Genetically encoded intrabody sensors report the interaction and trafficking of β-arrestin 1 upon activation of G protein – coupled receptors

Agonist stimulation of G protein–coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called β-arrestins (βarrs). The GPCR–βarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR–βarr complex formation can be used as a generic readout of GPCR and βarr activation. Although several methods are currently available to monitor GPCR–βarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated βarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR–βarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to singlechain variable fragments (ScFvs) and used them to monitor the localization and trafficking of βarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of βarr1, and the intrabodies co-localized with βarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report βarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for βarr1 recruitment and trafficking expands currently available approaches to visualize GPCR–βarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.


Abstract
Agonist stimulation of G protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called β-arrestins (βarrs). The GPCR-βarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-βarr complex formation can be used as a generic readout of GPCR and βarr activation. Although several methods are currently available to monitor GPCR-βarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated βarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp).
Here, we demonstrate that these FABs efficiently report the formation of a GPCR-βarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments (ScFvs) and used them to monitor the localization and trafficking of βarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of βarr1, and the intrabodies co-localized with βarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report βarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for βarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-βarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.

Introduction
G protein-coupled receptors (GPCRs) recognize a diverse set of ligands and initiate a broad spectrum of downstream signaling responses (1). Upon agonist-stimulation, GPCRs couple to three major sub-families of cellular proteins namely, the heterotrimeric G-proteins, GRKs (GPCR kinases) and β-arrestins (βarrs) (1). Of these, βarrs are multifunctional adaptor proteins, which play a central role in regulatory and signaling paradigms of GPCRs (2,3). βarrs are evenly distributed in the cytoplasm under basal condition, and upon agonist-stimulation, they typically translocate to the plasma membrane to interact with activated and phosphorylated receptors (4).
Binding of βarrs to GPCRs at the plasma membrane results in termination of G-protein coupling and desensitization of receptors through a steric hindrance based mechanism (5). Subsequently, βarrs either dissociate from the receptors and re-localize back in the cytoplasm, or they traffic into endosomal vesicles in complex with the receptors (2,4). These two different patterns are referred to as "class A" and "class B", respectively (4). βarrs also contribute in a number of downstream GPCR signaling pathways such as ERK1/2 MAP kinases activation although strict G-protein independence of such mechanisms are currently being discussed and debated (6)(7)(8)(9).
Considering the multifaceted roles of βarrs, understanding the details of their interaction with GPCRs continues to be a frontier area in GPCR research (10). The interaction of βarrs with GPCRs involves two distinct components (11,12). One is receptor phosphorylation, primarily in the carboxylterminus but also in the intracellular loops, and the other is the intracellular side of receptor transmembrane bundle, referred to as receptor core (11,12). There are several assays that are currently used to measure GPCR-βarr interaction including those based on resonance energy transfer (13)(14)(15), enzyme complementation (16) and reporter responses (17,18). Still however, developing novel sensors is desirable to expand the currently available toolbox and complement the existing assays.
Previous studies have suggested that receptor phosphorylation is not only sufficient to promote βarr binding but it can also induce βarr conformations capable of mediating receptor endocytosis and signaling (19)(20)(21). These findings raise the possibility that biochemical reagents such as antibodies, which selectively recognize βarr conformation triggered by the interaction of phosphorylated receptor, may serve as sensors for βarr recruitment and trafficking. Here, we develop and characterize intrabody sensors derived from synthetic antibody fragments (FABs) against βarr1 that report the formation of GPCR-βarr1 complexes and allow us to monitor βarr1 trafficking in cellular context.

Synthetic antibody fragments report the formation of β2V2R-βarr1 complex
Agonist-induced receptor phosphorylation is a key determinant for βarr recruitment (11). A phosphopeptide corresponding to the carboxylterminus of the human vasopressin V2 receptor, referred to as V2Rpp, has been used extensively as a surrogate to induce active βarr conformation in-vitro (22)(23)(24)(25). We have previously generated and characterized a set of synthetic antibody fragments (FABs) that selectively recognize V2Rpp-bound βarr1 (26). We have also used one of these FABs, referred to as Fab30, to monitor the interaction of βarr1 with a chimeric β2 adrenergic receptor harboring V2R carboxyl-terminus (referred to as β 2 V 2 R) and V 2 R (25). As the first step towards developing these FABs as potential sensors of GPCR-βarr interaction and trafficking, we first confirmed their ability to report the formation of β2V2R-βarr1 complex in-vitro ( Figure 1A-D). Here, we used lysates from cells expressing FLAG-β 2 V 2 R mixed with purified βarr1 and FABs, followed by co-immunoprecipitation (co-IP) and detection of the receptor as readout of complex formation. We observed that Fab30, and the additional FABs, selectively pull-down β2V2R upon agonist-stimulation through the formation of receptor-βarr1 complex ( Figure 1A-D). A control FAB that does not interact with βarr1 failed to yield any detectable signal in the co-IP experiment ( Figure 1A-B).

Fab30 reports the formation of βarr1 complex for multiple chimeric GPCRs
Before proceeding to generate potentially generic intrabody sensors from these FABs, we evaluated their ability to recognize βarr1 complex with other GPCRs. Considering that these FABs were selected against V2Rpp-bound βarr1, we reasoned that they should detect βarr1 complex for other chimeric GPCRs harboring the V2R carboxyl-terminus, similar to that in β2V2R. We generated six different chimeric GPCRs including the members from different sub-classes such as chemokine (CCR2-V 2 R), adrenergic (α2B-V 2 R), Complement (C5aR1-V2R), muscarinic (M5-V2R) and dopamine (D2-V2R and D5-V2R) receptors. Some of these receptors such as M5R, α2BR and D2R contain large 3 rd intracellular loop (ICL3) while others have relatively shorter ICL3. We tested the ability of Fab30, which was most effective among all the FABs, to report the formation of receptor-βarr1 complex in co-IP assay for these receptors. As presented in Figure 2A-F, we observed that Fab30 efficiently recognized βarr1 for every chimeric GPCR tested here, similar to that of β2V2R. This finding allowed us to conceive that these FABs should work as generic intrabody sensors of βarr1 interaction and trafficking in cellular context for a broad set of chimeric GPCRs.

Conversion of FABs into intrabodies and their expression analysis
In order to develop these FABs into cellular sensors of βarr1 activation and trafficking, it is required to express them in functional form in the cytoplasm as intrabodies. We therefore converted the selected FABs into ScFvs (single chain variable fragments) by connecting the variable domains of their heavy and light chains through a previously optimized flexible linker (12), and then expressed them in HEK-293 cells as intrabodies, either with a carboxyl-terminal HA tag or as YFP fusion ( Figure 3A-C). We observed robust expression of two of these intrabodies namely intrabody30 (Ib30) and intrabody4 (Ib4) in HEK-293 cells while others displayed relatively weaker expression ( Figure  3B). For YFP-tagged intrabodies, we observed cytoplasmic as well as nuclear localization ( Figure 3C). The underlying reason for nuclear localization of the intrabodies is not apparent to us although a previous study has also reported nuclear localization of an intrabody targeting β2 adrenergic receptor (27).

Ib30 and Ib4 report the interaction of βarr1 with β2V2R and trafficking
We next tested whether intrabodies can report the formation of receptor-βarr1 complex in cellular context. We first co-expressed β2V2R, βarr1 and HA-tagged intrabodies in HEK-293 cells, stimulated the cells with either an agonist (Isoproterenol) or inverse-agonist (carazolol) and immunoprecipitated the intrabodies using the HA tag. We observed that both intrabodies i.e. Ib30 and Ib4 recognized β2V2R-βarr1 complex upon agonist-stimulation although Ib30 was relatively more efficient ( Figure 4A-B). We also tested the ability of Ib30 to recognize β2V2R-βarr1 complex formed upon stimulation of the receptor with a set of ligands with varying efficacies. Importantly, we observed that the level of recognition of the β2V2R-βarr1 complex by Ib30 mirrors the efficacy of the ligands ( Figure 4C-D). This observation underscores the ability of Ib30 to report the formation of pharmacologically relevant receptor-βarr1 complex and corroborates its suitability as a reliable sensor of receptor-βarr1 interaction.
In order to probe the utility of intrabodies to monitor βarr1 trafficking upon receptor stimulation, we co-expressed β2V2R, βarr1-mCherry and YFP-tagged intrabodies in HEK-293 cells, and followed the localization of βarr1 and intrabodies using confocal microscopy after agonist treatment ( Figure 4E-F). As expected, activation of β2V2R resulted in a typical "class B" pattern of βarr1 translocation, and interestingly, the intrabodies followed the localization of βarr1 and displayed robust colocalization ( Figure 4E-F). We observed that Ib30 and Ib4 were first translocated to the cell surface from the cytoplasm, and upon sustained agonist-stimulation, they were localized in the intracellular vesicles. Taken together, these findings demonstrate the usefulness of intrabodies as yet another tool to monitor the formation of receptor-βarr1 complex in-vitro and βarr1 trafficking in the cellular context.
Intrabodies also report the interaction and trafficking of βarr1 upon V2R stimulation As the intrabodies are derived from FABs selected against V2Rpp-bound βarr1, we anticipated that they should be able to report agonist-induced βarr1 interaction and trafficking for V2R as well. Accordingly, we tested the ability of Ib30 and Ib4 to detect the formation of V2R-βarr1 complex in-vitro, and report agonist-induced translocation of βarr1 in cellular context ( Figure 5A-E). We observed a pattern very similar to that of β2V2R described above in both, the co-immunoprecipitation experiment and confocal microscopy ( Figure  5A-E). That is, Ib30 and Ib4 selectively recognized V2R-βarr1 complex upon agoniststimulation, and followed the localization pattern of βarr1 upon agonist-stimulation as reflected by translocation to the cell surface first followed by localization in intracellular vesicles. An additional band was observed on the Western blot in the co-IP experiment, which migrates below the V2R band, but its origin is currently not clear to us.
We also measured the ability of Ib30 to recognize endogenous βarr1 upon agoniststimulation of V2R, and observed a robust interaction in co-immunoprecipitation assay ( Figure 6A-B). Furthermore, we evaluated the translocation pattern of Ib30-YFP upon agoniststimulation for β2V2R and V2R in HEK-293 cells where βarr1 is overexpressed without any modification. As presented in Figure 6C, Ib30-YFP was robustly localized to intracellular vesicles after agonist-stimulation, which is reminiscent of typical translocation pattern of βarr1 for these receptors. These data further strengthen the utility of intrabody sensors described here in monitoring βarr1 recruitment and trafficking.

Intrabodies do not alter βarr recruitment, receptor endocytosis, G-protein coupling and ERK1/2 phosphorylation
In order for the intrabodies to be reliable sensors of βarr recruitment and trafficking, it is important that they do not significantly alter βarr recruitment, receptor endocytosis and Gprotein coupling. Therefore, we first measured agonist-induced recruitment of βarr1 to V2R in presence of either a control intrabody (Ib-CTL) or Ib30/Ib4 using an intermolecular BRET assay. As presented in Figure 7A, we did not observe any significant difference in βarr1 recruitment. Next, in order to probe whether V2R is colocalized with Ib30 and βarr1 on intracellular vesicles, we performed three-color confocal imaging on HEK-293 cells expressing Flag-V2R, βarr1-YFP and Ib30-HA after agonist-stimulation ( Figure 7B). Expectedly, we observed a robust co-localization of V2R, βarr1 and Ib30 on intracellular vesicles suggesting that Ib30 does not alter the normal trafficking pattern of receptor-βarr1 complex in cellular context. This is further corroborated by the pattern of V2R colocalization with the early endosomal markers EEA1 and APPL1 which remains unaltered in presence of Ib-CTL vs. Ib30 ( Figure  7C-D). Furthermore, we also measured βarr1 trafficking to endosomes upon V2R activation using an enhanced bystander BRET (ebBRET) set-up (15) in presence of either Ib-CTL or Ib4/Ib30. Although we did not observe a significant difference in EC50 values ( Figure 7E), Ib4/Ib30 appear to stabilize endosomal localization of βarr1 as reflected by ΔBRET signal ( Figure 7F). This observation is particularly relevant if the intrabody sensors are used in the context of receptor recycling where they might slow-down receptor recycling to the plasma membrane, and it would be interesting to probe this aspect further in future studies. We next measured the effect of intrabodies on Gαs-coupling to the V2R using cAMP response as readout. Once again, we did not observe any significant difference in cAMP dose response or time-kinetics for Ib-CTL vs Ib30/Ib4 conditions ( Figure 8A-B). Finally, we also evaluated the effect of intrabodies on agonist-induced ERK1/2 MAP kinase activation, a prototypical readout of V2R signaling, and did not detect a significant alteration by the intrabodies (Figure 8C-D). Taken together, these data establish that intrabodies do not have a major effect on transducer coupling and receptor endocytosis making them suitable sensors to record βarr1 interaction and trafficking for GPCRs.

Ib30 as a generic sensor of agonist-induced βarr1 trafficking for multiple chimeric GPCRs
Taking lead from the ability of Fab30 to recognize βarr1 complex with several chimeric GPCRs as presented in Figure 2, we next evaluated Ib30 as a sensor to report βarr1 trafficking for these chimeric GPCRs in cellular context. Similar to previous experiments, we coexpressed the chimeric receptors with βarr1-mCherry and Ib30-YFP in HEK-293 cells, and followed the localization of βarr1 and intrabodies using confocal microscopy after agonist treatment ( Figure 9A-F). We observed that similar to β2V2R, Ib30 followed βarr1 translocation pattern by first localizing to the cell surface followed by trafficking into intracellular vesicles for all of these chimeric receptors ( Figure 9A-F). It is worth noting here that the receptors used in Figure 9A-C contain most of the phosphorylation sites in their carboxyl-terminus while their 3 rd intracellular loops are relatively small. On the other hand, receptors included in Figure 9D-F, harbor a larger 3 rd intracellular loop, which also contains most of the potential phosphorylation sites, and their carboxyl-terminus is relatively smaller. Therefore, the data presented in Figure 9 not only demonstrate the generality of Ib30 as a sensor to monitor agonist-induced βarr1 recruitment and trafficking for chimeric GPCRs but also its versatility for receptors differing in terms of their carboxyl-terminus and intracellular loops.

Ib30 sensor suggests conformational diversity in GPCR-βarr1 complexes
Finally, we evaluated the ability of Ib30 sensor to report the trafficking of βarr1 for a set of GPCRs without the fusion of V2R-tail. We observed that Ib30-YFP followed agonistinduced translocation pattern of βarr1 for several different receptors including the complement C5a receptor 1 (C5aR1), the neurotensin receptor 1 (NTSR1), the muscarinic acetylcholine receptor subtype 2 (M2R), and the atypical chemokine receptor subtype 2 (ACKR2) ( Figure 10A-D). We also validated the ability of Ib30 to recognize receptor-bound βarr1 for C5aR1 and ACKR2 by coimmunoprecipitation experiment ( Figure 10E-F). These findings suggest that Ib30 can act as a sensor for monitoring agonist-induced βarr1 translocation for at least some GPCRs with their native carboxyl-terminus as well. Interestingly however, we observed that Ib30 did not robustly follow βarr1 translocation for the bradykinin subtype 2 receptor (B2R) upon agonist-agonist-stimulation ( Figure  10G) although there was clear translocation of βarr1, first to the plasma membrane and then in intracellular vesicles. Taken together, these data potentially hint at conformational differences in GPCR-βarr1 complexes, even if the overall recruitment patterns are apparently similar. Future studies focused on measuring conformational differences in different GPCRβarr complexes may provide additional insights and possibly link the conformational diversity to functional outcomes.

DISCUSSION
Monitoring βarr interaction and subsequent trafficking has been used extensively to study the activation and regulatory framework of GPCRs. A number of approaches are commonly utilized for this including direct fusion of fluorescent proteins to βarrs (4), resonance energy transfer (FRET/BRET) based assays (14,28), enzyme complementation methods (16) and reporter assays (17,18). Each of these methods necessitates a significant engineering and modification of the receptor, the βarr, or both. Intrabody sensors described here recognize receptor-bound βarr1 and report its trafficking in cellular context without the need for any modification of βarr1.
Although we observe that the intrabody sensors are capable of recognizing βarr1 for several GPCRs without the modification of their carboxyl-terminus, a potential drawback is that they are not likely to be universal for every GPCR as reflected for B2R in Figure 10G. On the other hand, these intrabody sensors are able to recognize βarr1 more generally in the context of chimeric GPCRs harboring V 2 R carboxylterminus. It is conceivable that a similar strategy can be employed for other GPCRs as well by using, for example, phosphopeptides derived from the corresponding receptors. It is also worth noting here that many of the βarr1 assays such as PRESTO-TANGO also utilize chimeric GPCRs with V2R carboxyl-terminus (V2R tail) (18). Engineering V2R tail typically imparts "class B" pattern on GPCRs and thereby, makes the detection of βarr1 interaction more robust compared to the unmodified receptors (29). It is also important to note that out of five different FABs tested here, only two expressed efficiently as intrabodies in the cytoplasm. Therefore, starting with a larger number of FABs may be desirable to obtain more functional intrabodies in future endeavors.
Considering that YFP fusion does not alter the ability of intrabodies to interact with βarr1 and follow their translocation, it is also conceivable that they can be adapted in resonance energy transfer assays, or even in NanoBit format, for quantitative measurements of receptor-βarr1 interaction. Such strategies may yield even more sensitive versions of these intrabody sensors compared to approaches utilized here. In addition, while the intrabody sensors developed here are specific to βarr1 (25), it is plausible to design and develop similar intrabodies for βarr2 as well. Such an effort may help uncover novel insights into the functional divergence of the two βarr isoforms (30).
Another interesting aspect of GPCR-βarr1 interaction is the ability of differential receptor phosphorylation patterns to induce distinct functional conformations in βarrs (31,32). For several GPCRs, different phosphorylation patterns arising in ligand-specific, cell-type specific and kinase-specific manners have been mapped and correlated with βarr mediated functional outcomes (33)(34)(35). Thus, it is tantalizing to hypothesize that intrabodies designed against different phosphopeptides derived from a given receptor may illuminate interesting attributes of receptor signaling and regulation in future.
In conclusion, our study expands the currently available toolbox to monitor GPCRβarr interaction and trafficking, and the intrabody sensors described here should facilitate drawing novel insights into GPCR signaling and regulatory paradigms.

Co-immunoprecipitation assay
In order to probe the reactivity of FABs towards β2V2R (Figure 1), Sf9 cells expressing FLAGtagged receptor were lysed and incubated with purified βarr1 and FABs. For the co-IP data presented in Figure 2, the plasmids encoding FLAG-tagged receptor and βarr1 were transfected in HEK-293 cells. 48h posttransfection, cells were serum-starved for 4-6h, stimulated with agonist, lysed by douncing and incubated with FAB30 for 1h at room temperature. Subsequently, the receptor-βarr1-FAB complex were solubilized with 1% MNG for 1h, centrifuged to collect the clarified solubilized complex, and 20μl of preequilibrated (in 20mM HEPES, 150mM NaCl, pH 7.4 buffer) Protein L beads (GE Healthcare) were added. After additional 1h incubation, beads were washed three times with wash buffer (20mM HEPES, 150mM NaCl, pH 7.4, 0.01% MNG) and eluted with 2XSDS loading buffer. Eluted samples were run on 12% SDS-PAGE, and the receptors were detected using HRP-coupled anti-FLAG M2 antibody while the FABs were visualized using Coomassie staining.

Confocal microscopy
In order to monitor the translocation of βarr1 and intrabodies by confocal microscopy ( Figure  3C-D, Figure 4E-F, Figure 5C-E, Figure 6C, Figure  9A-F, Figure 10A-D and Figure 10G), HEK-293 cells were transfected with plasmids encoding the indicated receptor, βarr1-mCherry and YFPtagged intrabodies. 24h post-infection, cells were seeded onto confocal dishes (GenetiX; Cat. No. 100350) pretreated with 0.01% poly-Dlysine (Sigma). After another 24h, cells were serum-starved for 4-6h prior to stimulation with saturating concentration of indicated agonists. For live cell confocal imaging, we used Zeiss LSM 710 NLO confocal microscope and samples were housed on a motorized XY stage with a CO2 enclosure and a temperature-controlled platform equipped with 32x array GaAsP descanned detector (Zeiss). YFP was excited with a diode laser at 488 nm laser line while mcherry was excited at 561 nm. Laser intensity and pinhole settings were kept in the same range for parallel set of experiments and spectral overlap for any two channels was avoided by adjusting proper filter excitation regions and bandwidths. Images were scanned using the line scan mode and images were finally processed in ZEN lite (ZEN-blue/ZENblack) software suite from ZEISS. Colocalization was analyzed by calculating Pearson's correlation coefficient (PCC) between the indicated channels using JACoP plugin in ImageJ software (37). At least three regions of interest (ROIs) per cell were analyzed and the mean±SEM of PCC are presented in the respective figure legends together with the number of cells and independent experiments.
For three color imaging ( Figure 7B) and colocalization with early endosomal markers ( Figure 7C), receptor imaging of live or fixed cells was monitored by ''feeding'' cells with anti-FLAG antibody (15 min, 37°C) in phenolred-free DMEM prior to agonist treatment. Fixed cells were washed three times in PBS/0.04% EDTA to remove FLAG antibody bound to the remaining surface receptors, fixed using 4% PFA (20 min at RT), permeabilized and stained using HA primary antibody followed by Alexa-Fluor 555 or 647 secondary antibodies. For co-localization of FLAG-V2R with endosomal markers, cells were treated as above except incubated with either of the following primary antibodies post-permeabilization; EEA1 (rabbit anti-EEA1 antibody from Cell Signaling Technology) or APPL1 (rabbit anti-APPL1 antibody from Cell Signaling Technology). Cells were imaged using a TCS-SP5 confocal microscope (Leica) with a 63x 1.4 numerical aperture (NA) objective and solid-state lasers of 488 nm, 561 nm, and/or 642 nm as light sources. Leica LAS AF image acquisition software was utilized. All subsequent raw-image tiff files were analyzed using ImageJ or LAS AF Lite (Leica), and colocalization was measured by calculating the Pearson's correlation coefficient (PCC) using JACoP plugin in ImageJ software as mentioned above.

GloSensor assay and ERK1/2 phosphorylation
In order to measure the effect of intrabodies on Gαs-coupling, if any, we measured agonistinduced cAMP response in GloSensor assay following a previously described protocol (25). Briefly, HEK-293 cells were transfected with plasmids encoding the V2R, the luciferase-based cAMP biosensor (pGloSensorTM-22F plasmid) and the intrabodies. 16h post-transfection, media was aspirated, cells were flushed and pooled together in assay buffer containing 1X Hanks balanced salt solution, pH 7.4 and 20 mM of 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid [HEPES]. Cell density was measured and adjusted such as to yield approximately 125,000 cells in 100μl. Cells were pelleted at 2000rpm for 3min to remove the assay buffer and then the pellet was resuspended in the desired volume of sodium luciferin solution prepared in the same assay buffer. After seeding the cells in a 96 well plate, the plate was incubated at 37°C for 90min followed by an additional incubation of 30 min at room temperature. Subsequently, various doses of the indicated ligand were added to the cells, and the luminescence reading was recorded using a micro-plate reader (Victor X4; Perkin Elmer). Agonist-induced phosphorylation of ERK1/2 MAP kinase was measured by Western blotting following a previously described protocol (38).

BRET assay
For measuring arr1 recruitment and endosomal localization by BRET ( Figure 8A and 8D), transfections were performed on HEK-293 cells seeded (40,000 cells/100 µl/well) in 96well white micro-plates (Greiner) using PEI at a ratio of 4:1 (PEI: DNA). In order to monitor V2R-arr1 interaction, we used arr1-RlucII and V2R-YFP plasmids described previously (39). To monitor endosomal translocation of βarr1, we used enhanced bystander BRET (ebBRET) where the BRET acceptor (Renilla green fluorescent protein; rGFP), is fused to the FYVE domain from endofin protein targeted to early endosomes (rGFP-FYVE) and βarr1 fusion with the BRET donor Renilla luciferase II (RlucII) (40). 48h post-transfection, culture media was removed, cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline) and replaced by HBSS (Hank's Balanced Salt Solution). Afterwards, cells were stimulated with increasing concentrations of AVP for 10min and 2.5µM coelenterazine H (BRET1) or coelenterazine 400a (BRET2) was added 5min before BRET measurement. BRET signals were recorded on a Mithras (Berthold scientific) micro-plate reader equipped with the following filters: 480/20 nm (donor) and 530/20 nm (acceptor) for BRET1 or 400/70 nm (donor) and 515/20 nm (acceptor) for BRET2. The BRET signal was determined as the ratio of the light emitted by the energy acceptor over the light emitted by energy donor. Raw BRET values are presented in Figure 7A and 7E while agonistinduced change in BRET signal (BRET) obtained by calculating the difference in BRET values for the highest and lowest concentrations of AVP is presented in Figure 7F.

Statistical analysis and data presentation
Quantified data were plotted and analyzed using GraphPad Prism software, and the details of experimental replicates and statistical analysis are mentioned in the corresponding figure legends.

Data availability
All data are available in the manuscript.

38.
Kumari       stimulation. HEK-293 cells expressing V 2 R and HA-tagged Ib30/Ib-CTL were stimulated with either inverse agonist (Tolvaptan; 100nM) or agonist (AVP; 100nM) followed by co-immunoprecipitation (co-IP) using anti-HA antibody agarose. Subsequently, the proteins were visualized by Western blotting using anti-βarr and anti-HA antibodies. B. Densitometry-based quantification of the data in panel A presented as mean±SEM from three independent experiments normalized with maximal response (treated as 100%) and analyzed using One-Way-ANOVA (****p <0.0001). C. HEK-293 cells expressing β 2 V 2 R/V 2 R and Ib30-YFP were stimulated with isoproterenol (10μM) and AVP (100nM), respectively, and the localization of Ib30-YFP was visualized using confocal microscopy. Representative images from three independent experiments are shown here. Scale bar is 10 μm.    For ACKR2, we observed significant membrane localization of βarr1 and Ib30, even before agonist-treatment, which results into higher PCC values for unstimulated condition. E-F. HEK-293 cells expressing the C5aR1 and ACKR2, respectively, together with βarr1 and Ib30 were stimulated with either respective agonists (100nM) for indicated time-points followed by co-immunoprecipitation (co-IP) using protein L agarose beads. Subsequently, the proteins were visualized by Western blotting using anti-FLAG M2 antibody and anti-HA antibody. The right panels show densitometry-based quantification of four independent experiments normalized with signal at 30min (treated as 100%) and analyzed using One-

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Agonist -