2302631) for SRC; and anti-JNK3 monoclonal antibody (1:5,000, Cell Signaling 2305, RRID:AB_2281744) for JNK3. Western blot images were taken using Bio-Rad ChemiDoc system and the densitometry analysis was performed by ImageLab v6.1 and statistical differences were determined by one-way ANOVA with Dunnett\u2019s post hoc test in GraphPad Prism software.<\/p>\n G protein GTPase assay<\/p>\n GTPase activity of purified heterotrimeric Gi protein was measured in vitro using the GTPase-Glo assay (Promega). HDL-reconstituted M2R (100\u2009nM) was pre-incubated with iperoxo (10\u2009\u03bcM) or DMSO for 15\u2009min at room temperature in assay buffer (20\u2009mM HEPES, pH 7.4, 100\u2009mM NaCl, 10\u2009mM MgCl2). \u03b2-arrestin modulators (Cmpd-5, Cmpd-46 or Cmpd-64; 10\u2009\u03bcM each) or DMSO control were subsequently added together with Gi heterotrimer (500\u2009nM) and GTP (2.5\u2009\u03bcM), and reactions were incubated for 1\u2009h at room temperature in the continued presence of iperoxo or DMSO. Reactions were terminated by addition of GTPase-Glo reagent, followed by detection reagent, according to the manufacturer\u2019s instructions. Luminescence was measured using a CLARIOstar plate reader (BMG Labtech).<\/p>\n Cytotoxicity assay<\/p>\n MTT assay was performed according to the manufacturer\u2019s instructions (Roche Diagnostics). HEK293 and U2OS cells were seeded in 96-well plates. The following day, cells were treated with the indicated concentration of modulator or vehicle for 8\u2009h. To evaluate the cytotoxic effects of the compounds, cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent at 37\u2009\u00b0C for 4\u2009h. The optical density was measured at 595\u2009nm (the absorbance of each sample was measured at 560 and 670\u2009nm). The optical density values of blue formazan formed in live cells based on the reduction of MTT were determined at 595\u2009nm. Cell viability was expressed as the percentage of MTT reduction in compound-treated cells compared to vehicle-treated cells.<\/p>\n Cryo-EM sample preparation and data acquisition<\/p>\n The expression construct for \u03b2ARR1\u2013BRIL was designed by inserting the thermostabilized apocytochrome b562 (M7W, H102I, K106L) from E. coli (BRIL) between residues 176\u2013182 at the hinge region of rat \u03b2ARR1\u00a0(ref. 64<\/a>) truncated at residue 393 and purified as wild-type \u03b2ARR1. \u03b2ARR1\u2013BRIL was incubated with 1.5-fold molar excess of anti-BRIL Fab (BAG2)42<\/a> and twofold molar excess of aFabNb43<\/a> for 1\u2009h at room temperature. The complex was subjected to size-exclusion chromatography on a Superdex 200 Increase column (Cytiva Life Sciences) in 20\u2009mM HEPES 7.5, 150\u2009mM NaCl buffer. Peak fractions were concentrated to 2\u2009mg\u2009ml\u22121 using Vivaspin 6 column with molecular weight cut-off of 30,000\u2009kDa (Sartorius). The complex was incubated for 2\u2009h on ice with 50-fold molar excess of Cmpd-5 solubilized in DMSO (\u03b2ARR1\u2013BRIL\u2013BAG2\u2013aFabNB\u2013Cmpd-5) or equivalent amount of DMSO (\u03b2ARR1\u2013BRIL\u2013BAG2\u2013aFabNB\u2013Apo), and then concentrated to 7\u20138\u2009mg\u2009ml\u22121. The sample was applied to glow-discharged 300-mesh holey-carbon grids (Quantifoil R1.2\/1.3, Electron Microscopy Sciences) using a Vitrobot Mark IV (Thermo Fisher Scientific) at 4\u2009\u00b0C and 100% humidity. The data were collected on a Titan Krios transmission electron microscope (Thermo Fisher) operating at 300\u2009kV equipped with a K3 direct electron detector (Gatan) in counting mode with a BioQuantum GIF energy filter (slit width of 20\u2009eV) at a magnification of 81,000\u00d7 corresponding to a pixel size of 1.08\u2009\u00c5 at the specimen level. Sixty-frame movies with a dose rate of about\u00a015\u2009e\u2212\u2009pixel\u22121\u2009s\u22121 and a total accumulated dose of about\u00a054\u201360\u2009e\u2212\u2009\u00c5\u22122 were collected using the Latitude-S (Gatan) single-particle data acquisition program. The nominal defocus values were set from \u22120.8 to \u22122.5\u2009\u00b5m.<\/p>\n Cryo-EM data processing<\/p>\n Movies were subjected to beam-induced motion correction using Patch Motion Correction in CryoSPARC v4.0.1\u00a0(ref. 77<\/a>) followed by determination of contrast transfer function parameters in Patch CTF. Micrographs with contrast transfer function fit better than 3.5\u2009\u00c5 were used for further analysis. Particles manually selected from 15 micrographs were used to train a model in the particle picking tool Topaz v0.2.5a78<\/a>. The trained model was used to pick particles in all micrographs generating 1,572,529 particle projections for \u03b2ARR1\u2013BRIL\u2013BAG2\u2013aFabNB\u2013Cmpd-5 and 1,307,318 particle projections for \u03b2ARR1\u2013BRIL\u2013BAG2\u2013aFabNB\u2013Apo. The particles were rescaled to the pixel size of 1.3824\u2009\u00c5 for further processing. A subset of particles (500,000) was subjected to ab initio model generation with 5 classes. All particles were then subjected to five rounds of heterogeneous refinement. The resulting particle stacks (396,911 particles for \u03b2ARR1\u2013BRIL\u2013BAG2\u2013aFabNB\u2013Cmpd-5 and 832,520 particles for \u03b2ARR1\u2013BRIL\u2013BAG2\u2013aFabNB\u2013Apo) were used in non-uniform refinement and local refinement with a mask excluding aFabNb and the constant region of BAG2. Then particles were subjected to 3D classification without alignment in CryoSPARC with a mask excluding aFabNb and the constant region of BAG2. Finally, the best classes with 177,430 particles (\u03b2ARR1\u2013BRIL\u2013BAG2\u2013aFabNB\u2013Cmpd-5) and 479,224 particles (\u03b2ARR1\u2013BRIL\u2013BAG2\u2013aFabNB\u2013Apo) were subjected to another round of local refinement in CryoSPARC, generating a map with a global resolution of 3.47\u2009\u00c5 and 3.52\u2009\u00c5, respectively. The cryo-EM maps were post-processed using DeepEMhancer with highRes deep learning model79<\/a>.<\/p>\n Model building and refinement<\/p>\n The initial models were built manually by fitting the crystal structure of \u03b2ARR1 (PDB: 1G4M<\/a>)80<\/a> into the experimental electron densities using UCSF Chimera v1.15\u00a0(ref. 81<\/a>). The BRIL\u2013BAG2\u2013aFabNb part of the model was derived from the cryo-EM structure of Frizzled5\u2013BRIL\u2013BAG2\u2013aFabNb (PDB: 6WW2<\/a>)82<\/a>. The structures were refined by combining manual adjustments in Coot v0.9.8.3\u00a0(ref. 83<\/a>) and ISOLDE84<\/a> in UCSF ChimeraX 1.6.1\u00a0(ref. 81<\/a>), followed by real-space refinement in PHENIX v1.20.1-4487\u00a0(ref. 85<\/a>) with Ramachandran, rotamer, torsion and secondary structure restraints enforced. The models were validated with MolProbity v4.5.1\u00a0(ref. 86<\/a>). Difference density maps between the Cmpd-5-bound and apo states were calculated using a local scaling-based density difference approach45<\/a>. Structural superpositions and pairwise r.m.s.d. and Q-score calculations were performed using the PDBeFold server87<\/a>. Cryo-EM refinement statistics are summarized in Extended Data Table 1<\/a>.<\/p>\n Modelling and molecular dynamics simulations<\/p>\n The apo \u03b2ARR1 model in the basal state and the \u03b22V2R\u2013\u03b2ARR1 complex (PDB code: 6TKO<\/a>)60<\/a> model were built and simulated as described previously88<\/a>. Due to a considerable number of missing residues in the Cmpd-5-bound \u03b2ARR1 cryo-EM structure, we constructed a Cmpd-5-bound \u03b2ARR1 model by placing Cmpd-5 in the MCL cleft of an equilibrated basal \u03b2ARR1 model according to the experimentally derived location of the bound Cmpd-5. A \u03b2ARR1 model in the active state was constructed by homology modelling with Modeller (v10.0)89<\/a> using the crystal structure of the V2Rpp-bound \u03b2ARR1 in the active state (PDB code: 4JQI<\/a>)44<\/a> as the template. Parts without any template\u2014that is, residues 1 to 5 and 307 to 311\u2014were ab initio modelled with Modeller. The resulting model with the lowest DOPE score was selected for the following steps. The selected \u03b2ARR1 models were processed by the Protein Preparation Wizard of Schrodinger Suite (v2023-1). The first and last residues of this model are in positively and negatively charged states, respectively, without capping, as assumed in their natural condition. The prepared model was then immersed in a simulation water box using the System Builder of Schrodinger Suite (v2023-1). A simple point charge water model was used to solvate the system, and Na+ and Cl\u2212 ions were added to neutralize the system and the salt concentration of the system was increased to 0.15\u2009M. The total system size was about\u00a0148,000 atoms.<\/p>\n Molecular dynamics simulations were carried out using Desmond MD System (v6.1; D.E. Shaw Research)90<\/a> with the OPLS4 force field91<\/a>. The simulation systems were minimized and equilibrated with restraints on the ligand heavy atoms and the protein backbone. For both the equilibrations and the following production runs, the constant temperature 310\u2009K was maintained by Langevin dynamics, 1\u2009atm constant pressure was achieved with the Langevin piston method92<\/a>. A cut-off distance of 9\u2009\u00c5 was used for the nonbonded interactions, and the particle-mesh Ewald summation method was used for the electrostatics interactions. The integration timestep was set to 2.5\u2009fs. The isothermal-isobaric (NPT) ensemble was used in a periodic boundary condition. The production runs of \u03b2-arrestin simulations are without any restraints. The analysis and visualization were performed with VMD93<\/a> and PyMol (Schrodinger).<\/p>\n Absolute protein\u2013ligand binding free energy perturbation calculations<\/p>\n We used absolute protein\u2013ligand binding free energy perturbation (AB-FEP) approach as implemented within Schrodinger suite (release 2025-2)94<\/a> which is based on the originally proposed double decoupling scheme95<\/a>. In this method, starting from the ligand in the water, the van der Waals and electrostatic interactions within the ligand, and between the ligand and water, were first gradually turned off, yielding a dummy ligand. The dummy ligand was then restrained to the protein binding site through a set of cross-link restraints. Finally, the van der Waals and electrostatic interactions within the ligand, and between the ligand and protein, were gradually turned on, while the cross-linked restraints were relaxed. The starting point for the AB-FEP run was a representative frame from the trajectory shown in Supplementary Videos\u00a01<\/a> and 2<\/a>.<\/p>\n Molecular docking of \u03b2-arrestin modulators<\/p>\n Molecular docking was performed to evaluate the binding of Cmpd-5, Cmpd-46 and Cmpd-64 at the \u03b2ARR1 allosteric site identified in the cryo-EM structure of the \u03b2ARR1\u2013Cmpd-5 complex. The \u03b2ARR1 structure was prepared from the cryo-EM model, with the Cmpd-5 binding site defined by residues forming the middle loop, lariat loop and C-loop. Docking was carried out using Schr\u00f6dinger Maestro (suite 2025-2). Ligands were prepared using LigPrep with ionization states assigned for physiological pH. A receptor grid was generated centred on the Cmpd-5-binding pocket. Cmpd-5 docked with a pose that closely aligned to the experimentally resolved conformation, validating the identified binding pocket. By contrast, Cmpd-46 and Cmpd-64 produced lower-scoring poses with less favourable shape and contact complementarity, consistent with their reduced binding and functional activity.<\/p>\n Graphing and statistical analyses<\/p>\n All graphs were generated and analysed using GraphPad Prism 10.0 (GraphPad Software). Dose\u2013response curves were fitted to a log(agonist) versus response model with parameters for span, baseline, and half-maximal effective concentration (EC50), and minimum baseline was corrected to zero. For statistical comparisons, one-way ANOVA with Dunnett\u2019s post hoc test was generally used for comparisons across more than two groups, and two-way ANOVA with Sidak\u2019s post hoc test or similar was used for comparisons involving multiple conditions, as specified in the figure legends. Most experiments were conducted with three biological replicates, and additional replicates served as controls. Replicates in the figure legends refer to biological replicates, with technical replicates included in some experiments for intra-replicate variation. Differences with P values\u2009<\u20090.05 were considered significant. Further statistical details and replicate information are provided in the figure legends.<\/p>\n Inclusion and ethics statement<\/p>\n All authors meet the authorship criteria required by Nature Portfolio journals and contributed meaningfully to the study. Authorship was determined collaboratively and was not influenced by gender, seniority, or institutional affiliation. Roles were agreed upon in advance, and the work was conducted responsibly and ethically, following institutional standards and inclusive research practices.<\/p>\n Reporting summary<\/p>\n