Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for βarrs, agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of βarr translocation to endosomes although βarrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like βarr1 conformation, rescues endosomal translocation of βarr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in βarr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of βarr1 with β2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of βarr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery. Significance The interaction of G protein-coupled receptors (GPCRs) with β-arrestins (βarrs) is a critical step in their regulatory and signaling paradigms. While intrabodies that bind to GPCRs, G proteins and βarrs have been utilized as biosensors and regulators of functional outcomes, allosteric targeting of receptor-transducer complexes to encode gain of function has not been documented so far. Here, we discover that a conformation-specific synthetic intrabody recognizing GPCR-bound βarr1 can allosterically enhance endosomal trafficking of βarr1 and agonist-induced ERK1/2 MAP kinase activation. This intrabody promotes an active-like βarr1 conformation and enhances the interaction of β2-adaptin with βarr1. Our findings establish a conceptual framework to allosterically modulate protein-protein interactions in GPCR signaling cascade to modulate their trafficking and signaling responses.


Significance 24
The interaction of G protein-coupled receptors (GPCRs) with β-arrestins (βarrs) is a critical step in 25 their regulatory and signaling paradigms. While intrabodies that bind to GPCRs, G proteins and βarrs 26 have been utilized as biosensors and regulators of functional outcomes, allosteric targeting of 27 receptor-transducer complexes to encode gain of function has not been documented so far. Here, 28 we discover that a conformation-specific synthetic intrabody recognizing GPCR-bound βarr1 can 29 allosterically enhance endosomal trafficking of βarr1 and agonist-induced ERK1/2 MAP kinase 30 activation. This intrabody promotes an active-like βarr1 conformation and enhances the interaction 31 of β 2 -adaptin with βarr1. Our findings establish a conceptual framework to allosterically modulate 32 protein-protein interactions in GPCR signaling cascade to modulate their trafficking and signaling 33 responses. G protein-coupled receptors (GPCRs) recognize a broad spectrum of ligands and play a critical role in 70 nearly every aspect of human physiology (1, 2). These receptors remain a major class of targets for 71 novel drug discovery (3). The spatio-temporal aspects of GPCR signaling are tightly regulated by 72 multifunctional β-arrestins (βarrs) (4, 5). Agonist-induced phosphorylation of GPCRs is a key 73 determinant of βarr interaction and their ensuing functional outcomes (6, 7). While some GPCRs 74 interact transiently with βarrs at the plasma membrane followed by rapid dissociation, others 75 display a prolonged interaction resulting in endosomal trafficking of receptor-βarr complexes (8). 76 These two patterns of βarr interaction and trafficking have been used to categorize the 77 corresponding receptors as class A or class B GPCRs, respectively (8). Interestingly, distinct 78 phosphorylation patterns on GPCRs have been linked to different βarr conformations, which in turn 79 determine the resulting functional responses (9-11). While cumulative phosphorylation on GPCRs is 80 typically believed to determine the affinity of βarr interaction, emerging evidence now suggests that 81 spatial positioning of even single phosphorylation sites may make decisive contributions to βarr 82 recruitment and subsequent functional outcomes (10,11). 83 We previously reported that mutation of a single phosphorylation site in the vasopressin 84 receptor (V 2 R) at Thr 360 in the carboxyl-terminus (i.e., V 2 R T360A ) dramatically altered βarr trafficking 85 patterns (10). V 2 R is a class B receptor in terms of βarr interaction and trafficking where agonist-86 stimulation results in membrane recruitment of βarrs first followed by endosomal localization (8). 87 Interestingly, upon agonist-stimulation of V 2 R T360A , βarrs efficiently translocate to the plasma 88 membrane, but do not traffic to endosomal compartments unlike the wild-type receptor even after 89 prolonged agonist-exposure ( Figure 1A) (10). V 2 R T360A also exhibits reduced levels of ERK1/2 90 activation compared to the wild-type receptor without any measurable effect on G protein-coupling 91 as assessed by measuring cAMP production (10). This mutation leads to the disruption of a salt-92 bridge with Lys 294 in βarr1 and consequently, reduces the fraction of active βarr1 conformation as 93 assessed using molecular dynamics simulation (10). 94 In order to better understand this intriguing effect of Thr 360 Ala mutation in V 2 R, we set out 95 to probe the conformation of βarr1 in complex with this receptor mutant, and compare it with the 96 wild-type V 2 R, using a previously described intrabody30 (Ib30) based sensor (12). We observed that 97 Ib30 robustly recognizes βarr1 recruited to the plasma membrane upon agonist-stimulation of 98 V 2 R T360A . Surprisingly, we also found that Ib30 also promotes endosomal localization of βarr1 for 99 V 2 R T360A and rescues ERK1/2 MAP kinase activation to almost V 2 R WT levels. We also discovered that 100 Ib30 enriched the active-like conformational population of βarr1, and interestingly, also enhances 101 the interaction of βarr1 with β 2 -adaptin. These findings establish the capability of Ib30 to 102 allosterically modulate βarr1 trafficking and activation for V 2 R T360A , and potentially open a novel 103 paradigm to modulate GPCR signaling using designer proteins. 104 Results 105 βarr1 conformations induced by V 2 Rpp T360 phospho-peptides 106 In order to probe whether the absence of Thr 360 phosphorylation influences βarr1 conformation, we 107 first synthesized two phospho-peptides corresponding to V 2 R T360 mutation, and used a previously 108 described limited trypsin proteolysis assay (13) to compare βarr1 conformation induced by V 2 Rpp T360 109 phospho-peptides with that of V 2 Rpp WT . These two phospho-peptides, referred to as V 2 Rpp T360-1 and 110 V 2 Rpp T360-2 contain a non-phosphorylated Thr or Ala at position 360, respectively, while the rest of 111 the sequence and phosphorylation patterns are identical to V 2 Rpp (referred to as V 2 Rpp WT ) ( Figure  112 1B). Similar to a previous study (13), we observed that activation of βarr1 by V 2 Rpp WT resulted in an 113 accelerated cleavage of the 48kDa band (Gly -8 -Arg 418 ), protection of 47kDa and 45kDa bands (Leu 1 -114 Arg 418 and Leu 1 -Arg 393 , respectively) and appearance of a 21kDa band (Leu 1 -Arg 188 ) ( Figure 1C-D, 115 Figure S1A-B). Interestingly, we observed that V 2 Rpp T360 phospho-peptides also induced a proteolysis 116 pattern qualitatively similar to that observed for V 2 Rpp WT . Still however, there were noticeable 117 differences, including relatively slower proteolysis rates of the 47kDa band and weaker intensity of 118 the 21kDa band. This observation indicates that V 2 R T360 phospho-peptides are capable of binding 119 βarr1; however, they do not induce a fully-active βarr1 conformation as generated by V 2 R WT . 120 In order to further probe the conformation of βarr1 induced by V 2 Rpp WT vs. V 2 Rpp T360 121 phospho-peptides, we measured the ability of conformationally-selective Fab30/ScFv30 sensors to 122 recognize βarr1 conformation upon binding of these phospho-peptides by co-immunoprecipitation 123 (co-IP) (Figure 2A-B, Figure S2). Fab30 and ScFv30 selectively recognize an active conformation of 124 βarr1 induced by V 2 Rpp, and thus, have been used as conformational biosensors to monitor βarr 125 activation in vitro (12,14). We observed that Fab30/ScFv30 robustly interact with the V 2 Rpp T360-1/2 -126 βarr1 complex albeit at lower levels than V 2 Rpp WT (Figure 2A-B, Figure S2). We carried out this assay 127 in presence of either 10-fold or 50-fold molar excess of the phospho-peptides compared to βarr1 but 128 the reactivity patterns of Fab30/ScFv30 did not change significantly (Figure 2A-B, Figure S2). Similar 129 to the limited proteolysis data presented in Figure 1, these data also suggest that V 2 Rpp T360 phospho-130 peptides induce a conformation in βarr1, which is qualitatively similar to that of V 2 Rpp WT but not 131 identical. 132 In order to further probe βarr1 conformation induced by different phospho-peptides, we 133 carried out the limited proteolysis assay in presence of ScFv30 ( Figure 2C-D, Figure S3A). We 134 observed that the 47kDa band (Leu 1 -Arg 418 ) is significantly protected in the presence of ScFv30, and 135 the bands at 32kDa and 21kDa (Leu 1 -Arg 285 and Leu 1 -Arg 188 , respectively) did not appear ( Figure 2C-136 D, Figure S3A). Interestingly, the proteolysis patterns observed in presence of ScFv30 were nearly-137 identical for V 2 Rpp WT and V 2 Rpp T360 phospho-peptides although an additional band at ~30kDa was 138 observed only with V 2 Rpp WT (Figure 2C-D). Taken together with the co-immunoprecipitation data in 139 Figure 2A-B, these data suggest that V 2 Rpp T360 phospho-peptides bind to βarr1 and ScFv30 140 potentially facilitates the transition of V 2 Rpp T360 -bound βarr1 toward an active-like conformation. 141 In order to gain further insights into βarr1 conformation, we analyzed the crystal structures 142 of βarr1 in basal, V 2 Rpp WT -and V 2 Rpp T360 -bound states. As the distal carboxyl-terminus of βarr1 is 143 not resolved in these structures, we focused primarily on Arg 285 and Arg 188 , which are the trypsin 144 cleavage sites yielding the 32kDa (Leu 1 -Arg 285 ) and 21kDa (Leu 1 -Arg 188 ) bands, respectively. Both of 145 these residues exhibit a reorientation of their side chains between the apo and phospho-peptide-146 bound conformations (Figure 2E-F). Furthermore, an analysis of the local interaction networks of 147 Arg 188 and Arg 285 calculated through CONTACT/ACT program within the CCP4 suite (15) revealed 148 differences between the V 2 Rpp WT vs. V 2 Rpp T360 -bound conformations ( Figure 2E-F, Figure S3B). 149 These structural differences provide a plausible explanation for the proteolysis patterns obtained for 150 V 2 Rpp WT vs. V 2 Rpp T360 , and support the hypothesis that V 2 R T360 induces an intermediate conformation 151 in βarr1 compared to apo-and V 2 R WT -bound state. 152 Intrabody30 rescues endosomal trafficking of βarr1 and ERK1/2 activation for V 2 R T360A 153 The experiments presented so far were carried out using isolated phospho-peptides in vitro. Thus, 154 we set out to measure the reactivity of Ib30, an intrabody derived from Fab30, that efficiently 155 recognizes active βarr1 in the cellular context, and also reports on βarr1 trafficking upon agonist-156 stimulation (11,12). We first co-expressed SmBiT-βarr1 and LgBiT-Ib30 constructs with V 2 R WT and 157 V 2 R T360A and measured agonist-induced changes in luminescence signal as a readout of βarr1-Ib30 158 interaction ( Figure 3A). We observed a robust interaction of βarr1 and Ib30 upon agonist-stimulation 159 of both, V 2 R WT and V 2 R T360A , suggesting that the conformation of βarr1 is qualitatively similar in the 160 cellular context, at least as reported by Ib30 sensor (Figure 3B, Figure S4A). We did not observe any 161 measurable effect of Ib30 on G protein-mediated cAMP responses in case of V 2 R T360A , similar to 162 V 2 R WT , suggesting that the intrabody does not significantly influence agonist-induced G αs coupling 163 ( Figure S5A-B). 164 In order to directly visualize the ability of Ib30 to recognize βarr1 upon recruitment to 165 V 2 R T360A , we co-expressed Ib30-mYFP construct together with βarr1-mCherry in HEK-293 cells 166 expressing V 2 R T360A , and monitored the localization of βarr1 and Ib30 by confocal microscopy. Ib30 167 translocated to the plasma membrane upon agonist-stimulation, similar to βarr1, and exhibited 168 robust colocalization with βarr1 ( Figure 3C-D), in accord with the NanoBiT data presented in Figure  169 3A-B. Surprisingly however, we also observed that upon prolonged agonist-exposure (10-30min), 170 both, βarr1 and Ib30 translocated to the endosomal vesicles and robustly co-localized ( Figure 3C-D). 171 This was unanticipated as βarr1 fails to translocate to endosomal vesicles even upon sustained 172 agonist stimulation for V 2 R T360A as reported previously (10). This observation led us to hypothesize 173 that Ib30 may potentially modulate the trafficking pattern of βarr1 for V 2 R T360A leading to endosomal 174 localization of βarr1. 175 In order to test this, we co-expressed βarr1-mYFP construct in HEK-293 cells expressing 176 either V 2 R WT or V 2 R T360A in presence or absence of HA-tagged Ib30. We monitored the localization of 177 βarr1 in these cells upon agonist-simulation and scored the localization pattern of βarr1 in terms of 178 plasma membrane vs. internalized vesicles. In line with data presented in Figure 3C To further corroborate these findings, we used an intermolecular bystander BRET assay to 183 monitor endosomal localization of βarr1 by using βarr1-R-Luc and GFP-FYVE constructs described 184 previously ( Figure 4A) (16). As shown in Figure 4B, we observed very low level of agonist-induced 185 BRET for V 2 R T360A in the presence of control intrabody (Ib-CTL) while V 2 R WT exhibited a robust 186 response as expected. Interestingly however, co-expression of Ib30 rescues the BRET signal (i.e., 187 endosomal trafficking of βarr1) to almost the same level as V 2 R WT ( Figure 4B). We also observed an 188 enhanced E max in BRET assay for V 2 R WT in the presence of Ib30, compared to Ib-CTL, although basal 189 BRET was also slightly higher. Taken together with the confocal microscopy observations, these data 190 establish that Ib30 not only recognizes V 2 R T360A -bound βarr1 conformation but also robustly 191 promotes its trafficking to endosomal vesicles and thereby, rescuing the trafficking pattern of βarr1 192 similar to the wild-type receptor. 193 We have previously reported that agonist-induced ERK1/2 MAP kinase activation is 194 significantly attenuated for V 2 R T360A compared to V 2 R WT . In order to measure whether Ib30 also 195 modulates agonist-induced ERK1/2 activation, we compared ERK1/2 responses for V 2 R WT and V 2 R T360A 196 with the control intrabody and Ib30 co-expression conditions. While Ib30 did not have a significant 197 effect on ERK1/2 activation for the V 2 R WT , it robustly enhanced the level of phosphorylated ERK1/2 198 upon agonist-stimulation, nearly to that of the V 2 R WT (Figure 4C-D, Figure S4B). Taken together with 199 the endocytosis data, these findings demonstrate an allosteric effect of Ib30 to positively modulate 200 βarr-mediated responses for the V 2 R T360A mutant in the cellular context. 201

Structural insights into βarr1 conformation and allosteric effect of Fab30 202
In order to better understand differences in the interaction pattern and binding mode of V 2 Rpp WT vs. 203 V 2 Rpp T360 , if any, we analyzed the crystal structures of V 2 Rpp WT -βarr1 (PDB: 4JQI) and V 2 Rpp T360-1 204 (PDB: 7DFA). Interestingly, a segment of the V 2 Rpp T360-1 containing residues Pro 353 to Thr 360 shows a 205 marked repositioning compared to the V 2 Rpp WT binding pose ( Figure 5A). In the V 2 Rpp WT -βarr1 206 crystal structure, pThr 360 engages Lys 294 , Lys 11 and Arg 25 in βarr1 through ionic interactions, which is 207 expectedly absent in case of V 2 Rpp T360 mutation. Of these, Lys 294 in the lariat loop and Lys 11 in the β-208 strand I of βarr1 are particularly noteworthy as they constitute a key part of the polar core and 209 phosphate sensor, respectively. These interactions are critical in the process of βarr1 activation upon 210 binding of phosphorylated carboxyl-terminus of GPCRs. Interestingly, pThr 359 in V 2 Rpp T360 phospho-211 peptide engages with Lys 11 but not with Lys 294 or Arg 25 . This interesting structural rearrangement 212 may in part explain an intermediate active-like conformation induced by V 2 Rpp T360 phospho-peptides 213 as observed in limited proteolysis and ScFv30 co-IP assay. 214 Next, in order to understand the effect of ScFv30 on βarr1 conformation in the context of 215 V 2 R T360 mutation, we used molecular dynamics (MD) simulation on V 2 Rpp WT -and V 2 Rpp T360A -bound 216 βarr1. We have previously reported that Thr 360 to Ala mutation results in a significant shift in the 217 population of βarr1 towards inactive-like conformation compared to the V 2 Rpp WT as assessed in 218 terms of the inter-domain rotation angle (10). In this study, we reproduce this behavior of V 2 Rpp T360A 219 compared to V 2 Rpp WT demonstrating that introducing Thr 360 Ala mutation into the V 2 Rpp-βarr1 220 complex leads to a dramatic shift towards inactive-like conformations with an inter-domain rotation 221 angles < 15° ( Figure 5B, V 2 Rpp WT : 36% vs. V 2 Rpp T360A : 58%). Strikingly, simulation of the V 2 Rpp T360A -222 βarr1 complex in presence of Fab30 demonstrates that antibody binding increases the population of 223 active-like βarr1 conformations ( Figure 5B, V 2 Rpp T360A +Fab30: 80% vs. V 2 Rpp T360A : 42%). Taken 224 together, these data underline the ability of ScFv30/Ib30 to promote active-like conformation in 225 βarr1 in the context of Thr 360 Ala mutation, and provide a plausible mechanism for the positive 226 allosteric effect of Ib30 on βarr1 trafficking to endosomes and ERK1/2 activation. 227

Intrabody30 enhances βarr1-β 2 -adaptin interaction 228
Next, we set out to identify a potential functional correlate of Ib30-induced enrichment of active-like 229 βarr1 conformation, and to reveal the mechanism of Ib30-mediated endosomal targeting of βarr1. 230 As the interaction of βarrs with β 2 -adaptin is a prominent mechanism that drives GPCR endocytosis, 231 we measured the effect of Ib30 on βarr1-β 2 -adaptin interaction. We used ear-domain of β 2 -adaptin 232 (592-951) tagged with GST at the N-terminus and assessed its interaction with βarr1 in presence of 233 V 2 R T360A . Here, we co-expressed βarr1 and V 2 R T360A in HEK-293 cells, prepared cellular lysates after 234 agonist stimulation, and used it for co-immunoprecipitation experiments. As presented in Figure 6A-235 B, we observed low but statistically significant interaction, compared to GST-alone control, between 236 βarr1 and β 2 -adaptin in presence of Ib-CTL. Interestingly, the presence of Ib30 enhances this 237 interaction several fold suggesting the ability of Ib30 to promote βarr1-β 2 -adaptin interaction ( Figure  238

6A-B). 239
In order to further corroborate this interesting finding in cellular context, we next used a 240 previously described BRET-based assay (17) to monitor agonist-induced interaction of βarr1 with β 2 -241 adaptin in the absence and presence of Ib30 for V 2 R WT and V 2 R T360A ( Figure 6C). There was robust 242 interaction between βarr1 and β 2 -adaptin for the V 2 R WT upon agonist-stimulation in the presence of 243 control intrabody, while the response was significantly lower for V 2 R T360A , which is in line with 244 significantly less endosomal trafficking of βarr1. Interestingly, co-expression of Ib30 significantly 245 enhanced βarr1-β 2 -adaptin interaction for V 2 R T360A , bringing to almost to the same level as V 2 R WT , 246 although basal BRET was also higher under Ib30 co-expression conditions for both, V 2 R WT and 247 V 2 R T360A . The enhanced BRET signal for V 2 R T360A in presence of Ib30 provides a plausible mechanism 248 for its ability to positively influence βarr1 trafficking to endosomes. Moreover, the elevated basal 249 BRET in the presence of Ib30 is also intriguing and it may result from the propensity of Ib30 to 250 enhance the interaction between βarr1 and β 2 -adaptin, even under basal conditions. In order to test 251 this hypothesis, we carried out a titration experiment, where we expressed Ib30 at increasing levels 252 and assessed βarr1-β 2 -adaptin interaction in the BRET assay. As presented in Figure 6D, increasing 253 expression of Ib30 indeed enhanced BRET in a saturable manner suggesting the ability of Ib30 to 254 promote basal interaction between βarr1 and β 2 -adaptin. Taken together, these observations 255 provide a mechanistic basis of Ib30-induced allosteric modulation of βarr1 trafficking pattern 256 observed for V 2 R T360A . 257

Discussion 258
In this study, we demonstrate that a synthetic intrabody (Ib30) can allosterically modulate agonist-259 induced trafficking patterns of βarr1 for V 2 R T360A lacking a key phosphorylation site in its carboxyl-260 terminus. Ib30 imparts this transition from a class A to a class B trafficking pattern by enriching the 261 fraction of active-like conformational populations of βarr1, and allosterically enhancing βarr1-β 2 -262 adaptin interactions. Moreover, Ib30 also rescues the reduced ERK1/2 activation for V 2 R T360A to levels 263 induced by the wild-type receptor. A previous study has demonstrated a critical role of βarr-β 2 -264 adpatin interaction in βarr-mediated ERK1/2 activation for V 2 R using a small molecule inhibitor of 265 this interaction (17). Therefore, an increase in βarr1-β 2 -adaptin interaction in presence of Ib30 may 266 provide a plausible mechanism for its ability to rescue agonist-induced ERK1/2 activation for V 2 R T360A . 267 However, additional mechanisms may also contribute to this intriguing observation, and it would be 268 interesting to probe this further in subsequent studies. While previous studies have used intrabodies 269 as biosensors of GPCR activation (18), βarr trafficking (12), inhibitors of GPCR endocytosis (19) and  270 Gβγ signaling (20), the current study provides yet another application of intrabodies to modulate 271 βarr trafficking and alter functional outcomes. 272 A recent study elegantly depicted the structures of βarr1 in complex with several different 273 phospho-peptides derived from the carboxyl-terminus of V 2 R including that corresponding to V 2 R T360-274 1 (21). Interestingly, the binding affinities of βarr1 to V 2 Rpp WT and V 2 Rpp T360 are comparable although 275 V 2 Rpp T360 exhibits a slightly altered binding mode compared to V 2 Rpp WT in the crystal structures (21). 276 Therefore, it is unlikely that distinct trafficking patterns of βarr1 for V 2 R WT vs. V 2 R T360A originate from 277 an affinity difference, and therefore, potentially point towards a conformational mechanism 278 underlying this phenomenon. This is in fact supported by MD simulation analysis of βarr1 structure 279 in complex with V 2 Rpp WT vs. V 2 Rpp T360A . Mutation of Thr 360 to Ala results in a significant reduction of 280 βarr1 conformational population in active-like state as assessed in terms of inter-domain rotation. 281 Strikingly however, simulations in the presence of Fab30 showed a dramatic enrichment of active-282 like conformational ensembles. 283 In the current conceptual framework, GPCR-βarr interactions are typically conceived to be 284 biphasic and involve the phosphorylated carboxyl-terminus of the receptor and the cytoplasmic face 285 of the activated transmembrane bundle (6,7,(22)(23)(24)(25). Previous studies have visualized such partially-286 engaged and fully-engaged GPCR-βarr complexes and deciphered functional outcomes associated 287 with these distinct conformations (23-25). A recent study using NMR spectroscopy demonstrated 288 that Fab30 binding to partially-activated βarr1 facilitates additional conformational changes in βarr1 289 leading to a fully-activated conformation (26). Our study therefore draws an interesting parallel 290 where Ib30 allosterically rescues a functionally-compromised βarr1 conformation to a functionally-291 competent conformation and corresponding cellular responses. It would be interesting to explore in 292 future studies whether the effect of Ib30 observed here for V 2 R T360A is somehow linked to the 293 transition between the partially-and fully-engaged βarr conformations in complex with the receptor. 294 The paradigm of βarr-AP2 interaction through β 2 -adaptin in driving GPCR endocytosis 295 through clathrin-mediated endocytosis is mostly conserved across GPCRs (5). Therefore, our study 296 also raises the possibility of using Ib30 to modulate the βarr1 trafficking for other GPCRs, and 297 decipher previously unknown functions. It is relevant to mention here that Ib30 efficiently 298 recognizes βarr1 in complex with several native GPCRs, although it was selected from a phage 299 display library using V 2 Rpp-βarr1 as the target (12, 22). An emerging paradigm suggests catalytic 300 activation of βarrs where they may continue to generate functional outputs even after dissociation 301 from activated receptors (27)(28)(29). It is therefore tempting to speculate if Ib30 may indeed recognize 302 such conformational "memory" and may help its visualization in the cellular context as well as at 303 high resolution using direct structural approaches. In case of wild-type V 2 R, agonist-stimulation 304 promotes co-localization of the receptor, βarr1 and Ib30 in endosomal vesicles (12); however, this 305 remains to be determined for the V 2 R T360A mutant in presence of Ib30. 306 In summary, we demonstrate that agonist-induced trafficking of βarrs can be allosterically 307 modulated using conformation-specific intrabodies targeting protein-protein interactions. These 308 findings open a new paradigm for modulating GPCR signaling in the cellular context and discovering 309 the interplay of distinct βarr functions.

Construct design and expression plasmids 353
The expression constructs for the wild-type human V 2 R and V 2 R T360A mutants have been described 354 previously (10). Briefly, the cDNA coding for V 2 R WT with an N-terminal HA signal sequence and FLAG 355 tag was PCR amplified and cloned in a customized pcDNA 3.1 (+) vector. This construct was also 356 cloned in pVL1393 vector for expression in Sf9 cells. The Thr 360 mutation was generated on the V 2 R WT 357 backbone using Q5 Site-Directed Mutagenesis Kit (NEB). The βarr1-mYFP plasmid used for confocal 358 imaging experiments was obtained from Addgene (cat. no. 36916). βarr1-mCherry plasmid was a gift 359 from Dr. Mark Scott, Institut Cochin, France. The plasmids encoding ScFv-CTL, ScFv30, Ib-CTL-HA, 360 Ib30-HA and Ib30-YFP have been described previously (12,19). The V 2 R WT and V 2 R T360A constructs 361 were also fused with a 15 amino-acid flexible linker to the small subunit of NanoLuc i.e., SmBiT at its 362 N-terminus. Similarly, Ib30 were N-terminally fused with LgBiT fragment in pCAGGS vector for 363 NanoLuc complementation-based NanoBit assay. For in-vitro assays, i.e., trypsin proteolysis and 364 ScFv30/Fab30 co-IP experiments, βarr1 was purified from BL21 cells by Glutathione Sepharose (GS) 365 affinity chromatography. All the constructs were sequence verified (Macrogen). V 2 R agonist AVP 366 (arginine-vasopressin) was synthesized by Genscript, and phospho-peptides V 2 Rpp WT , V 2 Rpp T360-1 and 367 V 2 Rpp T360-1 were synthesized by the peptide synthesis facility at Tufts University. The construct for 368 GST-tagged β 2 -adaptin (residues 592-951, Rat, isoform 2) in pGEX4T1 vector was received as a kind 369 gift from Dr. Thomas Pucadyil (Pune, India). 370

Limited trypsin proteolysis assay 371
To qualitatively assess the effect of different V 2 R phospho-peptides i.e., V 2 Rpp WT , V 2 Rpp T360-1 and 372 V 2 Rpp T360-2 on βarr1 conformation, limited trypsin proteolysis of βarr1 in the presence or absence of 373 these phospho-peptides was performed. The protocol for trypsin proteolysis of βarr1 has been 374 described previously (13). Briefly, βarr1 (5-10μM) was incubated in the absence or presence of (50:1 375 molar ratio, phospho-peptide: βarr1) the phospho-peptides for 30min at 4°C. Thereafter, L-1-376 Tosylamido-2-phenylethyl chloromethyl ketone (TPCK) treated Trypsin (Sigma-Aldrich; cat. no. 377 T1426) was added to the βarr1 phospho-peptide mixture at a ratio of 1:25 and 1:50 (w/w) and the 378 samples were incubated at 37°C for 5min. In addition to the indicated ratio of trypsin: βarr1, other 379 ratios like 1:10, 1:100 and 1:250 were also tried. At 1:10 ratio, βarr1 was completely digested while 380 at lower trypsin concentrations the resolution of the digested fragments was poor. At each time 381 point, 20μl of the reaction mix (5μg of βarr1) was withdrawn and transferred to a fresh 382 microcentrifuge tube containing 5μl of 5x SDS loading buffer in order to quench the proteolysis 383 reaction. The digested samples were separated on 12% SDS-polyacrylamide gels by electrophoresis 384 to determine the effect of phospho-peptides on the digestion pattern of βarr1. Additionally, to study 385 how ScFv30 affects the digestion pattern of βarr1 when activated with different phospho-peptides, a 386 50-fold molar excess of ScFv30 was added to the βarr1 samples prior to proteolysis. Samples without 387 ScFv30 were used as reference for comparison. After proteolysis with a 1:50 ratio of trypsin: βarr1, 388 the samples were quenched at 30min and resolved by SDS-PAGE as described earlier. 389

Surface expression of receptor mutants 390
The surface expression of V 2 R WT and V 2 R T360A used in different cellular assays was measured by 391 whole-cell surface ELISA. For this, HEK-293 cells transfected with either V 2 R WT or V 2 R T360A were 392 seeded at a density of 0.2 million per well in a 24-well plate precoated with 0.01% poly-D-Lysine 393 (Sigma-Aldrich; cat. no. P0899). After 24hr, cells were fixed with 4% (w/v) paraformaldehyde (pH 6.9) 394 on ice for 20min and washed three times with 1× tris-buffered saline (TBS) buffer [150mM NaCl and 395 50mM Tris-HCl (pH 7.4)]. Subsequently, nonspecific sites were blocked with 1% bovine serum 396 albumin (BSA; prepared in 1× TBS) for 90min, followed by the incubation of cells with horseradish 397 peroxidase (HRP)-coupled anti-FLAG M2 antibody (dilution-1:5000; Sigma-Aldrich; cat. no. A8592), 398 prepared in 1% BSA for 90min. Cells were then washed three times with 1% BSA in TBS, and 200μl of 399 tetramethylbenzidine (TMB) ELISA substrate (Thermo Fisher Scientific; cat. no. 34028) was added to 400 each well. Once blue color appeared in the wells, the reaction was stopped by transferring 100μl of 401 the solution to a different 96-well plate already containing 100μl of 1M H 2 SO 4 . Absorbance was 402 measured at 450nm in a multimode plate reader (Victor X4-Perkin-Elmer). For normalization of 403 signal across different wells, cell density was estimated using Janus Green (Sigma-Aldrich; cat. no. 404 201677) staining. TMB solution was removed from the wells; cells were washed with 1×TBS followed 405 by incubation with 0.2% (w/v) Janus Green for 20min. Thereafter, cells were washed three times 406 with distilled water and 800μl of 0.5N HCl was added to each well. 200μl of this solution was used 407 for measuring the absorbance at 595nm. Normalized surface expression of receptor constructs was 408 calculated as the ratio of absorbance at 450nm and 595nm. 409

Intrabody NanoBiT assay 410
Here, we measured the conformational change in ligand-induced βarr1 recognized by Ib30 using 411 NanoBiT assay. Ib30 and βarr1 were N-terminally fused to LgBiT and SmBiT respectively with the 15-412 amino acid flexible linker and inserted into the pCAGGS plasmid. For NanoBiT assay, HEK-293 cells at 413 a density of 3 million were transfected with V 2 R WT or V 2 R T360A receptor (5µg), LgBiT-Ib30 (5µg) and 414 SmBiT βarr1 (2µg) using PEI (Polyethylenimine); 1 mg ml -1 ) as transfection agent at DNA: PEI ratio of 415 1:3. After 16-18hr of transfection, cells were harvested in PBS solution containing 0.5mM EDTA and 416 centrifuged. Cells were resuspended in 3ml assay buffer (HBSS buffer with 0.01% BSA and 5mM 417 HEPES, pH 7.4) containing 10µM coelenterazine (GoldBio; cat. no. CZ05) at final concentration. The 418 cells were then seeded in a white, clear-bottom, 96-well plate at a density of 0.7-0.9 X 10 5 cells per 419 100μl per well. The plate was kept at 37:C for 90min in the CO 2 incubator followed by incubation at 420 room temperature for 30min. Basal readings were taken in luminescence mode of a multi-plate 421 reader (Victor X4-Perkin-Elmer). The cells were then stimulated with varying doses of ligand AVP 422 ranging from 1pM to 1μM (6x stock, 20µl per well) prepared in drug buffer (HBSS buffer with 5mM 423 HEPES, pH 7.4). Luminescence was recorded for 60min immediately after addition of ligand. The 424 initial counts of 4-10 cycles were averaged and fold increase was calculated with respect to vehicle 425 control (unstimulated values) and analyzed using nonlinear regression four-parameter sigmoidal 426 concentration-response curve in GraphPad Prism software. 427

Confocal microscopy 428
For visualizing the effect of intrabodies on βarr mediated receptor trafficking, HEK-293 cells were co-429 transfected with 3µg of either V 2 R WT or V 2 R T360A along with 2µg of βarr1-mYFP in presence or absence 430 of 2µg of Ib30 with help of polyethylenimine (Polysciences; cat. no. 23966) reagent (21µl) in 10cm 431 plates. Transfection was performed in FBS-deficient DMEM (Gibco; cat. no. 12800-017) after which 432 cells were replaced with DMEM supplemented with FBS (Gibco; cat. no. 10270-106). Post 24hr, cells 433 were seeded onto poly-D-lysine (Sigma-Aldrich; cat. no. P0899) precoated glass bottom confocal 434 dishes (SPL Lifesciences; cat. no. 100350) at a density of 1 million per dish. Cells were allowed to 435 adhere to confocal dishes for 24hr. The next day, cells were starved in FBS-deficient DMEM for 4hr 436 and then stimulated with 100nM AVP and live cells were visualized under the confocal microscope 437 (Zeiss LSM 710 NLO). The confocal microscope was equipped with a motorized XY stage along with a 438 temperature and CO 2 controlled platform. For visualizing Ib30 and βarr1 together, cells were 439 transfected with βarr1-mCherry (2µg) and Ib30-mYFP (2µg) along with V 2 R T360A (3µg). To excite 440 mYFP, a multi-line argon laser source was used and for the mCherry, a diode pump solid state laser 441 source was used. The emitted signal was detected with a 32x array GaAsP descanned detector 442

(Zeiss). For related experiments all microscopic settings including laser intensity and pinhole slit 443
were kept in the same range and for avoiding any spectral overlap between two channels filter 444 excitation regions and bandwidths were adjusted accordingly. Images were acquired in line scan 445 mode and were subsequently processed post imaging in ZEN lite (ZEISS) software suite. For 446 quantifying βarr trafficking to either membrane or endosomes, confocal images were categorized 447 into early (1 to 8min) and late time points (9 to 30min) post agonist stimulation. The cells with βarr1-448 mYFP fluorescence in the plasma membrane were scored as surface localized and the cells with 449 punctate structures in the cytoplasm were scored as internalized. In cases where βarrs were seen in 450 both, the membrane and in cytoplasmic punctate structures, cells having more than three puncta in 451 the cytoplasm were scored under internalized category. Biological replicates were imaged at least 452 three times independently on different days. Scored data from the cell count were plotted as 453 percentage of βarr recruitment from more than 500 cells for each condition. To avoid any 454 discrepancy in manual counting three different individuals counted the images in a blinded and 455 cross-checked fashion. All data were plotted in GraphPad Prism software. 456

Agonist-induced cAMP responses measured by GloSensor assay 457
To measure cAMP accumulation (as a readout for G protein activation), 50-60% confluent HEK-293 458 cells were co-transfected with either V 2 R WT or V 2 R T360A DNA (2µg), luciferase-based 22F cAMP 459 biosensor construct (3.5µg) and Ib-CTL (2µg) or Ib30 (1µg) DNA. After 18-20hr of transfection, cells 460 were washed with 1xPBS and treated with trypsin-EDTA (0.05%). Detached cells were harvested and 461 centrifuged at 1,000 rpm for 10min, and the cell pellet was resuspended in 0.5mg ml -1 luciferin 462 (GoldBio; cat. no. LUCNA) solution prepared in 1X HBSS buffer (Gibco; cat. no. 14065) containing 463 20mM HEPES (pH 7.4). Cells were then seeded at a density of 0.1-0.125 million per 100μl in 96 well 464 white plate. The same pool of cells was also seeded side by side for surface expression by whole cell 465 surface ELISA. The cells seeded in 96-well plate were incubated for 1.5hr in 5% CO 2 followed by 466 additional 30min at room temperature. Subsequently, the basal luminescence was recorded for 5 467 cycles using a plate reader (Victor X4-Perkin-Elmer), followed by addition of indicated concentrations 468 of agonist AVP and luminescence was recorded for 1hr (30 cycles

Effect of Ib30 on agonist induced ERK1/2 phosphorylation 495
To assess the effect of Ib30 on βarr mediated signaling downstream to V 2 R WT and V 2 R T360A mutant, 496 agonist induced ERK1/2 phosphorylation was measured. For this, 60-70% confluent HEK-293 cells 497 were co-transfected with 0.25μg of indicated V 2 R constructs and 1μg of HA-tagged Ib30. A control 498 intrabody (Ib-CTL) that does not recognize receptor bound βarr1 was also transfected in parallel at 499 levels comparable to Ib30 (3μg) to achieve normalized expression levels of both the intrabodies. 500 24hr after transfection, cells were seeded into six-well plates at a density of 1 million cells per well. 501 The next day, cells were serum-starved in DMEM for 6hr and were then stimulated with 100nM AVP 502 (agonist for V 2 R) for indicated time points. After stimulation for selected time points, the media was 503 aspirated and the cells were lysed in 100μl of 2× SDS protein loading buffer. Cellular lysates were 504 heated at 95°C for 15min, followed by centrifugation at 15,000 rpm for 15min. 10μl of samples were 505 loaded per well and separated by 12% SDS-polyacrylamide gel electrophoresis. Phosphorylated 506 ERK1/2 signal was detected by Western blotting using anti-phospho-ERK1/2 antibody (dilution-507 1:5000; CST; cat. no. 9101) followed by reprobing of the blots with anti-total-ERK1/2 antibody 508 (dilution-1:5000; CST; cat. no. 9102). The expression of Intrabody was confirmed by probing with 509 anti-HA antibody (dilution-1:5000; Santa-Cruz; cat. no. sc-805). Signal on the western blots was 510 detected using the ChemiDoc imaging system (Bio-Rad), and densitometry-based quantification was 511 carried out using Image Lab software (Bio-Rad). 512

Molecular dynamics simulations 513
Data without Fab30 was adapted from a previous study (10). To generate V 2 Rpp WT -βarr1, V 2 Rpp 360A -514 βarr1, and V 2 R T360A -βarr1-Fab30 complexes, we used previously determined crystal structure (22). 515 Missing fragments in the βarr1 and V 2 Rpp structures were modelled using the loop modeller module 516 available in the MOE package (www.chemcomp. com). In Fab30 we maintained residues 5 to 108 of 517 the light chain and residues 1 to 123 of the heavy chain. The complexes were solvated (TIP3P water) 518 and neutralized using a 0.15 concentration of NaCl ions. System parameters were obtained from the 519 Charmm36M forcefield (30). Simulations were carried out using the ACEMD3 engine (31). Both 520 systems underwent a 20ns equilibration in conditions of constant pressure (NPT ensemble, pressure 521 maintained with Berendsen barostat, 1.01325 bar pressure), using a timestep of 2fs. During this 522 stage restraints were applied to the backbone. This was followed with 3 x 2µs of simulation for each 523 system in conditions of constant volume (NVT ensemble) using a timestep of 4fs. This allowed us to 524 amass a total of 6µs simulation time per system. For each of the simulations we used a temperature 525 of 310K, which was maintained using the Langevin thermostat, hydrogen bonds were restrained 526 using the RATTLE algorithm. Non-bonded interactions were cut-off at a distance of 9Å, with a 527 smooth switching function applied at 7.5Å. The inter-domain rotation angle of βarr1 was analysed 528 using a script kindly provided by Naomi Latoracca (32). 529

Co-immunoprecipitation (co-IP) assay 530
Co-IP was performed to evaluate the interaction between V 2 Rpp WT , V 2 RppT 360-1 and V 2 Rpp T360-2 with 531 βarr1 in presence of Fab30 and ScFv30. 5μg of purified βarr1 was activated with 10-fold and 50-fold 532 molar excess of phospho-peptides for 1hr at room temperature (25 °C) in binding buffer (20mM 533 HEPES, pH7.4, 100mM NaCl). Thereafter, the activated βarr1 was incubated with 2.5μg of purified 534 Fab30 or ScFv30. Subsequently, 20μl of pre-equilibrated Protein L beads (GE Lifesciences;cat. no. 535 17547802) were added to the reaction mixture and incubated for an additional 1hr at room 536 temperature, which was followed by extensive washing (3-5 times) with binding buffer + 0.01% 537 LMNG. Elution was taken with 2X SDS loading buffer. Interaction of Fab30 and ScFv30 with βarr1 in 538 presence of phospho-peptides was visualized using Coomassie staining of the gels. Band intensity 539 was analysed by ImageJ gel analysis software. 540 To assess the effect of ScFv30 on V 2 R T360A induced βarr1-β 2 -adaptin interaction, we 541 performed co-immunoprecipitation assay (co-IP). The V 2 R T360A receptor was expressed in Sf9 cells, 542 stimulated with 100nM AVP and centrifuged to obtain receptor pellet. The receptor pellet was 543 resuspended in appropriate volume of lysis buffer having 20mM HEPES, 150mM NaCl, 1X PhosSTOP 544 (Roche; cat. no. 04906837001) and 1X protease inhibitor (Roche; cat. no. 04693116001), subjected 545 to Dounce homogenization and incubated with 1μg of purified βarr1 for 30min at room 546 temperature. The receptor-βarr complex was again incubated with 5μg of purified ScFv30 or ScFv-547 CTL for another 30min and solubilized with 1% LMNG for 1hr. Meanwhile, GST or GST-β 2 -adaptin 548 protein (2.5μg) was immobilized on 20μl buffer (20mM HEPES, 150mM NaCl) equilibrated GS beads 549 (1hr at room temperature) and washed once to remove any unbound protein. Subsequently, the 550 supernatant from solubilized complex was allowed to bind with protein bound GS beads (1hr at 551 room temperature) followed by three washes with wash buffer (20mM HEPES, 150mM NaCl, 0.01% 552 LMNG). The bead-bound complex was eluted in 2X SDS loading buffer. Eluted samples were 553 separated by 12% SDS-polyacrylamide gel electrophoresis and probed using βarr antibody (dilution-554 1:10000; CST; cat. no. 4674). After solubilization, 20μl of lysate was set aside for confirming equal 555 loading of βarr1 and ScFv. The lysate was run on separate 12% SDS-polyacrylamide gel and probed 556 using βarr antibody and HRP-coupled protein L antibody (dilution-1:2,000; GenScript; cat. No. 557 M00098) by western blotting. Band intensity was analysed by Image Lab software (Bio-Rad). 558

BRET assay for βarr1-β 2 -adaptin interaction 559
To monitor βarr1 and β 2 -adaptin interactions, BRET assays between βarr1-RlucII and β 2 -adaptin-YFP 560 were performed as described (17). HEK-293 cells were seeded at a density of 1x10 6 cells per 100mm 561 dish and transfected the next day with 250ng of V 2 R WT or V 2 R T360A along with 120ng of βarr1-RlucII, 562 1µg of β 2 -adaptin-YFP, and either 1.5µg Ib-CTL or 1µg Ib30 using PEI. Briefly, a total 6µg of DNA 563 (adjusted with pcDNA3.1/zeo(+)) in 0.5ml of PBS was mixed with 12µl of PEI (25kDa linear, 1mg ml -1 ) 564 in 0.5ml PBS and then incubated for 20min prior to applying to the cells. After 24hr, cells were 565 detached and seeded onto poly-ornithine-coated 96-well white plates at a density of ~35,000 cells 566 per well for the BRET assays, which were performed 48hr after transfection. For BRET assays, cells in 567 96-well plates were washed once with Tyrode's buffer (140mM NaCl, 2.7mM KCl, 1mM CaCl 2 , 12mM 568 NaHCO 3 , 5.6mM D-glucose, 0.5mM MgCl 2 , 0.37mM NaH 2 PO 4 , 25mM HEPES, pH 7.4) and left in 569 Tyrode's buffer for 1h at room temperature. Cells were stimulated with various concentrations of 570 AVP for 45min then BRET signals were measured using a plate reader (Victor X4-Perkin-Elmer). 571 Coelenterazine h (Nanolight™, final concentration of 5µM) was added 25min prior to BRET 572 measurement. The filter set used was 460/80nm and 535/30nm for detecting the RlucII Sequences of the V 2 R WT and Thr 360 mutant phospho-peptides (V 2 Rpp T360-1 and V 2 Rpp T360-2 ) used. 593 Phosphorylated S/T residues are highlighted in red. C. Limited trypsin proteolysis of V 2 Rpp T360-1 / 594 V 2 Rpp T360-2 activated βarr1 show a band pattern distinct from both peptide-free βarr1 and V 2 Rpp WT 595 activated βarr1 indicating an intermediate conformation for the V 2 R T360 mutant. Free (Apo) or 596 phosphopeptide-bound βarr1 was subjected to trypsin proteolysis at indicated trypsin: βarr1 ratio. 597 The proteolysis reaction was quenched at 5min post digestion with SDS buffer and the digestion 598 fragments were resolved on 12% SDS polyacrylamide gel. D. Densitometry based quantification of 599 the % protection of individual tryptic fragments generated from βarr1 at indicated trypsin: βarr1 600 ratio is shown. Data from four independent experiments, normalized with respect to V 2 Rpp WT 601 condition, and analyzed using one-way ANOVA is presented here (*p<0.1, **p<0.01, ***p<0.001, 602 **** p<0.0001). 603 Co-immunoprecipitation with ScFv30 shows that it recognizes the V 2 R T360A bound βarr1. V 2 Rpp T360-1 / 605 V 2 Rpp T360-2 activated βarr1 was incubated with ScFv30. The complex was subsequently pulled down 606 with Protein-L agarose beads. A representative blot from four independent experiments is shown 607 here. B. Densitometry-based quantification of βarr1-ScFv30 interaction normalized with V 2 Rpp WT -608 βarr1 control (taken as 100%) and analyzed using one-way ANOVA (*p<0.05, **p<0.01 ***p<0.001, 609 **** p<0.0001) is shown. C-D. ScFv30 alters the tryptic digestion pattern of V 2 Rpp T360-1 or V 2 Rpp T360-2 610 bound βarr1. βarr1 activated with 50-fold molar excess of different phospho-peptides was subjected 611 to limited trypsin proteolysis at a trypsin: βarr1 ratio of 1:50 in the presence or absence of ScFv30. 612 The proteolysis reaction was quenched with SDS buffer after 30min and the digested fragments 613 were separated by SDS-PAGE. The similarity in the digestion patterns of V 2 Rpp WT and V 2 Rpp T360-1 / 614 V 2 Rpp T360A-2 bound βarr1 in the presence of ScFv30 indicates that ScFv30 can indeed recognize the 615 V 2 R T360 mutant activated βarr1. However, the differences in their digestion patterns also indicate that 616 the V 2 R WT bound conformation of βarr1 is not identical to that of the V 2 R T360A bound βarr1. E-F. 617 Structural snapshots comparing the relative orientation and local interaction networks of trypsin 618 cleavage sites Arg 188 and Arg 285 in the crystal structures of basal (PDB: 1G4M, grey), V 2 Rpp WT (PDB: 619 4JQI, orange) and V 2 Rpp T360-1 (PDB: 7DFA, violet) bound βarr1 is shown. 620 Expression of Ib30 drives endosomal localization of βarr1 for V 2 R T360A . HEK-293 cells expressing V 2 R WT 631 or V 2 R T360A together with βarr1-YFP were stimulated with AVP (100nM) and the localization of βarr1 632 was monitored using confocal microscopy. F. The effect of Ib30 on localization of βarr1 as assessed 633 by manually scoring HEK-293 cells from multiple fields in three independent experiments. Captured 634 confocal images were grouped in two classes i.e., 1-8min and 9-30min post-agonist stimulation to 635 monitor membrane and endosomal localization, respectively. The bar graphs indicate the % of cells 636 showing βarr localization at the surface or in endosomal punctate structures. 637 BRET. HEK-293 cells expressing the V 2 R WT or V 2 R T360A, together with R-Luc-tagged βarr1 and Ib-641 CTL/Ib30 were stimulated with indicated doses of AVP followed by BRET measurement. Inset shows 642 the change in BRET between the Ib-CTL and Ib30 conditions for V 2 R WT and V 2 R T360A , respectively 643 (**p<0.01, unpaired t-test). Data from three independent experiments are presented here. C. HEK-644 293 cells expressing the indicated receptor construct together with Ib-CTL/Ib30 were stimulated 645 with AVP (100nM) followed by detection of ERK1/2 phosphorylation using western blot. Expression 646 of Ib-CTL/Ib30 is monitored using anti-HA antibody. D. Densitometry-based quantification from eight 647 independent experiments, normalized with respect to V 2 R WT +Ib-CTL condition (treated as 100%), 648 analyzed using one-way ANOVA is presented here (***p<0.001, **** p<0.0001). 649  Purified GST-β 2 -adaptin (592-951) was incubated with V 2 R T360A and βarr1 in presence of ScFv-CTL or 662 SvFv30 followed by co-IP and Western blotting. Unconjugated GST was used as a negative control. A 663 representative blot from three different experiments is shown here. The * symbol designates a non-664 specific band. B. Densitometry-based quantification of βarr1-β 2 -adaptin interaction from four 665 independent experiments normalized with GST control and analyzed using one-way ANOVA 666 (*p<0.05, ****p<0.0001). C. BRET between RlucII-tagged βarr1 and YFP-tagged β 2 -adaptin shows 667 enhanced interaction between βarr1 and β 2 -adaptin in presence of Ib30, as compared to Ib-CTL, for 668 both V 2 R WT and V 2 R T360A . D. Ib30 induced increase in βarr1-β 2 -adaptin interaction exists even in the 669 absence of either V 2 R WT and V 2 R T360A . βarr1-β 2 -adaptin interaction in presence of Ib30 exhibits a