Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling

GPCR–β-arrestin interaction and signaling are differentially regulated by distinct phosphorylation sites on the receptor.


INTRODUCTION
The interaction of -arrestins (arrs) with G protein-coupled receptors (GPCRs) is a versatile mechanism to regulate agonist-induced downstream signaling and trafficking of these receptors (1)(2)(3). In addition to their well-established contribution in terminating G-protein signaling and driving activated receptors to endocytic routes, arrs are now also appreciated to facilitate the formation of receptor-Gprotein-arr megaplexes (4,5). Furthermore, arrs also contribute positively toward downstream signaling cascades such as activation of MAP kinases, although a complete G-protein dependence of this phenomenon is currently discussed and debated (2,(6)(7)(8)(9). The recruitment of arrs involves two distinct but interlinked features of GPCRs, namely, agonist-induced receptor activation and receptor phosphorylation, which engage different interfaces on arrs (10,11). Recent studies have demonstrated an appreciable level of functional distinction associated with the two sets of interactions between GPCRs and arrs, i.e., through the receptor core and phosphorylated C terminus and resulting conformations of GPCR-arr complexes (12)(13)(14).
On the basis of the temporal stability of their interaction with arrs and trafficking patterns, GPCRs are typically categorized into two broad classes referred to as class A and B (15). While class A GPCRs have transient interaction with arrs resulting in rapid recycling, class B GPCRs exhibit a relatively stable and sustained interaction leading to their slow recycling and proteosomal degra-dation (15,16). Cumulative phosphorylation of GPCRs, especially in clusters of serine and threonine residues, is typically conceived to determine the stability of arr binding (15,17). A recent study has also proposed the presence or absence, and relative frequencies, of specific phosphorylation codes in the receptors as an important determinant of the stability patterns of GPCR-arr interaction (18). In addition, it is also established that specific phosphorylation patterns in GPCRs arising from phosphorylation by different kinases can drive distinct arr conformations leading to different functional outcomes, a framework that is referred to as phosphorylation "barcode" (19,20).
While these studies have collectively established the current conceptual framework of GPCR-arr interaction, a clear structural understanding of how specific receptor phosphorylation sites are linked to arr recruitment, activation, and conformational changes still remains relatively less well understood. A key limitation until recently has been the lack of structural templates of GPCR-arr complexes to design structure-guided systematic strategies, to probe and directly correlate the contribution of specific phosphorylation sites in arr recruitment and functional outcomes. However, there has been a notable progress on direct structural visualization of GPCR-arrestin interaction over the last few years using x-ray crystallography and cryo-electron microscopy (11,18,(21)(22)(23)(24)(25). These advances now allow structure-guided experimental design and interpretation of data to better understand the intricate details of GPCR-arr interaction and their functional relevance.
In this study, we set out to probe the contribution of different phosphorylation sites in the human vasopressin receptor (V 2 R), a prototypical GPCR, toward arr recruitment, trafficking, and extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation. We generate a set of systematically designed phosphorylation site mutants of the V 2 R and find that several phosphorylation sites can have distinct contribution in arr interaction and functional responses. Some phosphorylation sites work concertedly to affect arr recruitment, while others can have a decisive contribution on arr recruitment, trafficking, and signaling even at individual levels.
Molecular dynamics (MD) simulation provides structural insights into how specific phosphorylation sites on the receptor contribute toward the stability of arr interaction and the interdomain rotation in arrs upon activation. These findings help refine the conceptual framework of GPCR-arr interaction and have direct implications for the paradigm of biased agonism.

Phosphorylation site mutants of human V 2 R
Previous studies have measured the role of V 2 R phosphorylation site clusters in arr interaction and trafficking (26,27); however, the contribution of individual phosphorylation sites has not been explored. Therefore, we generated a series of V 2 R constructs with mutations of the potential phosphorylation sites either individually or in specific combinations, based on previously determined crystal structure of arr1 in complex with V 2 R phosphopeptide (V 2 Rpp) (21) (Fig. 1, A and B). In addition to the eight phospho-sites present in V 2 Rpp, we also generated a mutant for the C-terminal Thr 369 / Ser 370 /Ser 371 (V 2 R TSS/AAA ) cluster that is not phosphorylated in V 2 Rpp (Fig. 1B). We measured the surface expression of each of these mutants in human embryonic kidney (HEK) 293 cells coexpressing either arr1 or arr2 using a previously described whole-cell enzymelinked immunosorbent assay (ELISA) assay (28), and we observed that these mutants are expressed at comparable levels (fig. S1A). We then measured the interaction of V 2 R TSS/AAA mutant with arr1 and arr2 using a cross-linking-based coimmunoprecipitation (co-IP) assay and observed that it interacts with arrs at similar levels as the wild-type receptor (V 2 R WT ) (Fig. 1, C and D). We further corroborated the similar pattern of arr2 interaction of this mutant with V 2 R WT using the Tango assay (Fig. 1G). We also evaluated the trafficking of arrs upon stimulation of V 2 R TSS/AAA mutant and observed a typical "class B pattern" similar to that of V 2 R WT (Fig. 1, E and F, and fig. S2). Furthermore, agonist-induced ERK1/2 phosphorylation downstream of V 2 R TSS/AAA was comparable to V 2 R WT (Fig. 1, H and I). Together, these experiments suggest that the distal "TSS cluster" does not significantly contribute toward arr recruitment, trafficking, and ERK1/2 phosphorylation.

Contribution of Thr 347 , Ser 350 , and Ser 357 in arr recruitment and trafficking
In addition to phospho-site clusters, i.e., TT cluster (Thr 359 Thr 360 ), SSS cluster (Ser 362 Ser 363 Ser 364 ), and TSS cluster (Thr 369 Ser 370 Ser 371 ), there are three scattered phosphorylation sites present in the C terminus of the V 2 R, which were also phosphorylated in V 2 Rpp, i.e., Thr 347 , Ser 350 , and Ser 357 . Of these, only Ser 357 interacts with Lys 11 on  strand I of arr1 in the crystal structure of V 2 Rpp-arr1 complex (Figs. 1A and 3A). We generated phospho-site mutants of V 2 R corresponding to each of these sites, i.e., Thr 347 , Ser 350 , and Ser 357 , and measured the interaction and trafficking of arrs. We observed that V 2 R T347A and V 2 R S350A interacted efficiently with arr1 and arr2, similar to V 2 R WT (Fig. 2, A to D). Moreover, the overall trafficking pattern of arrs for the V 2 R T347A and V 2 R S350A was similar to that of V 2 R WT (Fig. 2, E and F, and fig. S2). However, V 2 R S357A exhibits a significant attenuation of arr interaction compared to V 2 R WT as measured by co-IP assay (Fig. 3, B and C). We further confirmed the interaction pattern of V 2 R S357A with arr2 using Tango assay and observed a significant reduction compared to V 2 R WT (Fig. 3D), similar to that observed by co-IP (Fig. 3, B and C). We next measured agonist-induced trafficking of arrs for V 2 R S357A using confocal microscopy. While the trafficking patterns of arrs were qualitatively similar to V 2 R WT , i.e., surface translocation followed by robust internalization (Fig. 3E), we observed a reduced level of arr trafficking to internalized vesicles for V 2 R S357A compared to V 2 R WT ( fig. S2). To exclude the possibility of arr internalization independent of the receptor (i.e., after dissociation from the receptor), as observed for a couple of different GPCRs previously (29,30), we also measured the colocalization of V 2 R S357A with arr2 in internalized vesicles. As presented in Fig. 3F, V 2 R S357A was colocalized with arr2 in internalized vesicles, suggesting that despite a reduced level of overall recruitment, the trafficking pattern of the receptor is not substantially altered. On the basis of the reduced level of arr interaction, we anticipated a decrease in agonist-induced ERK1/2 phosphorylation for V 2 R S357A . Unexpectedly, we did not observe a significant difference compared to V 2 R WT , although a slight reduction in some experimental replicates was noticeable (Fig. 3G). Together, these data suggest that Thr 347 and Ser 350 are dispensable for arr recruitment, at least in HEK-293 cells, but Ser 357 plays an important role in arr recruitment and trafficking without affecting ERK1/2 phosphorylation.
Ser 362 and Ser 363 of SSS cluster are critical for arr recruitment, trafficking, and ERK1/2 activation Although previous studies have suggested a critical role of SSS cluster in V 2 R-arr interaction and functional outcomes (26,27), a systematic analysis of the contribution of each of these phospho-sites individually has not been reported. Therefore, we generated five different constructs with mutations at either individual phospho-sites or in combination (Fig. 4A). While Ser 362 interacts with Arg 7 on  strand I in arr1, Ser 363 and Ser 364 both are in direct contact with Lys 107 on  helix I (Fig. 4A). We observed that Ser 362 and Ser 363 are important for arr recruitment, while Ser 364 does not seem to have a major role, when tested individually either by co-IP ( fig. S3) or Tango assay (Fig. 4B). The double mutant, i.e., V 2 R S362A/S363A (V 2 R SS/AA ), is affected more markedly with respect to arr recruitment compared to individual mutations ( Fig. 4B and fig. S5A), while the triple mutant, i.e., V 2 R S362A/S363A/364A (V 2 R SSS/AAA ), is completely deficient in arr recruitment ( Fig. 4B and fig. S5B). We also observed that each of the individual phospho-site mutants exhibited typical "class B" pattern of arr trafficking ( fig. S4), similar to V 2 R WT . Quantification of confocal images, however, suggests a noticeable decrease in arr localization, particularly arr2, to internalized vesicles for V 2 R S362A and V 2 R S363A ( fig. S2). The double mutant, i.e., V 2 R SS/AA displays a "class A" pattern of arr recruitment reflected by translocation of arrs to the surface at early time points followed by redistribution in the cytoplasm (Fig. 4C). The triple mutant, i.e., V 2 R SSS/AAA , failed to exhibit any detectable translocation of arrs (Fig. 4C), which also agrees with the lack of interaction observed in co-IP and Tango assays. We also measured agonist-induced ERK1/2 MAP kinase phosphorylation upon agonist stimulation of the double (V 2 R SS/AA ) and the triple (V 2 R SSS/AAA ) mutants and observed a significant reduction in V 2 R SSS/AAA -mediated ERK1/2 phosphorylation compared to V 2 R WT at 5-min time point (Fig. 4D). There was no significant change in ERK1/2 phosphorylation mediated by the double mutant (V 2 R SS/AA ) (Fig. 4E). Together, these data suggest that Ser 362 and Ser 363 contribute toward arr interaction, and their collective contribution is more pronounced than individual sites. Furthermore, while Ser 364 appears to be less important when tested individually, Representative images from four independent experiments (five for arr2), and densitometry-based quantification of data (means ± SEM), normalized with respect to arr co-IP for the V 2 R WT at 30 min agonist stimulation (treated as 100%), are shown. (E and F) Agonist-induced trafficking of arrs for the V 2 R TSS/AAA mutant is similar to that of V 2 R WT as assessed by confocal microscopy in HEK-293 cells expressing the receptor and arr-mYFP. Cells were stimulated with 100 nM AVP, and representative images from three independent experiments at indicated time points are shown. Scale bars, 10 m. (G) Agonist-induced recruitment of arr2 for V 2 R TSS/AAA mutant is also measured by Tango assay and found to be similar to that of V 2 R WT . Data (means ± SEM) from six independent experiments, each performed in duplicate and normalized with respect to the signal for V 2 R WT at 1 M AVP concentration (treated as 100%), are shown here. (H and I) Agonist-induced (100 nM AVP) ERK1/2 phosphorylation for V 2 R TSS/AAA mutant is comparable to V 2 R WT in HEK-293 cells at 5-min time point of agonist stimulation. A representative image from six independent experiments and densitometry-based quantification of the data, normalized with respect to the signal at 5 min for V 2 R WT (treated as 100%). Data in (C), (D), and (I) are analyzed using two-way analysis of variance (ANOVA). ns, nonsignificant. Representative images from four independent experiments (five for S 350 + arr2), and densitometry-based quantification of data, normalized with respect to arr co-IP for the V 2 R WT at 30-min agonist stimulation (treated as 100%), are shown. Data are analyzed using two-way ANOVA. (E and F) Agonist-induced trafficking of arrs for the V 2 R T347A and V 2 R S30A is similar to that of V 2 R WT as assessed by confocal microscopy. HEK-293 cells expressing the indicated receptor mutant and arr-mYFP were stimulated with 100 nM AVP, and representative images from three independent experiments at indicated time points are shown. Scale bars, 10 m. (B and C) V 2 R S357A mutant exhibits significant reduction in agonistinduced (100 nM AVP) arr recruitment compared to V 2 R WT as assessed by co-IP assay in HEK-293 cells. A representative image from four independent experiments and densitometry-based normalized data (means ± SEM) with respect to the signal for V 2 R WT at 30 min (treated as 100%) is shown. Data are analyzed using two-way ANOVA (***P < 0.001 and ****P < 0.0001). (D) The reduction in agonist-induced arr2 recruitment for V 2 R S357A mutant compared to V 2 R WT is further corroborated by Tango assay. Data (means ± SEM) from seven independent experiments, each performed in duplicate and normalized with respect to the signal for V 2 R WT at 1 M AVP concentration (treated as 100%), are shown here. (E) S 357 A mutation does not significantly alter the agonist-induced trafficking pattern of arrs as measured qualitatively by confocal microscopy in HEK-293 cells expressing the receptor and arr-mYFP. Cells were stimulated with 100 nM AVP, and representative images from three independent experiments at indicated time points are shown. Scale bars, 10 m. (F) V 2 R S357A exhibits agonist-induced (100 nM AVP) trafficking and colocalization with arr2 in endosomal vesicles in HEK-293 cells. As visualized by confocal microscopy. Scale bar, 10 m. (G) Agonist-induced (100 nM AVP) ERK1/2 phosphorylation downstream of V 2 R S357A is similar to that of V 2 R WT as measured in HEK-293 cells at indicated time points. A representative image from nine independent experiments, and densitometry-based quantification of data (means ± SEM), normalized with respect to the signal at 5 min for V 2 R WT , is shown in the bottom panel. Data are analyzed using two-way ANOVA. Rpp-arr1 crystal structure depicting the interaction of receptor-bound phosphate groups with K 107 /R 7 in arr1. The bottom panel shows the C-terminal sequences of V 2 R mutants with mutated S/T residues highlighted in red. (B) Tango assay reveals a prominent contribution of S 362 and S 363 , but not of S 364 , in arr2 recruitment. Simultaneous mutation of S 362 /S 363 results in near-complete loss of arr2 recruitment, which is reduced even further for the S 362 /S 363 /S 364 (SSS/AAA) mutation. Data represent means ± SEM of eight independent experiments (six for V 2 R SS/AA ), each carried out in duplicate and normalized with respect to the response at 1 M concentration of AVP for V 2 R WT . (C) V 2 R SS/AA mutant exhibits class A pattern of arr translocation, and V 2 R SSS/AAA displays no detectable translocation of arr. Representative images from three independent experiments on HEK-293 cells expressing the indicated receptor mutants and arr-mYFP, stimulated with 100 nM AVP, are shown. Scale bars, 10 m. (D and E) Agonist-induced (100 nM AVP) ERK1/2 activation downstream of V 2 R SS/AA is similar to that of V 2 R WT but V 2 R SSS/AAA exhibits a significant reduction at 5-min time point. Representative images from 12 (V 2 R SSS/AAA ) and 6 (V 2 R SS/AA ) independent experiments, and densitometry-based quantification of data (mean ± SEM), normalized with respect to the signal at 5-min time point for V 2 R WT (treated as 100%), are shown. Data are analyzed using two-way ANOVA (****P < 0.0001). (F) Agonist-induced cAMP response for V 2 R SSS/AAA is similar to that of V 2 R WT as measured in HEK-293 cells using the GloSensor assay. Data (means ± SEM) from six independent experiments, each performed in duplicate, and normalized with respect to the response at 1 M concentration of AVP for V 2 R WT (treated as 100%). Two-way ANOVA suggests that the apparent difference in the cAMP dose-response curves for V 2 R WT and V 2 R SSS/AAA is not statistically significant.

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in the context of the triple mutant (V 2 R SSS/AAA ), it seems to act concertedly with the other sites toward overall arr recruitment, trafficking, and ERK1/2 activation.

SSS 362/363/364 AAA mutant yields a G-protein-biased receptor
As the V 2 R SSS/AAA mutant exhibits near-complete loss of arr recruitment, it may potentially behave as a G-protein-biased mutant, if it maintains efficient G-protein coupling. To test this hypothesis, we measured agonist-induced cyclic adenosine 3′,5′-monophosphate (cAMP) response for this mutant and observed that it indeed exhibited a robust cAMP response, similar to V 2 R WT (Fig. 4F). At a low agonist dose, this mutant is even more efficient in producing cAMP response compared to V 2 R WT , and the cAMP response appears to be more sustained, as expected, due to lack of arr-mediated desensitization ( fig. S7A). Therefore, V 2 R SSS/AAA represents a arr couplingdeficient, Gs-biased V 2 R mutant that can be used in the future to delineate the specific contributions of G-protein and arrs downstream of V 2 R.
Thr 360 , but not Thr 359 , is critical for overall arr recruitment, trafficking, and ERK1/2 phosphorylation We next focused on the TT cluster and generated three different mutants as depicted in Fig. 5A. While Thr 359 is not involved in any interaction with Lys/Arg in arrs, Thr 360 interacts with Arg 25 in  strand II and Lys 294 in the lariat loop (Fig. 5A). We observed that V 2 R T359A exhibits efficient interaction with arrs ( fig. S6A); however, V 2 R T360A displays significantly reduced interaction with arrs (Fig. 5, B to D). The combination of these two phospho-sites, i.e., V 2 R T359A/T360A (V 2 R TT/AA ), exhibits even more pronounced loss of arr interaction compared to V 2 R T360A ( Fig. 5D and fig. S6B). Notably, we also observed that V 2 R T360A exhibits a typical class A pattern in terms of arr trafficking as reflected by the surface translocation of arrs followed by its redistribution in the cytoplasm (Fig. 5E). The double mutant V 2 R TT/AA exhibited a pattern similar to V 2 R T360A (Fig. 5E). On the other hand, V 2 R T359A displayed a typical class B pattern of arr translocation upon agonist stimulation ( fig. S6C), although there appears to be a noticeable increase in the localization of arr2 in internalized vesicles, compared to V 2 R WT during the early time frame ( fig. S2). We also measured agonist-induced ERK1/2 phosphorylation by V 2 R T360A and V 2 R TT/AA and observed a significant reduction compared to V 2 R WT for both of these mutants ( Fig. 5F  and fig. S6D). Together, these data suggest that Thr 360 plays a critical role in driving the interaction of V 2 R with arrs as well as in determining the class B pattern of arr trafficking and ERK1/2 phosphorylation, while Thr 359 appears to be less important, at least in HEK-293 cells. Moreover, V 2 R T360A also maintains an efficient G-protein coupling profile, as measured using cAMP responses via the GloSensor assay ( fig. S7, B and C), and thus represents another G-protein-biased V 2 R mutant, similar to V 2 R SSS/AAA .

Structural insights into receptor-arr interaction and conformation
To gain structural and mechanistic insights into our findings, we used MD simulation using the V 2 Rpp-arr1 crystal structure as a template (21). We first carried out classical unbiased simulation to monitor the dynamics of V 2 Rpp in the context of phospho-site mutations. Here, a quantitative measure of V 2 Rpp dynamics is obtained by computing the root mean square fluctuation (RMSF) per residue. We observed that the WT and mutated phosphopeptides corresponding to the mutants described above exhibited an overall similar RMSF profile ( fig. S8). Expectedly, we observed higher RMSF at the N-terminal (346 to 348) and the C-terminal ends (366 to 372) of the phosphopeptide, while two stretches in the middle that adopt an extended  strand and pack against the  strand I of arr1 via backbone interactions displayed much lower RMSF profile ( fig. S8).
We found that Thr 360 is repeatedly the most stable position in all simulated systems (fig. S8). This indicates that Thr 360 is an anchor point for the binding of phosphorylated receptor tail to arrs and provides a potential mechanistic basis for a marked reduction in arr recruitment. Thr 360 is a part of the extended  strand in the middle of V 2 Rpp, and it interacts with Lys 294 in the lariat loop of arr1 through a strong electrostatic interaction (Fig. 6A). Structurally, Thr 360 is at the center of a three-way connection between the N-domain, the V 2 Rpp, and the C-domain of arr1 through the Thr 360 -Lys 294 ionic lock (Fig. 6A). Thus, it is tempting to speculate that the Thr 360 -Lys 294 ionic lock may be a crucial determinant for the interdomain rotation between the N-and C-domain observed upon V 2 Rpp binding and activation of arr1.
To test this possibility, we first assessed the interdomain rotation angle of the arr1 in complex with the V 2 Rpp and observed an average rotation angle of 17°, which agrees well with experimental observation (21) and previous simulation experiments (31) (Fig. 6B). The average interdomain rotation angle changed to about 11° for V 2 Rpp T360A (Fig. 6C). In complex with V 2 Rpp, arr1 is able to sample a broad spectrum of conformations during activation where larger interdomain rotation occurs at high probability, while the smaller interdomain rotation has relatively lower probability. However, in the context of V 2 Rpp T360A , conformations with smaller interdomain rotation become markedly more populated (Fig. 6C). This marked alteration is quantitatively visible upon comparison of active-like populations (i.e., with an interdomain rotation angle >15°) between V 2 Rpp and V 2 Rpp T360A analysis (Fig. 6, B and C).
We further computed the stability of the ionic lock (Thr 360 -Lys 294 ) across all sampled activation states of the arr1-V 2 Rpp complex (Fig. 6D). We observed that the ionic lock stability directly correlates with the interdomain rotation angles (Fig. 6D). There is a marked reduction in the ionic lock formation in inactive-like arr1 conformations with interdomain rotation angles <15°. This is in agreement with the difference in average interdomain rotation angles and conformational distribution between V 2 Rpp (17°; ionic lock present) and V 2 Rpp T360A (11°; ionic lock absent) as mentioned above (Fig. 6, B and C). Together, these simulation data underscore the role of Thr 360 -Lys 294 ionic lock as an important element in stabilizing the relative orientation of the N-and the C-domain in arr1 upon activation, which may, in turn, fine-tune the functional responses.

DISCUSSION
GPCR phosphorylation is a key determinant of arr interaction and imparting specific conformational signatures linked to distinct functional responses (19,32). Previous studies have proposed a direct link between the receptor phosphorylation patterns and ensuing functional outcomes; however, integrating these findings in a structural framework still remains somewhat preliminary. Here, we find that even a single phosphorylation site in V 2 R, i.e., Thr 360 can have a decisive contribution in arr recruitment by serving as an anchor point for stable interaction. Moreover, it can also critically influence (B and C) V 2 R T360A mutant exhibits near-complete loss of arr recruitment as measured in HEK-293 cells, stimulated with 100 nM AVP, using the co-IP assay. Representative images from three independent experiments (four for arr2) and densitometry-based quantification of data (mean ± SEM), normalized with respect to the signal for V 2 R WT at 30-min time point (treated as 100%), are shown. Data are analyzed using two-way ANOVA (**P < 0.01 and ****P < 0.0001). (D) Tango assay corroborates a major reduction in agonist-induced arr2 recruitment for V 2 R T360A , which is reduced even further in the double phospho-site mutant, i.e., V 2 R TT/AA . Mutation of T 359 alone does not lead to a significant reduction in arr2 recruitment. Data (means ± SEM) from of seven independent experiments (eight for V 2 R S360A ), normalized with respect to the response at 1 M concentration of AVP for the V 2 R WT (treated as 100%), are shown. (E) Mutation of T 360 alone or in combination T 359 (i.e., V 2 R TT/AA ) confers a class A pattern of agonist-induced translocation of arrs, and significant endosomal trafficking of arrs is not observed even after prolonged agonist stimulation (100 nM AVP). Representative images from three independent experiments on HEK-293 cells expressing the indicated receptor mutants and arr-mYFP, stimulated with 100 nM AVP, are shown. Scale bars, 10 m. (F) V 2 R T360A mutant displays a significantly reduced level of agonist-induced ERK1/2 phosphorylation compared to V 2 R WT in HEK-293 cells stimulated with AVP (100 nM). A representative image from nine independent experiments and densitometry-based quantification of data (means ± SEM), normalized with respect to the signal at 5 min after agonist stimulation for V 2 R WT (treated as 100%). Data are analyzed using two-way ANOVA (**P < 0.01 and ****P < 0.0001).
the activation-dependent conformational changes such as interdomain rotation angle via the formation of an important ionic lock with Lys 294 in arr1. This observation underscores the importance of spatial positioning of key phosphorylation sites in the receptor as crucial parameter, in addition to previously determined phosphoclusters (27) and phospho-codes (18). The importance of spatial localization of the key phospho-sites is further corroborated by the observation that Thr 359 positioned right next to Thr 360 has no measurable effect on arr recruitment or trafficking. The phosphate group on Thr 359 points away from the Lys 294 in V 2 Rpp-arr1 crystal structure, suggesting that its spatial positioning is unsuitable for in-teracting with the lariat loop, even in the context of Thr 360 phosphosite mutation. As Lys 294 is conserved in arrs, it is possible that its interaction with suitably positioned receptor phosphates may contribute generally toward arr activation, conformational change, and functional responses, although its mutation does not appear to significantly affect the overall interaction between the selected GPCRs and arrs as reported in a recent study (33). Future studies designed to probe this in detail with a set of different GPCRs may shed further light on this interesting conjecture.
Although previous studies have reported a collective role of triple serine cluster, i.e., Ser 362/363/364 in arr trafficking (26, 27, 34), interdomain rotation angles adopted by arr1 in complex with the V 2 Rpp as measured by MD simulation. We observed a peak with an interdomain rotation of about 17° which agrees well with the experimental data and previous simulation studies. (C) Distribution of the interdomain rotation angles adopted by arr1 upon V 2 R T360A mutation where the peak is shifted to about 11°. For V 2 R T360A mutation, the fraction of active conformers with larger interdomain rotation is also reduced compared to V 2 Rpp. (D) The stability of the ionic lock between T 360 and K 294 as a function of the interdomain rotation angle reveals that the ionic lock formation reduces with the decrease in the interdomain rotation. arr1 conformers with an interdomain rotation angle of less than 11° display a lower frequency of ionic lock formation. Note that the frequencies do not reach more than 30% due to a large flexibility of the lariat loop, which gives the ionic lock a rather transient character.
our study reveals a concerted contribution of individual phosphosites present in this cluster. While individual mutation of Ser 362 and Ser 363 significantly reduces arr binding but not the trafficking pattern, Ser 364 is mostly dispensable. However, a combination of Ser 362 and Ser 363 diminishes arr recruitment further and also changes the trafficking pattern from class B to class A. Although Ser 364 by itself does not appear to have a major role, in conjunction with Ser 362/ Ser 363 mutation, it facilitates complete abrogation of arr recruitment. This observation implies that contribution of some phospho-sites present in a cluster may be evident only upon a combinatorial analysis. Moreover, as the G-protein coupling of the V 2 R SSS/AAA mutant remains primarily unaltered, it essentially imparts G-protein bias on V 2 R. Thus, it may serve as a promising tool to further investigate structural and functional aspects of V 2 R-effector coupling and signaling responses (35). An intriguing pattern that emerges from our study is that the extent of arr recruitment and ERK1/2 phosphorylation do not necessarily correlate with each other. For example, V 2 R S357A and V 2 R SS/AA mutants have significantly reduced levels of arr recruitment; however, their agonist-induced ERK1/2 phosphorylation patterns are mostly similar to V 2 R WT . While the contribution of both G-proteins and arrs in ERK1/2 activation downstream of GPCRs is well established, our data suggest that even a transient interaction or an overall lesser extent of arr interaction is sufficient to drive robust ERK1/2 activation. This notion is also confirmed by previous studies on the 1 adrenergic receptor system (29,30).
A recent study using the rhodopsin-visual-arrestin system has proposed that phospho-sites can be categorized as the key sites, modulatory sites, and inhibitory sites and hypothesize that a similar pattern may exist for other GPCRs as well (36). While we do observe that Ser 357 and Thr 360 mutation significantly decreases arr recruitment, we did not find an inhibitory role of any of the phosphosites in the V 2 R. Nonetheless, future studies with additional receptor systems may provide experimental evidence, or lack thereof, for this provocative hypothesis. Moreover, recent studies using intrabody sensors have suggested conformational diversity in GPCR-arr complexes despite an overall similar recruitment profile and trafficking patterns (37)(38)(39). Therefore, it would be very interesting to analyze the conformational signatures of arrs in complex with these V 2 R mutants in further studies. It is also worth noting that although we observe that the mutation of some putative phosphorylation sites do not have a significant effect on arr recruitment and trafficking, we cannot discern whether these sites are phosphorylated, or not, in HEK-293 cells or if they are completely dispensable. This remains an open question for future investigation especially considering the emerging evidence for cell type-and tissue-specific GPCR phosphorylation and signaling mechanisms (40). Furthermore, a kinetic analysis of agonist-induced arr recruitment for these receptor mutants may yield additional insights into the potential contribution of different phosphorylation sites in transient interactions between the receptor and arrs. It is also worth noting that the crystal structure of arr1 in complex with V 2 Rpp was determined using rat arr1, while the constructs used here for co-IP experiments are of bovine origin. Although the sequences of arr1 are highly similar across different species, arr2 displays slightly higher sequence divergence, and a minor effect of such sequence differences on arr conformation and functional outcomes cannot be completely ruled out.
In conclusion, we find that even single phosphorylation sites on GPCRs may encode critical determinants for arr interaction and trafficking. Moreover, individual sites in a cluster may act in a concerted fashion to impart distinct arr interaction and trafficking patterns. Our data also reveal that a single phospho-site may act as an anchor point for the stability of interaction and directing the degree of interdomain rotation during the activation process. This study provides a missing piece in the paradigm of GPCR-arr interaction using V 2 R as a model system, and it also offers a framework that may potentially have general applicability for other GPCRs as well.

General reagents, cell culture, and expression plasmids
Most of the general chemicals used here for molecular biology, biochemistry, and cell biology experiments were purchased from Sigma-Aldrich. Trypsin-EDTA, Hank's balanced salt solution (HBSS), and penicillin-streptomycin solution were purchased from Thermo Fisher Scientific. The expression constructs for the wild-type human V 2 R , bovine arr1, and arr2 have been described previously (39), and rat arr1/2-mYFP plasmids were obtained from Addgene (cat. nos. 36916 and 36917). The phosphorylation site mutants were generated using Q5 Site-Directed Mutagenesis Kit (NEB) and sequence-verified (Macrogen). V 2 R agonist AVP (arginine-vasopressin) was either purchased from Sigma-Aldrich or synthesized (GenScript). HEK-293 cells (American Type Culture Collection) were maintained and cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 g/ml). Cells were cultured in 10-cm dishes (Corning) at 37°C under 5% CO 2 and passaged at 70 to 80% confluency using 0.05% trypsin-EDTA for detachment.

DNA transfection and surface expression of V 2 R mutants
For various assays described in the manuscript, HEK-293 cells at 60 to 70% confluency were transfected with the indicated constructs using polyethylenimine (PEI) as the transfection reagent at a typical DNA:PEI ratio of 1:3. Surface expression of V 2 R constructs was measured using whole-cell surface ELISA as described previously (28). Briefly, 24 hours after transfection, 0.2 million transfected cells were seeded into each well of 24-well plates, precoated with 0.01% poly-d-lysine. After another 24 hours, cells were fixed with 4% (w/v) paraformaldehyde (pH 6.9) on ice for 20 min and washed three times with 1× tris-buffered saline (TBS) buffer [150 mM NaCl and 50 mM tris-HCl (pH 7.4)]. Subsequently, nonspecific sites were blocked with 1% bovine serum albumin (BSA; prepared in 1× TBS) for 90 min, followed by the incubation of cells with horseradish peroxidase (HRP)-coupled anti-Flag M2 antibody (Sigma-Aldrich; cat. no. A8592) at a dilution of 1:10,000, prepared in 1% BSA for 90 min. Cells were then washed three times with 1× TBS, and 200 l of tetramethylbenzidine (TMB) ELISA substrate (GenScript) was added to each well. Once the blue color appeared in the wells, the reaction was stopped by transferring 100 l of the solution to a different 96-well plate already containing 100 l of 1 M H 2 SO 4 . Absorbance was measured at 450 nm in a multimode plate reader (PerkinElmer, Victor X4). For normalization of signal across different wells, cell density was estimated using Janus Green staining. TMB solution was removed from the wells; cells were washed three times with 1× TBS followed by incubation with 0.2% (w/v) Janus Green for 20 min. Afterward, cells were washed three times with distilled water, 800 l of 0.5 N HCl was added to each well, and 200 l of this solution was used for measuring the absorbance at 595 nm. Normalized surface expression of V 2 R constructs was calculated as the ratio of absorbance at 450 and 595 nm.

Chemical cross-linking and co-IP
For measuring agonist-induced V 2 R-arr interaction, HEK-293 cells expressing the corresponding proteins were starved using incomplete DMEM for 6 hours, followed by stimulation with AVP (100 nM) for indicated time points. Afterward, cells were collected, lysed by douncing in lysis buffer [20 mM Hepes (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, and 1× PhosStop], followed by the addition of freshly prepared 1 mM DSP (dithiobis succinimidyl-propionate) (Sigma-Aldrich; cat. no. D3669). After 40 min of DSP cross-linking with continuous tumbling, the reaction was quenched with 100 mM tris-HCl (pH 8.5), and then cellular lysate was solubilized with 1% (v/v) MNG (maltose neopentyl glycol) for 1 hour at room temperature. Subsequently, the solubilized proteins were separated by centrifugation at 15,000 rpm for 30 min, and pre-equilibrated anti-Flag M1 antibody sepharose beads were added. Samples were supplemented with 2 mM CaCl 2 , and bead binding was allowed to occur for 2 hours at 4°C with gentle tumbling. The beads were washed three times each with low-salt buffer [20 mM Hepes (pH 7.4), 150 mM NaCl, 2 mM CaCl 2 , and 0.01% (v/v) MNG] and high-salt buffer [20 mM Hepes (pH 7.4), 350 mM NaCl, 2 mM CaCl 2 , and 0.01% (v/v) MNG], alternatively, to remove unbound and nonspecifically bound proteins. Last, the bound proteins were eluted using elution buffer [20 mM Hepes (pH 7.4), 150 mM NaCl, 2 mM EDTA, 0.01% MNG, and Flag peptide (250 g/ml)]. A similar protocol was followed for the control co-IP experiment (presented in fig. S1B), except that anti-HA antibody agarose beads were used, instead of M1 antibody agarose. Receptor and arrs in co-IP samples were detected by Western blotting by first using rabbit anti-arr antibodies (1:5000; CST, cat. no. 4674), followed by reprobing the blots with HRPconjugated anti-Flag M2 antibody (1:5000; Sigma-Aldrich, cat. no. A8592). Protein bands on the Western blots were visualized using a ChemiDoc imaging system (Bio-Rad). For densitometrybased quantification of co-IP samples, the band intensities on the Western blots were measured using either the Image Lab software (Bio-Rad), or ImageJ, and plotted in GraphPad Prism. The anti-Flag M2 antibody blots detecting the immunoprecipitation of various V 2 R constructs typically exhibited two bands, and both bands were used for densitometry. These two bands presumably indicate mature (fully glycosylated) and immature (partially glycosylated) receptor populations.

GloSensor assay for measuring agonist-induced cAMP response
For measuring cAMP response for V 2 R constructs, HEK-293 cells were cotransfected with the indicated receptor construct and 22F plasmid (Promega). Twenty-four hours after transfection, cells were detached from the plates, centrifuged, and resuspended in buffer [1× HBSS supplemented with 20 mM Hepes (pH 7.4)] containing luciferin (0.5 mg/ml; GoldBio). Cells were seeded in white, glassbottom 96-well plates at a density of 80,000 to 100,000 cells per well in 100 l volume per well. Afterward, the 96-well plate was kept at 37°C for 1.5 hours under 5% CO 2 , followed by an additional incubation at room temperature for 30 min. Subsequently, the basal luminescence readings were recorded using a plate reader (Victor X4, PerkinElmer), followed by the addition of indicated concentrations of agonist (AVP) and recording of luminescence for up to 1 hour. Data were corrected for baseline signal and normalized with respect to highest concentration (1 M) of AVP and plotted in GraphPad Prism. The GloSensor experiments were performed at an endogenous level of arrs, i.e., without arr overexpression, and only the indicated receptor constructs together with 22F plasmid were transfected for overexpression.

Agonist-induced ERK1/2 phosphorylation
Agonist-induced ERK1/2 phosphorylation was measured as a readout of arr signaling downstream of V 2 R mutants following the previously described protocol (41). Briefly, HEK-293 cells were transfected with 0.5 g of indicated V 2 R constructs, and 24 hours after transfection, cells were seeded into six-well plates at a density of about 1 million cells per well. The next day, cells were serumstarved in DMEM for 6 hours followed by stimulation with 100 nM AVP for indicated time points, culture medium was aspirated, and cells were lysed in 100 l of 2× SDS gel loading buffer. Cellular lysates were heated at 95°C for 15 min, followed by centrifugation at 15,000 rpm for 10 min, and 10 l of samples was used for SDSpolyacrylamide gel electrophoresis. Phosphorylated ERK1/2 signal was detected by Western blotting using anti-phospho-ERK1/2 antibody (1:5000; CST, cat. no. 9101) followed by reprobing of the blots with anti-total-ERK1/2 antibody (1:5000; CST, cat. no. 9102). Signal on the Western blots was detected using the ChemiDoc imaging system (Bio-Rad), and densitometry-based quantification was carried out using Image Lab software or ImageJ. ERK1/2 phosphorylation experiments were performed at an endogenous level of arrs, i.e., without arr overexpression, and only the indicated receptor constructs were transfected for overexpression.

Confocal microscopy
To visualize the agonist-induced trafficking of arrs upon stimulation of V 2 R mutants, HEK-293 cells were cotransfected with the indicated V 2 R construct and arr1/2-mYFP. Twenty-four hours after transfection, 1 million cells were seeded in glass bottom confocal imaging plates, precoated with 0.01% poly-d-lysine. After another 24 hours, cells were serum-starved for 2 to 3 hours and then subjected to live cell imaging using Carl Zeiss LSM780NLO confocal microscope fitted with 32× array GaAsP descanned detector (Zeiss) under 63×/1.40 numerical aperture objective with oil immersion. First, the cytoplasmic distribution of arrs was recorded under basal conditions, followed by stimulation of cells and recording of arrs localization in indicated time frame. For the two-color confocal imaging to measure the colocalization of the V 2 R S357A and arr2 (presented in Fig. 3F), transfected cells (24 hours after transfection) were seeded onto glass coverslips, precoated with 0.01% poly-d-lysine, and allowed to grow for another 24 hours. The next day, cells were serum-starved for 2 hours followed by stimulation with AVP (100 nM) for 0, 10, and 30 min. Subsequently, the cells were fixed with 4% paraformaldehyde prepared in 1× phosphate-buffered saline (PBS), permeabilized with 0.01% Triton X-100 for 10 min. For staining the receptor, cells were incubated with DyLight 594 conjugated anti-Flag M1 antibody (at 1:100 dilution prepared in 1% BSA solution) for 1 hour at room temperature. Afterward, cells were washed several times with 1× PBS, and then the coverslips containing fixed cells were mounted onto glass slides using VectaShield H-1000 mounting medium (VectaShield). The slides were air-dried for 20 to 30 min before imaging by confocal microscopy. Multiline argon laser source is used for green channel (mYFP), and for the red channel (DyLight 594), a diode pump solid state laser source was used. All the settings including laser intensity and pinhole settings were maintained in the same range for parallel set of experiments, and the filter excitation regions and bandwidths were adjusted for the channels to avoid any spectral overlap.
For the quantification of agonist-induced localization of arrs for different V 2 R mutants, confocal images from multiple fields in at least three independent experiments were manually scored. Confocal images captured during 1 to 8 and 9 to 60 min after agonist stimulation were grouped under early and late time frames, respectively. The localization of arrs was scored as surface and internalized on the basis of YFP fluorescence in the plasma membrane and punctate structures in the cytoplasm, respectively. In other words, cells with arr-YFP in the plasma membrane are scored under "surface" category, while the cells displaying arr-YFP in punctate structures in the cytoplasm are counted under "internalized" category. All images in the field were used for counting, and the data are plotted as percentage of arr localization pattern from more than hundred cells for each condition. In a scenario where arrs were present in both, the membrane and in punctate structures, cells with more than three punctae in the cytoplasm were scored under internalized category. To minimize any bias in scoring, the same set of images was analyzed by three different individuals and cross-checked. Data were plotted using GraphPad Prism software.
Tango assay for arr2 recruitment Tango assay was used to measure agonist-used arr2 recruitment following a previously described protocol (42). Briefly, HTLA cells expressing a tTA-dependent luciferase reporter and arr2-TEV fusion protein were transfected with indicated V 2 R constructs. The V 2 R constructs for Tango assay compose of a receptor-coding region, followed by a TEV cleavage site and the tTA transcription factor coding sequence. Approximately 3 million HTLA cells were seeded onto a 10-cm cell culture plate, transfected with indicated receptor constructs, and 24 hours after transfection, cells were detached using trypsin-EDTA solution. Cells were resuspended in complete DMEM and seeded into 96-well white polystyrene plates at a density of about 50,000 cells per well. After another 24 hours, cells were stimulated with indicated concentrations of AVP for 7 to 8 hours. Subsequently, the growth medium was removed from the wells, and 100 l of luciferin solution (0.5 mg/ml in 1× HBSS buffer) was added to each well. The luminescence signal was measured at 450 nm, and data were baseline-corrected, plotted, and analyzed using nonlinear regression in GraphPad Prism software.

MD simulation System setup and simulation
To generate all simulated complexes, we used the structure of V 2 Rpp in complex with arr1 [Protein Data Bank (PDB) code: 4JQI]. The cocrystallized Fab30 antibody was removed, and missing fragments in the arr1 and V2Rpp structures were modeled using the loop modeler module available in the MOE package (www.chemcomp. com). The complexes were solvated (TIP3P water) and set to an ionic strength of 0.15 M sodium chloride. Simulation parameters were obtained from the Charmm36M force field (43). In the simulation protocol, we adhere to the guidelines of the GPCRmd consortium (44). Systems generated this way were simulated using the ACEMD software (45). To allow rearrangement of waters and side chains, we carried out a 25-ns equilibration phase in NPT conditions with restraints applied to backbone atoms. The time step was set at 2 fs, and the pressure was kept constant, using the Berendsen barostat. After NPT equilibration, systems were subjected to production runs (NVT ensemble) for 1 s in four parallel runs. Simulation runs of the V 2 R WT and V 2 R T360A systems were extended to 2 s, amassing a total of 8 s per system. For each NVT run, we used a 4-fs time step. In all runs, temperature was kept at 300 K using the Langevin thermostat, and hydrogen bonds were restrained using the RATTLE algorithm. Nonbonded interactions were cut off at 9 Å with a smooth switching function applied at 7.5 Å.

Analysis
To evaluate C-terminal tail stability, we aligned the system using backbone atoms of arrestin. Afterward, RMSF values were calculated for the C atoms of the C-terminal tail. The interdomain rotation angle was used as a metric to assess the activation state of arr1.
We computed the displacement of the C-domain relative to the N-domain between the inactive (PDB code: 1G4R) and active arr1 crystal structures (PDB code: 4JQI) as previously described (31). The corresponding script was provided by N. Latorraca. Using obtained values of the rotational angles, we divided the simulation frames into groups with a bin width of 1. For each bin of rotation angle, we assessed the stability of the ionic lock between residue T360 of the peptide and K294 of the lariat loop. A salt bridge was defined as the distance between heavy polar atoms of those residues with less than 4 Å.

Statistical analysis and data presentation
Experiments were repeated at least three times, and data were plotted and analyzed using GraphPad Prism software. The details of data normalization, statistical analysis, and P values are included in the corresponding figure legends.

SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/6/37/eabb8368/DC1 View/request a protocol for this paper from Bio-protocol.