Functional and structural insights into interactions between β-Arrestin 1 and Gαs or Gαi1

Domain: chemistry
Published: 2026-01-20
Editorial outcome: major_revision

Abstract
G proteins and arrestins are key transducers for G protein-coupled receptor (GPCR) signaling, mediating distinct downstream pathways. Recent evidence suggests that G proteins and β-arrestins (βarrs) can directly or functionally interact. However, the molecular details and functional consequences of Gα–βarr interactions remain poorly understood. Here, we quantify the binding affinities between βarr1 and Gαs or Gαi1 in various activation states using microscalethermophoresis (MST). βarr1 in the active conformational ensemble state favors binding, whereas Gα activation status is less determinant. Hydrogen/deuterium exchange mass spectrometry reveals distinct conformational changes between Gαs versus Gαi1 upon βarr1 binding, suggesting differential binding mechanism between Gαs–βarr1 and Gαi1–βarr1 complexes. Both the Ras-like domain and the α-helical domain of Gα contribute to complex formation. Functionally, a BODIPY-FL–GTPγS assay shows that βarr1 does not alter GDP/GTP turnover of Gαs or Gαi1, whereas β-strand XX (βXX) release assays demonstrate that Gαs enhances βarr1 C-tail release. Together, these results propose molecular mechanism of the interaction and asymmetric functional coupling within Gα–βarr complexes and uncover a previously underappreciated layer of GPCR signal transduction.

Introduction
G proteins and arrestins are the two major downstream signaling molecules of G protein-coupled receptors (GPCRs) 1–3. Activated GPCRs interact with the Gα subunit of the heterotrimeric G protein, releasing guanosine diphosphate (GDP) from Gα. The empty Gα is rapidly occupied with guanosine triphosphate (GTP), activating the G proteins (Fig. 1a) 4–6. Based on sequence homology and signaling properties, Gα subunits are classified into four subfamilies: Gαs, Gαi/o, Gαq/11, and Gα12/13 4.

The activated GPCRs are also phosphorylated and interact with arrestins; the receptor-bound arrestins desensitize the G protein-mediated signaling 7,8, regulate GPCR internalization, and/or initiate G protein-independent signal transduction (Fig. 1a) 8–11. Arrestins are composed of four subtypes, arrestin 1–4; arrestin 1 and 4 are primarily expressed in the visual system, while arrestin 2 and 3 (also called β-arrestin 1 and 2 [βarr1 and βarr2], respectively) are expressed ubiquitously 7,10,12.

Most studies have focused on the interaction between GPCRs and their downstream signaling molecules; however, emerging evidence suggests that G proteins and arrestins can physically or functionally interact 13–16, βarr1 directly interacts with Gαs 16, GPCRs can form a “mega-complex” comprising both G proteins and βarrs 15, and receptor-mediated Gαi–βarr complexes facilitate extracellular signal-regulated kinase 1/2 (ERK1/2) activation 14. Despite these findings, the precise functional and structural mechanisms of βarr–Gα interactions remain poorly defined.

Herein, we studied the functional and structural interaction between βarr and Gα. As previous reports suggest that βarr1 can directly or functionally interact with Gαs or Gαi1 13–16, we selected βarr1 as a representative βarr and investigated its interaction with Gαs or Gαi1. We measured the binding affinities between βarr1 and Gαs or Gαi1 under various activation states using microscalethermophoresis (MST). The functional implications of these interactions were investigated using BODIPY-FL–GTPγS for Gα activation analyses and using the house-developed β-strand XX (βXX) release assay and an intramolecular bioluminescence resonance energy transfer (BRET) sensor for βarr1 activation analyses. The structural mechanism and binding interfaces between βarr1 and Gαs or Gαi1 interactions were analyzed using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Together, these approaches provide a comprehensive framework for understanding βArr–Gα interaction at the molecular level.

Results
Interaction between different activation states of βarr1 and Gα
To test whether βarr1 physically interacts with Gαs or Gαi1, we measured the binding affinity (Kd) between purified βarr1 and purified Gαs or Gαi1 using MST. To assess how Gα activation state influences these interactions, we prepared Gα in GDP-bound (inactive) or GTPγS-bound (active) conformations.

The arrestin activation status can be modulated by introducing mutations or adding phosphorylated GPCRs. Basal state arrestins are stabilized by the interactions between the N- and C-domains, specifically through the interaction between βI, αI, and C-terminal βXX (three-elements interaction, Fig. 1b, blue circle) as well as the ionic interactions between the residues in βIII, βX, gate loop, and C-tail (polar core interaction, Fig. 1b, orange circle) 17,18. Upon phosphorylated GPCR binding to the N-domain of arrestins, βXX is released from the N-domain, and the polar core is disrupted (Fig. 1c). Disruption of the interdomain interactions induces domain rotation and conformational changes of the loops between the two domains (such as finger, middle, lariat, and middle loops; Fig. 1c) 19,20. These structural changes are canonical features of arrestin activation.

However, arrestin activation does not follow a simple binary switch between basal and active states. Instead, arrestins undergo dynamic conformational states that are finely tuned by the activating receptor, its ligand, and the receptor’s phosphorylation pattern. These inputs shape globally and locally distinct active states that selectively recruit downstream partners, as proposed in the barcode or flute model of arrestin signaling 21–24.

Truncation of the C-terminus, including βXX, transforms arrestins into an “active conformational ensemble state” by mimicking βXX release but without occupying the phosphorylated receptor-binding pockets. This active conformational ensemble state is relieved from autoinhibition but not locked into a specific active conformation and therefore has been widely used to study the mechanism of arrestin activation 19,25–27.

In this study, we used full-length βarr1 to represent the basal state, C-terminally truncated βarr1 (βarr1_ΔC) to represent the active conformational ensemble state rather than a single active state, and V2Rpp-bound βarr1 (V2Rpp, a phosphopeptide from the vasopressin type-2 receptor) as one specific receptor-tail–stabilized active conformer 13,19,28.

When the interaction between the basal state βarr1 and GDP-bound or GTPγS-bound Gαs was analyzed using MST, the interactions were insignificant (Fig. 1d, e). Similarly, GTPγS-bound Gαi1 did not significantly interact with the basal state βarr1 (Fig. 1g). However, the GDP-bound Gαi1 interacted with basal state βarr1 (Kd = 290.48 ± 111.99 nM) with relatively low MST response amplitude (approximately 5‰) (Fig. 1f). When the interaction between the V2Rpp-bound βarr1 and GDP-bound or GTPγS-bound Gα was analyzed using MST, the interactions were insignificant for all tested conditions (Fig. 1h–k). Conversely, βarr1_ΔC significantly interacted with all tested Gα proteins (GDP-bound Gαs, GTPγS-bound Gαs, GDP-bound Gαi1, and GTPγS-bound Gαi1) with similar affinities (Kd: 80.68–161.92 nM; Fig. 1l–o).

These results indicate that Gαs and Gαi1, whether in their inactive or active states, preferentially interact with the active conformational ensemble state βarr1 (βarr1_ΔC) but not with basal or V2Rpp-specific active states. V2Rpp occupancy at phosphate-binding pockets may favor a particular active-like conformer that does not engage Gα efficiently. On the contrary, C-tail truncation permits interconversion among multiple active-like sub-states, increasing the probability of populating a Gα-compatible conformation. Of note, GDP-bound Gαi1 also engages basal βarr1, albeit with significantly lower affinity than with βarr1_ΔC (Supplementary Fig. 1b).

HDX analyses upon complex formation between βarr1_ΔC and Gαs or Gαi1
To identify interaction interfaces and conformational changes associated with βarr1–Gα complex formation, we performed HDX-MS. HDX-MS probes the conformational dynamics of proteins by measuring the exchange rate between the amide hydrogens in the proteins and deuterium in the solvent 29,30. This method is particularly effective in detecting protein–ligand interfaces and conformational changes, even in systems with weak or transient interactions 31,32. Upon complex formation, solvent accessibility typically decreases at the protein–protein interface, reducing HDX rate. Additionally, an altered HDX rate indicates altered backbone flexibility or changes in secondary structure stability.

Given the stronger binding affinity of βarr1_ΔC, we co-incubated purified βarr1_ΔC with GTPγS-bound Gαs or Gαi1, followed by HDX at various durations (10, 100, 1000, and 10,000 s) in D₂O buffer. Control samples included βarr1_ΔC, GTPγS–Gαs, and GTPγS–Gαi1 alone. After HDX, the samples were digested with pepsin, and peptide-level deuterium uptake was analyzed. The peptic peptides used for HDX-MS analyses are summarized in Supplementary Fig. 2, and the detailed information about the HDX-MS conditions and the peptic peptides is described in Supplementary Data 1 and 2, respectively.

Upon βarr1_ΔC binding, Gαs exhibited altered HDX in specific regions. Notably, the αD/αE loop showed enhanced deuterium uptake (Fig. 2a, red; Supplementary Fig. 3a, peptide 169–176), suggesting increased conformational flexibility in this region. In contrast, HDX decreased in multiple regions, including the αA/αB loop, αE/αF loop, switch I, and αG/α4 loop (Fig. 2a, blue; Supplementary Fig. 3a, peptides 108–118, 190–197, 201–208, and 316–331), indicating reduced solvent accessibility or increased structural stabilization upon complex formation.

Co-incubation with βarr1_ΔC decreased the HDX of Gαi1 in the αA/αB loop extending through αB, αD/αE loop, switch II, α3 helix, and adjacent α3/β5 loop, and the C-terminal part of α5 (Fig. 2b, blue; Supplementary Fig. 3b), suggesting that these regions may participate in complex formation or undergo conformational stabilization.

We next assessed the conformational changes in βarr1_ΔC upon complex formation. Binding to GTPγS–Gαs-induced minimal HDX changes in βarr1_ΔC. A small increase in HDX was observed in the βIV/βV loop (Fig. 2c, red; Supplementary Fig. 3c, peptide 43–54), suggesting localized flexibility, while a minor decrease in the lariat loop (Fig. 2c, blue; Supplementary Fig. 3c, peptide 282–289) indicated slight structural stabilization, although the effect on the lariat loop was modest (<0.2 Da). A similar pattern was observed upon binding to GTPγS–Gαi1, where modest HDX reductions were detected in the finger loop (Fig. 2d, blue; Supplementary Fig. 3d, peptide 69–75) and lariat loop (Fig. 2d, blue; Supplementary Fig. 3d, peptide 279–289), again with low magnitude changes (<0.2 Da).

Collectively, the HDX-MS results indicate that complex formation with βarr1_ΔC distinctly changes deuterium uptake across multiple regions of both Gαs and Gαi. Conversely, βarr1_ΔC exhibited only modest changes in HDX upon binding to either Gαs or Gαi1, with localized effects observed in loop regions, such as the βIV/βV, finger, and lariat loops. While the magnitude of these changes was small, they may reflect Gα binding-associated subtle conformational rearrangements and warrant further structural investigation.

Domain-level contributions of Gαs and Gαi1 to βarr1 binding
While HDX-MS provides valuable insights into regions affected by βarr1–Gα complex formation, the observed changes may reflect either direct contact surfaces or allosteric rearrangements. To directly assess the structural domains of Gα subunits contributing to βarr1 binding, we dissected the interaction using individual domains of Gαs and Gαi1. Gα subunits are composed of a Ras-like GTPase domain (RD) and an α-helical domain (AHD), which together coordinate nucleotide binding and hydrolysis (Fig. 2e). Nucleotide exchange is closely linked to conformational rearrangements between these two domains, particularly AHD displacement.

Our HDX-MS data showed βarr1_ΔC-induced HDX changes in both RD and AHD regions of Gαs and Gαi1 (Fig. 2a, b), suggesting that both domains might contribute to the interaction interface. To test this, we employed truncated constructs: miniGα variants (lacking the AHD) 33 and isolated AHD domains (residues 68–192 for Gαs AHD and 61–183 for Gαi1 AHD) (Supplementary Fig. 4). We then quantified their binding to βarr1_ΔC using MST.

The miniGαs construct displayed a significantly reduced binding affinity to βarr1_ΔC (Kd = 463.96 ± 140.49 nM) than the full-length GDP- or GTPγS-bound Gαs (Fig. 3a; Supplementary Fig. 1a). Conversely, the isolated Gαs AHD retained strong binding to βarr1_ΔC (Kd = 47.24 ± 14.98 nM), comparable to the full-length Gαs under both nucleotide states (Fig. 3b). These findings suggest that the Gαs AHD plays a dominant role in mediating the interaction with βarr1_ΔC, whereas the Gαs RD is less critical.

However, both miniGαi1 and Gαi1 AHD constructs exhibited Kd values comparable to that of the full-length GDP- or GTPγS-bound Gαi1 (Kd = 107.97 ± 27.08 and 62.54 ± 17.99 nM, respectively; Fig. 3c, d; Supplementary Fig. 1b). These data indicate that both the RD and AHD of Gαi1 contribute to βarr1 binding, and neither domain alone is solely responsible for the interaction.

HDX-MS analysis of βarr1 interaction interfaces within individual Gα domains
Building on our domain-level binding analysis, we next used HDX-MS to further map the potential interaction interfaces between βarr1_ΔC and the RD or AHD domains of Gαs and Gαi1 (Supplementary Data 3).

Upon co-incubation with βarr1_ΔC, miniGαs exhibited decreased deuterium uptake in the αG/α4 loop extending into α4 (Fig. 3e and Supplementary Fig. 5a), suggesting a conformational stabilization or reduction in solvent accessibility in this region. For the isolated AHD (Gαs AHD), reduced HDX was observed in the αA helix, αA/αB loop, αD/αE loop, and αE/αF loop (Fig. 3f and Supplementary Fig. 5b; peptides 68–99, 110-119, 169–176, and 190–198). Notably, the αG/α4 loop in RD and αA/αB and αE/αF loops in AHD were also protected upon βarr1_ΔC binding to full-length Gαs (Fig. 2a and Supplementary Fig. 3a), identifying these regions as strong candidates for the direct βarr1 binding interface (Fig. 3g, green-highlighted). Other regions affected in full-length Gαs, such as switch I and the αD/αE loop (Fig. 2a), were not consistently observed across the constructs and may reflect allosteric conformational changes rather than direct contacts.

Co-incubating βarr1_ΔC with miniGαi1 reduced HDX in the C-terminal part of α5 (Fig. 3h and Supplementary Fig. 5c). Gαi1 AHD displayed protection in the αA/αB loop extending through αB, αD/αE loop, and αE/αF loop (Fig. 3i and Supplementary Fig. 5d). Among these, the C-terminal part of α5 in the RD and the αA/αB through αB and αD/αE regions in the AHD were similarly protected in full-length Gαi1 upon βarr1_ΔC interaction (Fig. 2b and Supplementary Fig. 3b), reinforcing their likely involvement in complex formation (Fig. 3j, green-highlighted). Regions, such as switch II, α3, and α3/β5 loop, altered in the full-length Gαi1 complex (Fig. 2b), were not consistently affected in domain-specific constructs and may reflect allosteric effects.

Contrary to the HDX profiles of Gα domains, those of βarr1_ΔC remained largely unchanged upon co-incubation with miniGα or AHD constructs (Supplementary Data 3). The absence of significant protection in βarr1_ΔC may be because the binding occurs through side chains without affecting the backbone dynamics, which may not be sufficient to induce measurable HDX level changes within βarr1. Further structural characterization, such as crosslinking-mass spectrometry or cryo-EM, may be needed to define βarr1’s contact surfaces at higher resolution.

Collectively, the HDX-MS results indicate that although both RDs and AHDs are involved in both βarr1–Gαs and βarr1–Gαi1 complex formation, the binding interfaces (compare Fig. 3g and 3j) and allosteric conformational changes (compare Fig. 2a and 2b) might differ between these complexes.

βarr1 does not alter the activation status of Gα subunits
Having established that βarr1_ΔC directly binds both Gαs and Gαi1 through interfaces spanning RD and AHD, we next asked whether this interaction influences G protein activation. To assess the GDP/GTP exchange rate, we employed BODIPY-FL–GTPγS, a BODIPY fluorescence-labeled GTP analog (Fig. 4a). In solution, BODIPY fluorescence is quenched by the guanine ring; however, this quenching is relieved upon incorporation into Gα 34,35. Thus, an increased fluorescence corresponds to GDP release and BODIPY-FL–GTPγS binding and serves as a proxy for Gα activation.

We measured fluorescence changes over time upon incubating BODIPY-FL–GTPγS with purified Gαs or Gαi1 in the presence or absence of βarr1_ΔC. In both cases, GTPγS incorporation rate and extent were unaffected by βarr1_ΔC (Fig. 4b, c), indicating that βarr1 binding does not significantly alter the nucleotide exchange activity of either Gα isoform under these conditions. These results suggest that while βarr1_ΔC engages both Gαs and Gαi1 directly, the interaction does not modulate their activation status, at least in terms of GDP/GTP turnover.

Gαs–βarr1 interactions modulate βarr1 activation status
A previous study has suggested that the interaction of Gαi1 with βarr1 regulates ERK1/2 activation 14. Building on our observation of direct binding between βarr1_ΔC and Gαs or Gαi1 (Fig. 1l–o), we investigated whether these interactions affect βarr1 activation status. As βarr1 activation is associated with the displacement of its C-terminal βXX, we assessed this structural hallmark using a fluorescence-based βXX release assay 36. This method leverages the environment-sensitive nature of bimane fluorescence and its quenching by nearby aromatic residues, such as tryptophan 37.

In our βXX release assay system, residue 385 (adjacent to βXX) was labeled with bimane, and a tryptophan was introduced at residue 5 (Fig. 5a, yellow and cyan circles, respectively). In the basal state, proximity between these residues quenches bimane fluorescence (Fig. 5a, upper panel). Upon βXX release, the increased separation between residues 5 and 385 reduces the quenching effect (Fig. 5a, lower panel), resulting in an increase in fluorescence intensity accompanied by a slight red shift in the emission spectrum (Supplementary Fig. 6).

Co-incubation with V2Rpp increased bimane fluorescence, reflecting βXX release upon V2Rpp-binding (Fig. 5b, gray solid bars). Conversely, GTPγS-bound Gαi1 or Gαs alone did not alter bimane fluorescence (Fig. 5b, green and red empty bars), suggesting that neither Gα directly activates βarr1 in the absence of receptor input in this in vitro system.

Interestingly, GTPγS-bound Gαs, but not Gαi1, enhanced V2Rpp-induced βXX release when added before or after V2Rpp co-incubation (Fig. 5b, red solid bars), indicating that active Gαs can potentiate receptor-mediated βarr1 activation. These findings suggest a cooperative mechanism where Gαs amplifies the conformational activation of βarr1 in response to receptor-derived phosphopeptides, while Gαi1 does not share this capability.

Both RD and AHD of Gαs are required for potentiating βarr1 activation
As βarr1_ΔC directly interacts with both miniGαs and Gαs AHD (Fig. 3), we tested which domain is responsible for the potentiation of V2Rpp-induced βXX release. Neither miniGαs nor Gαs AHD alone enhanced V2Rpp-induced βXX release (Fig. 5c, d). These findings indicate that while both domains contribute to βarr1 binding, with AHD playing the predominant role, they are individually insufficient to facilitate βarr1 activation. Instead, full-length Gαs requires concerted action between RD and AHD to promote βarr1 conformational response to V2R-derived signals.

Validation of the physical and functional interaction between Gαs and βarr1 in the cell
To validate the functional interaction between Gαs and βarr1, we first examined whether these proteins associate in cells using the proximity ligation assay (PLA) (Fig. 6a). HEK293S cells were transfected with V2R, and PLA signals representing endogenous Gαs–βarr1 complex formation were measured in the presence or absence of arginine vasopressin (AVP) stimulation. Upon AVP treatment, we observed a significant increase in PLA puncta, with 26.27 ± 3.20 puncta per nucleus, compared to untreated cells (P < 0.0001) (Fig. 6b, c). These results indicate that endogenous Gαs and βarr1 interact in cells, and that this interaction is enhanced upon activation. As our MST analyses showed that the activation state of Gαs does not affect the interaction with βarr1 (Fig. 1l, m), the AVP-dependent increase in complex formation is most likely explained by activation of βarr1 rather than by changes in Gαs.

Notably, our MST with purified proteins showed that Gαs does not interact with V2Rpp-bound βarr1 (Fig. 1h, i), suggesting a discrepancy between in vitro and cellular observations. This difference can likely be attributed to the distinct conformational states of βarr1 induced in each system. In cells, receptor activation leads to dual engagement of βarr1 through both the phosphorylated C-terminal tail and the receptor core 38, whereas V2Rpp alone promotes only tail engagement, which may not fully stabilize the Gαs-interacting conformation. In addition, β-arrestins can remain signaling-competent after receptor dissociation, sustaining an active-like ensemble that can engage other downstream signaling molecules 39,40. This receptor-free, signaling-competent ensemble likely resembles βarr1_ΔC, providing a basis for its interaction with Gαs in cells.

As an next step, we investigated whether the interaction affects the activation status of βarr1 using an intramolecular BRET sensor (Fig. 6d). In this sensor, the basal (βXX–engaged) conformation yields a higher BRET signal, whereas βXX release reduces BRET. Gαs overexpression significantly decreased the BRET signal, indicating that Gαs alone can promote βarr1 activation (Fig. 6e). As expected, AVP reduced the BRET signal as V2R phosphorylated C-tail engages at the N-domain and replacing βXX (Fig. 6e). Co-expression of Gαs potentiated this AVP-induced βXX release (Fig. 6e).

By contrast, in our bimane fluorescence quenching assay with purified proteins, Gαs alone did not trigger βXX release (Fig. 5b). This discrepancy likely reflects cell-specific factors that prime βarr1 in vivo. For example, overexpressed V2R can exhibit basal activity, generating a subpopulation of phosphorylated receptors that biases βarr1 toward activation. In addition, plasma-membrane lipids such as phosphatidylinositol 4,5-bisphosphate (PIP) can allosterically favor pre-active arrestin ensembles even in the absence of receptor engagement 36,41–43. Thus, cellular βarr1 likely exists as an ensemble of basal and active-like states, with Gαs overexpression shifting the equilibrium toward βXX release.

In summary, convergent evidence from cell-based and in vitro assays support a model in which Gαs preferentially engages the active conformational ensemble state of βarr1 and promotes or stabilizes the βXX-released conformation (Fig. 6g) while the precise βarr1 sub-state favored by Gαs remains to be defined.

Conformational changes of full-length βarr1 or Gαs upon interaction
To explore the structural mechanism by which Gαs facilitates βarr1 activation (Figs. 5b and 6e, f), we performed HDX-MS on full-length βarr1 in the absence or presence of V2Rpp, with or without GTPγS-bound Gαs co-incubation. After 60 min co-incubation, HDX was initiated by transferring samples into D₂O. The samples were pepsin-digested and analyzed by mass spectrometry (Supplementary Data 4).

When GTPγS-bound Gαs was co-incubated, full-length βarr1 either with or without V2Rpp did not show HDX changes. Similarly, we could not detect HDX changes in Gαs. The lack of Gαs-induced HDX changes in full-length βarr1 either with or without V2Rpp can be explained by the lack of stable binding between Gαs and the basal or V2Rpp-bound state βarr1 (Fig. 1d, e, h, i).

Nevertheless, Gαs facilitated βXX release from V2Rpp-bound state in in vitro (Fig. 5b) and both basal and V2R-activated βarr1 in cells (Fig. 6e, f). We interpret this as a transient, low-occupancy allosteric effect. Gαs briefly engages βarr1 when the protein samples active conformational ensembles, thereby promoting βXX release, but dissociates as βarr1 transitions to subsequent conformations (Fig. 6g). In the in vitro setting, V2Rpp may populate only a small fraction of such Gαs-competent ensembles, yielding functional potentiation without sufficient complex lifetime or abundance for detection by MST or HDX-MS. In cells, receptor activity and membrane factors likely enrich these active-like ensembles even within subpopulations of basal or V2R-activated βarr1, enabling the observed effect.

Methods
Gα, GαAHD, and miniGα expression and purification
As described previously 33,46–48, the cDNA encoding human Gαi1 containing an N-terminal His-tag and TEV cleavage site was constructed in pET21a. Human Gαs cDNA with an N-terminal His-tag and an HRV3C protease cleavage site was inserted into pET28a. Recombinant Gαs AHD and Gαi1 AHD with an N-terminal His-tag followed by a thrombin cleavage site, and a C-terminal FLAG tag were synthesized and constructed in pET28a. Recombinant miniGαs and miniGαi1 containing an N-terminal His-tag and TEV cleavage site were cloned into pET21a.

All constructs were transformed into Escherichia coli LOBSTR cells and grown in Terrific Broth (TB) medium in the presence of an antibiotic at 37°C until OD600 reached 0.6–0.8. Protein expression was induced with 0.03 mM IPTG at 16°C for 24 h for full-length Gα proteins, 0.5 mM IPTG at 37°C for 4 h for AHD constructs, and 0.5 mM IPTG at 16°C for 24 h for miniGα proteins. Cell pellets were harvested and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.4, 300 mM NaCl, 2 mM MgCl2, 20 µM GDP, 1:1000 protease inhibitor cocktail, 2.5 µg/mL leupeptin, 10 µg/mL benzamidine, 100 µM TCEP) in three-fold cell pellet volume. Lysozyme was added at 3 mg/mL and incubated at room temperature (22–25°C) for 30 min, followed by incubation with 10 µg/mL DNase I for another 30 min. Lysates were collected by centrifugation at 18,000 × g for 30 min at 4°C, supplemented with 20 mM imidazole, and loaded on an Ni-IDA column equilibrated with lysis buffer containing 20 mM imidazole. The resin was washed with wash buffer (20 mM HEPES pH 7.4, 300 mM NaCl, 2 mM MgCl2, 20 µM GDP, 1:1000 protease inhibitor cocktail, 2.5 µg/mL leupeptin, 10 µg/mL benzamidine, 100 µM TCEP, and 20 mM imidazole). The proteins were eluted with elution buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl2, 20 µM GDP, 1:1000 protease inhibitor cocktail, 2.5 µg/mL leupeptin, 10 µg/mL benzamidine, 100 µM TCEP, 200 mM imidazole) and further purified using a Superdex 200 (10/300) column with ÄKTA FPLC. For GTPγS-bound Gα preparation, 20 μM GTPγS was added instead of GDP during the wash and elution steps of Ni-IDA purification. Subsequently, 1 mM GTPγS was added to the Ni-IDA eluate and incubated on ice for 1 h, followed by gel filtration in buffer containing 20 μM GTPγS.

βarr1 expression and purification
Rat βarr1 cDNA with an N-terminal His-tag and a thrombin cleavage site was cloned into the pET28a vector. A C-terminal truncated variant (βarr1_ΔC, residues 1–365) was generated by introducing a stop codon at residue 365 via site-directed mutagenesis. A cysteine-free rat βarr1 variant (C59V/C125S/C140I/C150V/C242V/C251V/C269S) containing an N-terminal His-tag and a thrombin cleavage site was cloned into the pET28b vector. A site-directed mutant (G5W/D385C) was subsequently generated from this construct.

βarr1 constructs were expressed in E. coli BL21 (DE3) cells. Cultures were grown in Luria Bertani (LB) medium containing antibiotics at 37°C and induced with 0.03 mM IPTG when OD600 reached 0.6, followed by 24 h incubation at 16°C. After induction, the cells were harvested, centrifuged, and resuspended in the lysis buffer (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 1:1000 protease inhibitor cocktail, 2.5 µg/mL leupeptin, 10 µg/mL benzamidine, 100 µM TCEP) in five-fold cell pellet volume. Lysozyme was added at 3 mg/mL and incubated at room temperature for 30 min, followed by incubation with 10 µg/mL DNase I for another 30 min. The lysates were collected by centrifugation at 18,000 × g for 30 min at 4°C. The supernatant was supplemented with 20 mM imidazole and loaded on a Ni-IDA column pre-equilibrated with lysis buffer containing 20 mM imidazole. The resin was washed with wash buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1:1000 protease inhibitor cocktail, 2.5 µg/mL leupeptin, 10 µg/mL benzamidine, 100 µM TCEP, and 20 mM imidazole). The proteins were eluted with elution buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1:1000 protease inhibitor cocktail, 2.5 µg/mL leupeptin, 10 µg/mL benzamidine, 100 µM TCEP, 200 mM imidazole). The Ni-IDA purified βarr1 and βarr1_ΔC proteins were further purified using a HiTrap Q column or an SP column (GE Healthcare), respectively, and subsequently buffer-exchanged using a Zeba desalting column to reduce salt concentration for further use. For the cysteine-free βarr1 G5W/D385C variant, Ni-IDA purified proteins were further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column on an ÄKTA FPLC system.

Synthetic peptide
The V2Rpp peptide (ARGRpTPPpSLGPQDEpSCpTpTApSpSpSLAKDTSS), derived from the phosphorylated C-terminus of the human V2 vasopressin receptor, was synthesized by the Tufts University Core Facility.

Microscale thermophoresis (MST)
βarr1 was labeled using the Monolith NT™ Protein Labeling Kit RED-MALEIMIDE 2nd Generation (NanoTemper) according to the manufacturer’s instructions. Labeled βarr1 (100 nM) was mixed with serially diluted unlabeled proteins (Gα, GαAHD, or miniGα) in interaction buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM MgCl2, 100 µM TCEP, 0.05% Tween-20, and 20 µM GDP or GTPγS. For V2Rpp-bound βarr1, labeled βarr1 was pre-incubated with a 10-fold molar excess of V2Rpp for 30 min at room temperature to pre-form the V2Rpp–βarr1 complex. This complex was then titrated with unlabeled Gα, Gα_AHD, or miniGα in the same interaction buffer supplemented with 30 µM V2Rpp to maintain the peptide-bound state during the binding assay. After incubating for 15 min at room temperature, the samples were loaded into premium capillaries (NanoTemper). MST measurements were performed on a Monolith NT.115 (NanoTemper) using 20% excitation power and medium MST power at 25°C. Data were collected from the MST-on phase (5 s), and best-fit Kd values were calculated using MO.Affinity Analysis software (v2.3) with a defined 68% confidence interval (~1 standard deviation) for uncertainty. All measurements with a signal-to-noise ratio of ≥5, as well as response amplitude ≥5, were accepted as binding events.

HDX-MS
To form βarr1–Gα complexes, βarr1 and G protein (Gα, GαAHD, or miniGα) were mixed at 1:1 (40 µM: 40 µM) and 1:2 (40 µM: 80 µM) molar ratios, followed by incubation for 40 min at room temperature. Hydrogen/deuterium exchange was initiated by mixing 5 µL G protein, βarr1, and complex samples with 25 µL D2O buffer (20 mM HEPES pD 7.4, 150 mM NaCl, 2 mM MgCl2, 20 µM GTPγS or GDP, 100 µM TCEP, 10% glycerol in D2O) and incubating on ice for 10, 100, 1000, or 10,000 s. The deuterated samples were quenched by adding 30 µL ice-cold quench buffer (60 mM NaH2PO4, pH 2.01, 10% glycerol, 20 mM TCEP) at each time point. Non-deuterated control samples were prepared in parallel using H2O buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl2, 20 µM GTPγS or GDP, 100 µM TCEP, 10% glycerol in H2O), followed by quenching and freezing as above.

The quenched samples were sent for digestion by passing through an immobilized pepsin column (2.1 × 30 mm2; Life Technologies, Carlsbad, CA, USA) at a 100 µL/min flow rate with 0.05% formic acid in H2O at 12°C. The peptide fragments were subsequently collected on a C18 VanGuard trap column (1.7 µm × 30 mm; Waters, Milford, MA, USA) and desalted with 0.05% formic acid in H2O. Peptic peptides were then separated using ultra-pressure liquid chromatography (UPLC) on an ACQUITY UPLC C18 column (1.7 µm, 1.0 × 100 mm2; Waters) at 40 µL/min with an acetonitrile gradient created by two pumps: mobile phase A (0.15% formic acid in H2O) and B (0.15% formic acid in acetonitrile). The gradient started at 8% B and increased to 85% B over 8.5 min. To minimize the back-exchange of deuterium to hydrogen, the sample, solvents, trap, and UPLC column were all maintained at pH 2.5 and 0.5°C during analysis. Mass spectrometry analyses were performed using a Xevo G2-XS QTof (Waters) as established previously 49,50. The peptides were identified in the ND samples using the Protein Lynx Global Server 2.4 (Waters). To process the HDX-MS data, the amount of deuterium in each peptide was determined by measuring the centroid of the isotopic distribution using DynamX 3.0 (Waters). The average back-exchange in our system was approximately 30–40%. Back-exchange correction was not performed, as the proteins exhibited aggregation in fully deuterated samples, and the analyses focused on comparative assessments between different conformational states.

BODIPY-FL–GTPγS assay
Nucleotide exchange function of Gα was assessed by monitoring the binding of BODIPY-FL–GTPγS (Invitrogen, Waltham, MA, USA) to GDP-bound Gα proteins. BODIPY-FL–GTPγS (final concentration: 500 nM) was prepared in imaging buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 100 µM TCEP. Fluorescence baseline was recorded for 120 s using a Synergy Neo or Neo2 plate reader (BioTek, Winooski, VT, USA) in a 96-well black plate, with excitation at 485 nm (3 nm bandwidth) and emission at 510 nm (20 nm bandwidth). After baseline acquisition, 1.5 µM GDP-bound Gα or Gα–βarr1 complex was added to the wells, and fluorescence was recorded every 10 s for 80 min. All experiments were performed at room temperature and repeated in triplicate. The resulting kinetics spectra were plotted using GraphPad Prism 8.0.

β-arrestin βXX release assay
Cys-free βarr1 G5W/D385C (final concentration: 20 µM) was prepared in 20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl2, and labeled by co-incubation with a 10-fold molar excess of bromobimane for 1 h on ice. Excess dye was removed using a Zeba spin desalting column. The labeled βarr1 was then diluted to 5 µM final concentration and incubated with 15 µM Gα, GαAHD, or miniGα for 1 h at room temperature in the presence or absence of pre-incubated 50 µM V2Rpp. Fluorescence measurements were performed in a MicroFluor 96-well plate, with excitation at 390 nm and emission spectra recorded from 420 to 600 nm in 1-nm increments using a Synergy Neo or Neo2 plate reader (BioTek, Winooski, VT, USA). Fluorescence changes were quantified by measuring the emission intensity at the peak wavelength and normalized as a fold change relative to the vehicle-treated control.

Proximity ligation assay (PLA)
PLA experiment was done using the Duolink® in situ detection reagent green kit (Cat# DUO92008, Sigma-Aldrich) following the provided protocol. Briefly, cells were fixed and permeabilized before incubation with primary antibodies (rabbit anti-Gαs, #ab235959, Abcam; mouse anti-βarr1 (ZA005), Cat# 39-5000, Invitrogen) for 1 h at RT, followed by incubation with the provided oligonucleotide-conjugated secondary antibodies for 30 min at 37°C with washes after each step. Ligation and amplification were performed by incubating cells with the provided ligase, polymerase, and detection solutions. PLA images were acquired on a Leica Thunder imager (Leica), equipped with a 40x/1.3 NA objective and a Leica K8 sCMOS (Leica) controlled by LAS X software (Leica). The DAPI channel was acquired using a 391/32-nm excitation filter, a DFT51010 filter cube (11525418, Leica), a 415-nm dichroic mirror, and a 435/30-nm emission filter. PLA signal was acquired using a 479/33-nm excitation filter, a DFT51010 filter cube (11525418, Leica), a 500-nm dichroic mirror, and a 519/25-nm emission filter. PLA fluorescence images were analyzed using FIJI. Raw fluorescence images were corrected by background subtraction and converted to grayscale. Images of PLA puncta and cell nuclei were then adjusted for brightness and converted to binary mode. PLA puncta and nuclei were counted using the analyze particle plugin in FIJI based on the size criteria 0.3–10 pixel2 for PLA puncta and 30-infinite pixel2 for nuclei.

Bioluminescence resonance energy transfer (BRET) assay
A BRET-based β-arrestin 1 conformational biosensor was generated by fusing mCitrine and NanoLuc at the N- and C-terminus of rat β-arrestin 1, respectively, as described previously 51. Upon activation, conformational changes of β-arrestin can be monitored by detecting changes in BRET ratios in living cells. For the BRET assay, ΔGαs cells were seeded at a density of 4 × 105 cells per well in 6-well plates and co-transfected with plasmids encoding the β-arrestin 1 BRET sensor, human V2R, and human Gαs using Lipofectamine™ 2000 (Invitrogen, #11668019) according to the manufacturer’s instructions. After 6 h, the transfected cells were harvested and replated into white 96-well plates (4 × 104 cells per well; Greiner, #655083) for 24 h. On the day of the assay, culture medium was replaced with HBSS buffer (pH 7.4) containing 20 mM HEPES, and NanoLuc substrate (Promega, #N1662) was added at a 1:1000 dilution. BRET signals were recorded using a CLARIOstar plate reader (BMG) in monochromator mode, with donor emission collected at 440–480 nm and acceptor emission at 515–555 nm. Before measurement, cells were dark-adapted in the reader for 5 min, followed by five baseline readings. After AVP addition, BRET changes were monitored for 20 min. The BRET ratio was calculated as the value of the acceptor channel divided by that of the donor channel.

Statistics and reproducibility
For HDX-MS analysis, statistical significance was determined using a two-tailed unpaired Student’s t-test (P < 0.05) combined with a mass difference > 0.2 Da to assess the differences between samples with and without the binding partner. For the C-tail release assay, a one-way ANOVA followed by Tukey’s post-hoc test was applied to analyze the differences among multiple conditions, with statistical significance set at P < 0.05. For the PLA assay, a two-tailed unpaired Student’s t-test was used to assess the differences in PLA signals with or without AVP treatment, and the statistical significance was set at P < 0.0001. For BRET assays, a two-tailed unpaired Student’s t-test was used to compare BRET signals in the absence or presence of Gαs, whereas a one-way ANOVA followed by Tukey’s post-hoc test was performed to analyze BRET responses across conditions with or without AVP treatment in the absence or presence of Gαs, with statistical significance set at P < 0.0001. More than three independent experiments were conducted for each dataset.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Duan et al seeks to examine the interaction between bArr1 and G proteins using purified proteins and biophysical techniques. This is to be commended, for such approach yielded quantitative measurement including estimations of interaction affinities. However, there are issues with the study both technical and related to the biological significance of the findings.\n1. Non-physiological protein concentrations. Fig. 1. Measured affinities range from 80 to 160 nM based on MXT. HDX was performed at 40 uM bArr1 and 40 uM or 80 uM Ga, diluted to ~6.6 uM for bArr1 (or ~13 uM for Ga). Other measurements were also performed with bARR1 in micromolar range. To evaluate biological relevance of the results the authors should compare these to the concentrations of endogenous Gs, Gi, and bArr1 in cells.\n2. Fig. 1. Full-length bArr1 shows no detectable interaction with either Gas or Gai active or inactive. The authors instead use truncated bArr1, bArr1_DC, with C-tail deleted that does show interaction. The authors claim that bArr1_DC represents the active form of bARR1, which is not precisely true. bArr1_DC is known for its lower selectivity towards phosphorylated over unphosphorylated receptors, but it lacks functional characteristic of receptor-bound arrestins such as the ability to activate ERK.\n3. Suppl. Figs. 2 and 4. Most changes in H/D exchange rates are detectable after 100 sec, a few are detected after 10 sec. Half-life of the complex with KD ~ 100 nM (Fig. 1) is less than 7 sec. Conformational changes in proteins (the authors repeatedly mention “conformational flexibility”) occur in femtosecond to microsecond range (e.g., as shown for rhodopsin). The authors should take this into account discussing their data and explain why they believe that their results are biologically relevant, especially considering that bArr1 with C-terminal deletion, not the full-length protein present in cells, was used in these experiments, and detected differences were relatively small.\n4. Figs. 1 and 6. Fig. 1 shows that full-length bArr1 does not interact with Gs heterotrimer or GTP S-liganded Gas subunit, only demonstrates weak interaction with Gi heterotrimer. Fig 6b suggests that GTPgamaS-liganded Gas (but not GTPgamaS-liganded Gai) facilitates C-tail release in full-length bArr1 induced by V2Rpp. The authors should explain this controversy.\n5. There is no clear description of statistical models or statistical analysis employed. For example, in Fig. 6 the legend states that one-way ANOVA followed by Tukey’s post hoc test was used. But it is unclear how that was done, for there are two factors here at least, V2Rpp and Ga.\nIn short, the data were obtained at super-physiological concentrations of proteins studied, which makes their biological relevance questionable. Even in these conditions detected changes were relatively small. Thus, the data do not unambiguously support authors’ conclusions. This work, upon addition of clear discussion of these points, belongs in a more specialized journal.", "reviewer_2": "I have been called in to comment on the HDX only. I have looked through the method and the data the authors are using fairly standard continuous labelling HDX over a decent range of timepoints there’s nothing in the protocols to make me worried.\nI wouldn’t have done the HDX experiments like this as I really think you need a dmax sample at least the control for back exchange this is probably more important when the difference are very small as in this m/s. However, I don’t really think its going to going to change the story – but maybe consider in the future.\nFigures 2 and 4 need scale bar to indicate the %D for the colour changes. I’m also surprised that so much of the structure is uncoloured in these images. It may be that there are many regions in the protein for which there were no corresponding peptides detected and the authors should also indicate this in the images.", "reviewer_3": "The manuscript by Longhan Duan et al. presents biochemical and biophysical data demonstrating a direct interaction between arrestins and G proteins, which appears to be sensitive to their conformational state (i.e., inactive or active) and may lead to distinct functional outcomes. Specifically, the authors analyzed the interaction between βarr1 and either Gαs or Gαi1 using microscale thermophoresis (MST) and hydrogen/deuterium exchange mass spectrometry (HDX-MS), with proteins prepared to favor either the active or inactive state. A deletion mutant of βarr1, referred to as βarr1_ΔC, was used to represent the \"active\" conformation of βarr1, while G proteins were prepared in GDP-bound (inactive) or GTPγS-bound (active) forms.\nThe functional consequences of these complexes were examined using fluorescence-based assays, which notably revealed that the active, GTPγS-bound Gαs can potentiate βarr1 activation in response to receptor-derived phosphopeptides.\nOverall, the manuscript is clearly written, and the experiments have been conducted and interpreted using established, validated methodologies.\nI have several questions and comments:\n• Have the authors tested the effect of receptor-derived phosphopeptides on the affinity between arrestins and G proteins in the MST experiments?\n• Upon deletion of the C-terminal region of arrestin, what do the authors propose occurs structurally? Is arrestin fluctuating between active and inactive conformations? In other words, do the authors believe that in the absence of receptor-derived phosphopeptides, all βarr1_ΔC molecules adopt an active-like state? If so, what would be the expected impact of receptor-derived phosphopeptides in the MST or functional assays?\n• The discussion in lines 242–244 could benefit from clarification regarding the lack of HDX changes. The probes used in HDX and bimane fluorescence experiments are quite different, and this distinction should be explicitly discussed.\n• Could the addition of scFv30 assist by further stabilizing the active conformation of arrestin in these experiments?\nMinor corrections:\n• Line 63: The word “muations” should be corrected to “mutations”.\n• Figure 6a: The label “D386C” should be corrected to “D385C”.\nA final technical point:\n• It is unclear whether the bimane spectra exhibited a shift in λ_max, as the authors report only fold changes in fluorescence intensity. Additional clarification on whether spectral shifts were observed and how the fluorescence intensity was quantified would be helpful."}, "author_response": "We greatly appreciate the time and effort the reviewers have dedicated to evaluating our manuscript. We understand the importance of addressing the comments raised and are thankful for the opportunity to clarify and enhance our study on Gα and βarr1 interaction.\nReviewer #1 (Remarks to the Author):\nThe manuscript by Duan et al seeks to examine the interaction between bArr1 and G proteins using purified proteins and biophysical techniques. This is to be commended, for such approach yielded quantitative measurement including estimations of interaction affinities. However, there are issues with the study both technical and related to the biological significance of the findings.\n1. Non-physiological protein concentrations. Fig. 1. Measured affinities range from 80 to 160 nM based on MXT. HDX was performed at 40 uM bArr1 and 40 uM or 80 uM Ga, diluted to ~6.6 uM for bArr1 (or ~13 uM for Ga). Other measurements were also performed with bARR1 in micromolar range. To evaluate biological relevance of the results the authors should compare these to the concentrations of endogenous Gs, Gi, and bArr1 in cells.\nResponse:\nWe thank the reviewer for raising this important point regarding the protein concentrations used in our assays. We acknowledge that the concentrations employed in our HDX-MS and other biophysical experiments are higher than the average endogenous cytosolic concentrations typically found in cells. However, the use of micromolar-range protein concentrations is a widely accepted approach in structural and biophysical studies, especially when investigating weak, transient, or dynamic protein–protein interactions, where higher concentrations are often necessary to drive complex formation in vitro and achieve sufficient signal-to-noise for meaningful detection.\nIndeed, both our group and others have routinely used super-physiological concentrations of GPCRs, G proteins, or arrestins to dissect conformational dynamics and binding interfaces (doi: 10.1038/s44319-024-00239-x, 10.1073/pnas.2026491118, 10.1038/s41467-020-16975-2, 10.1016/j.cell.2019.04.022). These in vitro systems allow precise control of stoichiometry and binding conditions, enabling mechanistic insights that are difficult to obtain in cellular systems.\nWhile average cytosolic concentrations of βarr1 and Gα subunits are generally estimated to be in the low nanomolar range, local concentrations can reach low micromolar levels near activated receptors or within membrane microdomains due to localized recruitment, diffusion barriers, and scaffolded interactions. This compartmental enrichment is particularly relevant for GPCR signaling, where protein crowding at the plasma membrane facilitates interactions that may be too transient or low-affinity to detect in bulk measurements.\nMoreover, the biological relevance of these interactions in a cellular context is supported by prior studies (doi: 10.1126/science.aay1833, 10.1016/j.febslet.2013.01.027, 10.1126/scisignal.abg5203) and by our companion manuscript by Lee et al., which demonstrates βarr1–Gα interactions in the cellular context. To further address the reviewer’s concern, we have now included cell-based validation experiments in the revised manuscript to directly test whether our in vitro findings hold in a more physiological setting. Given that βarr1–Gi interactions has already been characterized in prior studies and in Lee et al.'s work, we focused our in-cell validation on the βarr1–Gαs interaction.\nWe detected the interaction between endogenous Gαs and βarr1 in cells using proximity ligation assay (PLA) (Fig. 6a-c). To test whether this interaction influences the C-tail release of βarr1, we employed a BRET-based sensor system previously developed to monitor βarr C-terminal movement (doi: 10.1038/sj.embor.7400373; 10.1074/jbc.M306589200) (Fig. 6d). Notably, overexpression of Gαs alone was sufficient to promote βarr1 C-tail release (Fig. 6e), and it further enhanced V2R-induced C-tail release (Fig. 6f), suggesting that Gαs can allosterically modulate βarr1 conformation both independently and in conjunction with receptor activation.\nWe hope that these additions provide direct evidence that complements our in vitro findings and support the physiological plausibility of the observed βarr1–G protein interactions\n2. Fig. 1. Full-length bArr1 shows no detectable interaction with either Gas or Gai active or inactive. The authors instead use truncated bArr1, bArr1_DC, with C-tail deleted that does show interaction. The authors claim that bArr1_DC represents the active form of bARR1, which is not precisely true. bArr1_DC is known for its lower selectivity towards phosphorylated over unphosphorylated receptors, but it lacks functional characteristic of receptor-bound arrestins such as the ability to activate ERK.\nResponse:\nWe thank the reviewer for this important clarification. As Reviewer #3 raised a similar point, we have taken this opportunity to revise the manuscript and elaborate on the conformational complexity of arrestin activation.\nWe agree that βarr1_ΔC does not represent the fully active state of receptor-bound βarr1 and, thus, should not be termed as the \"active\" form. Arrestins are now understood to adopt a range of dynamic active conformations, rather than transitioning between simple basal and active structures. These conformational states are influenced by receptor identity, ligand type, and phosphorylation pattern, as demonstrated in multiple recent structural and biophysical studies (e.g., DOI: 10.1038/s41467-020-18433-5; 10.1038/ncomms9202; 10.1126/science.adj3347). This conformational diversity supports the “Barcode theory” and “Flute model” of arrestin signaling, which propose that different phosphorylation patterns of receptors encode distinct arrestin conformations that selectively engage downstream effectors. (e.g., DOI: 10.1126/scisignal.2001707; 10.1124/mol.116.107839).\nOur use of βarr1_ΔC was intended to study a more conformationally dynamic state that is primed for interaction with binding partners but not fixed in a single receptor-stabilized conformation. Therefore, we clarify that βarr1_ΔC should be considered a “pre-active” rather than fully active state. We have revised the manuscript accordingly and now refer to βarr1_ΔC as a “pre-active” form.\nFurthermore, in the revised manuscript, we added data of MST analyses between V2Rpp-bound fully-active βarr1 and Gαs/i1. Interestingly, all tested Gαs/i1 did not show interaction with V2Rpp-bound fully-active βarr1 (Fig. 1 h-k). These data suggest that Gαs/i1 interact better with conformationally dynamic pre-active state than basal or a V2Rpp-specific active states.\nPage 4:\n“However, arrestin activation does not follow a simple binary switch between basal and active states. Instead, arrestins undergo dynamic conformational states that are finely tuned by the activating receptor, its ligand, and the receptor’s phosphorylation pattern. These inputs shape globally and locally distinct active states that selectively recruit downstream partners, as proposed in the barcode or flute models of arrestin signaling21-24.\nTruncation of the C-terminus including βXX transforms arrestins into conformationally dynamic pre-active state by mimicking βXX release. This pre-active state is relieved from autoinhibition but not locked into a specific active conformation and therefore has been widely used study the mechanism of arrestin activation19,25-27. In this study, we used full-length βarr1 to represent the basal state, and excess amount of V2Rpp, the phospho-peptide derived from the vasopressin receptor type 2, was used to activate βarr113,19,28. To mimic the conformationally flexible pre-active state, we used C-terminally truncated βarr1 (βarr1_ΔC).\nWhen the interaction between the basal state βarr1 and GDP-bound or GTPγS-bound Gαs were analyzed using MST, the interactions were insignificant (Fig. 1d,e). Similarly, GTPγS-bound Gαi1 did not significantly interact with the basal state βarr1 (Fig. 1g). However, the GDP-bound Gαi1 interacted with basal state βarr1 (KD=290.48±111.99 nM) with relatively low MST response amplitude (approximately 5‰) (Fig. 1f). When the interaction between the V2Rpp-bound βarr1 and GDP-bound or GTPγS-bound Gα were analyzed using MST, the interactions were insignificant for all tested conditions (Fig. 1h-k). Conversely, βarr1_ΔC significantly interacted with all tested Gα proteins (GDP-bound Gαs, GTPγS-bound Gαs, GDP-bound Gαi1, and GTPγS-bound Gαi1) with similar affinities (KD: 80.68–161.92 nM; Fig. 1l–o).\nThese results indicate that Gαs and Gαi1, whether in their inactive or active states, preferentially interact with conformationally dynamic pre-active βarr1 (βarr1_ΔC) but not with basal or V2Rpp-specific active states. Of note, GDP-bound Gαi1 also engages basal βarr1, albeit with significantly lower affinity than with βarr1_ΔC (Supplementary Fig. 1b).”\n3. Suppl. Figs. 2 and 4. Most changes in H/D exchange rates are detectable after 100 sec, a few are detected after 10 sec. Half-life of the complex with KD ~ 100 nM (Fig. 1) is less than 7 sec. Conformational changes in proteins (the authors repeatedly mention “conformational flexibility”) occur in femtosecond to microsecond range (e.g., as shown for rhodopsin). The authors should take this into account discussing their data and explain why they believe that their results are biologically relevant, especially considering that bArr1 with C-terminal deletion, not the full-length protein present in cells, was used in these experiments, and detected differences were relatively small.\nResponse:\nWe appreciate the reviewer’s thoughtful comment regarding the temporal resolution of HDX-MS and the interpretation of dynamic structural changes. We agree that conformational transitions in proteins occur on timescales ranging from femtoseconds to microseconds. However, we respectfully clarify that the x-axis in HDX-MS plots does not represent the duration of binding or complex lifetime, but rather the duration of D₂O exposure used to monitor backbone amide hydrogen exchange.\nIn our experimental design, the βarr1–Gα complexes were pre-formed prior to D₂O labeling, as detailed in both Results and Methods sections:\nPage 5:\n“Given the stronger binding affinity of βarr1_ΔC, we co-incubated purified βarr1_ΔC with GTPγS-bound Gαs or Gαi1, followed by HDX at various durations (10, 100, 1 000, and 10 000 s) in D₂O buffer”\nPage 15:\n“To form βarr1–Gα complexes, βarr1 and G protein (Gα, GαAHD, or miniGα) were mixed at 1:1 (40 μM: 40 μM) and 1:2 (40 μM: 80 μM) molar ratios, followed by incubation for 40 min at room temperature. Hydrogen/deuterium exchange was initiated by mixing 5 μL G protein, βarr1, and complex samples with 25 μL D O buffer (20 mM HEPES pD 7.4, 150 mM NaCl, 2 mM MgCl₂, 20 μM GTPγS or GDP, 100 μM TCEP, 10% glycerol in D O) and incubated on ice for 10, 100, 1 000, or 10 000 s.”\nThus, the current HDX-MS measurements do not track complex formation kinetics but rather probe structural differences in the conformational ensemble of proteins in different states, which can be probed either with short D O incubation or longer D O incubation.\nAs for the biological relevance of the observed differences, although the detected HDX differences were modest in magnitude (which is typical for subtle allosteric effects), they were localized, reproducible, and state-specific—consistent with biologically meaningful rearrangements. Furthermore, we used C-terminally truncated βarr1 (βarr1_ΔC) as a pre-active model to allow conformational sampling without locking arrestin in a specific conformation (see also our response to the comment #2).\n4. Figs. 1 and 6. Fig. 1 shows that full-length bArr1 does not interact with Gs heterotrimer or GTPγS-liganded Gas subunit, only demonstrates weak interaction with Gi heterotrimer. Fig 6b suggests that GTPgamaS-liganded Gas (but not GTPgamaS-liganded Gai) facilitates C-tail release in full-length bArr1 induced by V2Rpp. The authors should explain this controversy.\nResponse:\nWe have discussed the potential explanation for this controversy as below.\nPage 11:\n“Nevertheless, Gαs facilitated βXX release (Fig. 5b and 6e,f). We interpret this as a transient, low-occupancy allosteric effect. Gαs may form short-lived complexes with full-length βarr1 that are sufficient to promote βXX release but dissociate rapidly, yielding low affinity that cannot be measured by MST and minimal time-averaged protection below HDX sensitivity. In this sense, Gαs acts allosterically modulating the βarr1 conformational ensemble without requiring a persistent, high-affinity complex.”\nPage 12:\n“Notably, full-length βarr1 did not exhibit measurable HDX changes upon Gαs binding in the presence or absence of V2Rpp, possibly reflecting transient interactions or localized structural effects below HDX-MS detection thresholds.”\n5. There is no clear description of statistical models or statistical analysis employed. For example, in Fig. 6 the legend states that one-way ANOVA followed by Tukey’s post hoc test was used. But it is unclear how that was done, for there are two factors here at least, V2Rpp and Ga.\nResponse:\nWe have now clearly stated the statistical analyses in the figure legends as below.\nDeuterium uptake plots (Supplementary Fig. 3 and 5):\n“Results were derived from 3 independent experiments, and data are presented as mean ± SEM. The statistical significance of the differences between two conditions at each D O incubation time was determined using Student’s t-test.”\nβXX release assay (Fig. 5):\n“Statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc test (*P<0.05) comparing all tested conditions. For simplicity, the statistical significance was noted only for the pairs with functional importance. Data are presented as mean ± SD from 6–7 independent experiments.”\nPLA assay (Fig. 6b)”\n“Data represent pooled counts from 42 (NT) and 44 (AVP) randomly selected fields of view across 3 independent experiments. Bars indicate mean ± SEM. Scatter plots show individual data points. *P < 0.0001; statistical analysis performed using unpaired two-tailed Student’s t-test.”\nBRET assay (Fig. 6e-f):\n“corresponding luminescence intensities are presented as mean ± SD with individual points from 27 wells per condition (right panel); unpaired Student’s t-test, *P < 0.001.”\n“data are from 9 wells per condition; one-way ANOVA with Tukey’s post-hoc test, *P < 0.0001.”\nIn short, the data were obtained at super-physiological concentrations of proteins studied, which makes their biological relevance questionable. Even in these conditions detected changes were relatively small. Thus, the data do not unambiguously support authors’ conclusions. This work, upon addition of clear discussion of these points, belongs in a more specialized journal.\nResponse:\nWe appreciate the reviewer’s concern regarding the biological relevance of our findings at the protein concentrations used. As described in our previous responses, we fully acknowledge the limitations of using super-physiological concentrations in vitro, which are often necessary to resolve transient or low-affinity interactions and conformational changes with sufficient sensitivity in biophysical assays such as HDX-MS. Importantly, to directly address this concern, we have now performed additional cell-based experiments that support the relevance of the observed βarr1–Gα interactions in a cellular context as discussed above, which provide in-cell validation of our in vitro observations and strengthen the physiological relevance of our conclusions.\nWe also respectfully disagree with the suggestion that this work is only suitable for a more specialized journal. Gα and β-arrestins are two central signaling effectors downstream of GPCRs, the largest and most clinically targeted family of membrane receptors. While the activation mechanisms of G proteins and arrestins have been individually well studied, their direct interaction remains poorly understood and underexplored. Our study provides new structural and mechanistic insight into this emerging aspect of GPCR signaling, which we believe will be of broad interest to the diverse readership of Nature Communications.\nReviewer #2 (Remarks to the Author):\nI have been called in to comment on the HDX only. I have looked through the method and the data the authors are using fairly standard continuous labelling HDX over a decent range of timepoints there’s nothing in the protocols to make me worried.\nI wouldn’t have done the HDX experiments like this as I really think you need a dmax sample at least the control for back exchange this is probably more important when the difference are very small as in this m/s. However, I don’t really think its going to going to change the story – but maybe consider in the future.\nResponse:\nWe sincerely thank the reviewer for their careful evaluation of the HDX-MS methodology and for the insightful comment regarding back-exchange correction. We fully agree that including a fully deuterated control is ideal for estimating and correcting for back-exchange, especially in cases where HDX differences are subtle. Unfortunately, in our system, arrestins exhibited significant aggregation under fully deuterated conditions, even in the presence of denaturants, which precluded reliable acquisition of D data.\nmax\nAs a result, we opted to proceed without back-exchange correction, with the understanding that our analyses are based on relative comparisons between different conformational or binding states rather than absolute uptake values. We have now clarified this limitation explicitly in the revised manuscript as follows:\nPage 16:\n“The average back-exchange in our system was approximately 30–40%. Back-exchange correction was not performed, as the proteins exhibited aggregation in fully deuterated samples, and the analyses focused on comparative assessments between different conformational states.”\nWe appreciate the reviewer’s suggestion and will certainly consider incorporating d control samples in future studies, should protein solubility permit\nFigures 2 and 4 need scale bar to indicate the %D for the colour changes. I’m also surprised that so much of the structure is uncoloured in these images. It may be that there are many regions in the protein for which there were no corresponding peptides detected and the authors should also indicate this in the images.\nResponse:\nIn the original versions of Figures 2 and 4, the color coding was intended to indicate qualitative differences in deuterium uptake relative to the control, rather than absolute %D values. Specifically, blue represents regions with decreased HDX, red indicates increased HDX, and white denotes no significant difference. We have now clarified this explicitly in the revised figure legends. We opted not to include a continuous %D scale because the majority of observed differences were relatively small (typically <10%), making a gradient scale potentially misleading.\nRegarding the uncolored regions, we fully agree that it is important to distinguish between areas showing no HDX change and those lacking peptide coverage. To address this, we have added peptide coverage maps in a new Supplementary Figure 2, which clearly indicate the regions for which no peptides were detected. This addition provides essential context for interpreting the HDX data and improves the transparency of our structural mapping.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Longhan Duan et al. presents biochemical and biophysical data demonstrating a direct interaction between arrestins and G proteins, which appears to be sensitive to their conformational state (i.e., inactive or active) and may lead to distinct functional outcomes. Specifically, the authors analyzed the interaction between βarr1 and either Gαs or Gαi1 using microscale thermophoresis (MST) and hydrogen/deuterium exchange mass spectrometry (HDX-MS), with proteins prepared to favor either the active or inactive state. A deletion mutant of βarr1, referred to as βarr1_ΔC, was used to represent the \"active\" conformation of βarr1, while G proteins were prepared in GDP-bound (inactive) or GTPγS-bound (active) forms.\nThe functional consequences of these complexes were examined using fluorescence-based assays, which notably revealed that the active, GTPγS-bound Gαs can potentiate βarr1 activation in response to receptor-derived phosphopeptides.\nOverall, the manuscript is clearly written, and the experiments have been conducted and interpreted using established, validated methodologies.\nI have several questions and comments:\n1. Have the authors tested the effect of receptor-derived phosphopeptides on the affinity between arrestins and G proteins in the MST experiments?\nResponse:\nYes, we investigated the interaction between V2Rpp-bound, fully active βarr1 and Gαs or Gαi1 using MST. Interestingly, none of the tested Gα subunits exhibited detectable binding to V2Rpp-bound βarr1 (Fig. 1h–k). These results suggest that Gαs and Gαi1 interact preferentially with the conformationally dynamic, pre-active state of βarr1 such as the C-terminally truncated βarr1 rather than with the V2Rpp-specific active conformation.\nWe have now clarified this point in the revised manuscript and expanded the discussion on the conformational flexibility of the C-terminal truncated βarr1, contrasting it with the more structurally constrained, receptor-bound active state.\nPage 4:\n“However, arrestin activation does not follow a simple binary switch between basal and active states. Instead, arrestins undergo dynamic conformational states that are finely tuned by the activating receptor, its ligand, and the receptor’s phosphorylation pattern. These inputs shape globally and locally distinct active states that selectively recruit downstream partners, as proposed in the barcode or flute models of arrestin signaling21-24.\nTruncation of the C-terminus including βXX transforms arrestins into conformationally dynamic pre-active state by mimicking βXX release. This pre-active state is relieved from autoinhibition but not locked into a specific active conformation and therefore has been widely used study the mechanism of arrestin activation19,25-27. In this study, we used full-length βarr1 to represent the basal state, and excess amount of V2Rpp, the phospho-peptide derived from the vasopressin receptor type 2, was used to activate βarr113,19,28. To mimic the conformationally flexible pre-active state, we used C-terminally truncated βarr1 (βarr1_ΔC).\nWhen the interaction between the basal state βarr1 and GDP-bound or GTPγS-bound Gαs were analyzed using MST, the interactions were insignificant (Fig. 1d,e). Similarly, GTPγS-bound Gαi1 did not significantly interact with the basal state βarr1 (Fig. 1g). However, the GDP-bound Gαi1 interacted with basal state βarr1 (KD=290.48±111.99 nM) with relatively low MST response amplitude (approximately 5‰) (Fig. 1f). When the interaction between the V2Rpp-bound βarr1 and GDP-bound or GTPγS-bound Gα were analyzed using MST, the interactions were insignificant for all tested conditions (Fig. 1h-k). Conversely, βarr1_ΔC significantly interacted with all tested Gα proteins (GDP-bound Gαs, GTPγS-bound Gαs, GDP-bound Gαi1, and GTPγS-bound Gαi1) with similar affinities (KD: 80.68–161.92 nM; Fig. 1l–o).\nThese results indicate that Gαs and Gαi1, whether in their inactive or active states, preferentially interact with conformationally dynamic pre-active βarr1 (βarr1_ΔC) but not with basal or V2Rpp-specific active states. Of note, GDP-bound Gαi1 also engages basal βarr1, albeit with significantly lower affinity than with βarr1_ΔC (Supplementary Fig. 1b).”\n2. Upon deletion of the C-terminal region of arrestin, what do the authors propose occurs structurally? Is arrestin fluctuating between active and inactive conformations? In other words, do the authors believe that in the absence of receptor-derived phosphopeptides, all βarr1_ΔC molecules adopt an active-like state? If so, what would be the expected impact of receptor-derived phosphopeptides in the MST or functional assays?\nResponse:\nWe thank the reviewer for this important clarification. We agree that βarr1_ΔC does not represent the fully active state of receptor-bound βarr1 and, thus, should not be termed as the \"active\" form. As Reviewer #1 raised a similar point, we have taken this opportunity to revise the manuscript and elaborate on the conformational complexity of arrestin activation.\nArrestins are now understood to adopt a range of dynamic active conformations, rather than transitioning between simple basal and active structures. These conformational states are influenced by receptor identity, ligand type, and phosphorylation pattern, as demonstrated in multiple recent structural and biophysical studies (e.g., DOI: 10.1038/s41467-020-18433-5; 10.1038/ncomms9202; 10.1126/science.adj3347). This conformational diversity supports the “Barcode theory” and “Flute model” of arrestin signaling, which propose that different phosphorylation patterns of receptors encode distinct arrestin conformations that selectively engage downstream effectors. (e.g., DOI: 10.1126/scisignal.2001707; 10.1124/mol.116.107839).\nOur use of βarr1_ΔC was intended to study a more conformationally dynamic state that is primed for interaction with binding partners but not fixed in a single receptor-stabilized conformation. Therefore, we have revised the manuscript to clarify that βarr1_ΔC should be considered a “pre-active” rather than fully active state. We have revised the manuscript accordingly and now refer to βarr1_ΔC as a “pre-active” form.\nAs discussed above response to comment #1, Gαs and Gαi1 interact preferentially with the conformationally dynamic pre-active state of βarr1 rather than with the basal or V2Rpp-specific active states. In line with this observation, in the Discussion section of the original manuscript, we already suggested that Gα-binding would induce distinct conformation, which may be different from V2Rpp-bound form.\n“Notably, our findings raise the possibility that Gαs induces a previously uncharacterized βarr1 conformation—distinct from classical “active” or “basal” states—thereby contributing to signaling diversity. This hypothesis also aligns with the \"barcode\" theory of arrestins, which posits that that discrete arrestin conformations can encode specific downstream signaling outcomes”\nIn the revised manuscript, we discussed further about this Gα-induced distinct conformation.\nPage 12:\n“Notably, our findings raise the possibility that Gα induces a previously uncharacterized βarr1 conformation—distinct from classical “active” or “basal” states—thereby contributing to signaling diversity. Moreover, arrestins can remain active or signaling-competent state even after dissociation from the receptor39,40, potentially corresponding to the βXX-released “pre-active” conformation. It is possible that Gαs or Gαi1 can engage with βarr1 in this pre-active state within cells, promoting distinct conformational rearrangements, as captured by HDX-MS (Fig. 2c and 2d), which may underlie divergent downstream signaling profiles. This hypothesis also aligns with the \"barcode\" theory of arrestins, which posits that discrete arrestin conformations can encode specific downstream signaling outcomes46”\n3. The discussion in lines 242–244 could benefit from clarification regarding the lack of HDX changes. The probes used in HDX and bimane fluorescence experiments are quite different, and this distinction should be explicitly discussed.\nResponse:\nWe have discussed as below.\nPage 11:\n“Nevertheless, Gαs facilitated βXX release (Fig. 5b and 6e). We interpret this as a transient, low-occupancy allosteric effect. Gαs may form short-lived complexes with full-length βarr1 that are sufficient to promote βXX release but dissociate rapidly, yielding low affinity that cannot be measured by MST and minimal time-averaged protection below HDX sensitivity. In this sense, Gαs acts allosterically modulating the βarr1 conformational ensemble without requiring a persistent, high-affinity complex.”\nPage 12:\n“Notably, full-length βarr1 did not exhibit measurable HDX changes upon Gαs binding in the presence or absence of V2Rpp, possibly reflecting transient interactions or localized structural effects below HDX-MS detection thresholds.”\nResponse:\nWe appreciate the reviewer’s suggestion. As discussed in our response to comment #2, arrestins are known to adopt a range of active conformations depending on the receptor, ligand, and phosphorylation pattern. The Gα-interacting conformation may differ from the specific receptor-bound state stabilized by scFv30.\nIn fact, our newly added data indicate that the V2Rpp-bound active state of βarr1 is less compatible with Gα interaction compared to the pre-active state represented by C-terminally truncated βarr1 (βarr1_ΔC). Given that scFv30 is designed to stabilize a particular receptor-specific active conformation, it is likely that its presence would further constrain βarr1 into a conformation that is suboptimal for Gα binding, and may even inhibit the interaction.\nMinor corrections:\n5. Line 63: The word “muations” should be corrected to “mutations”.\nResponse: Thank you so much for careful review. We have corrected the error.\n6. Figure 6a: The label “D386C” should be corrected to “D385C”.\nResponse: We have corrected the error.\nA final technical point:\n7. It is unclear whether the bimane spectra exhibited a shift in λ_max, as the authors report only fold changes in fluorescence intensity. Additional clarification on whether spectral shifts were observed and how the fluorescence intensity was quantified would be helpful.\nResponse:\nIn the revised manuscript, we now include the full bimane fluorescence spectra comparing the basal and V2Rpp-induced active state of βarr1, presented in Supplementary Figure 6. The spectra reveal a slight red shift (right-shift in λmax) upon activation of βarr1, in addition to an overall increase in fluorescence intensity. This observation is now addressed in the Results section of the revised manuscript as follows:\nPage 9:\n“Upon βXX release, the increased separation between residues 5 and 385 (Fig. 6a, yellow star) reduces quenching, resulting in an increase in fluorescence intensity accompanied by a slight red shift in the emission spectrum (Supplementary Fig. 6).”\nAdditionally, we have clarified the quantification method in the Methods section (Page 17):\n“Fluorescence changes were quantified by measuring the emission intensity at the peak wavelength and normalized as fold change relative to the vehicle-treated control.”\nWe hope these additions address the reviewer’s concerns and improve the clarity and completeness of our fluorescence analysis."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is a revised version of the manuscript by Duan et all aimed at elucidation of potential functionally significant interaction between beta-arrestin-1 (bArr1) and G s or G i.\nIn the revised version, the authors added studies with bArr1 coupled with phosphopeptide derived from V2 receptor (to represent as they say an active form of bArr1, in addition to previously used basal bArr1 and βarr1_ΔC, an bArr2 with C-tail truncated at the 365 residue.\nThey also incorporated studies with proximity ligation assay (PLA) and a BRET-based sensor system to further demonstrate interaction between G proteins and bArr1.\nThe overall conclusion of the work is that “these results define structural determinants and asymmetric functional coupling within Gα–βarr complexes”.\nWith MST data, the authors showed that neither basal bArr1 not V2Rpp-bound bArr1 interact with active or inactive Galpha. Therefore, the only bArr1 form that shows interaction remains βarr1_ΔC. Thus, the controversy is casting doubt on biological relevance of the results. In the revised version, the authors attempt to resolve the controversy by claiming that βarr1_ΔC is a “conformationally dynamic pre-active state by mimicking βXX release”. Although bArr1 undoubtedly can assume multiple conformation, there is no evidence that such specific pre-active state exists. Furthermore, V2Rpp most certainly releases bArr1 C-tail, which should be enough to push bArr1 into a hypothetical pre-activated state, so it is unclear why this form shows no interaction in MST.\nThe in vitro βXX release assay data are inconsistent with the MST measurements (Fig. 1d-k) and so are cell-based BRET experiments. The authors attempt to reconcile the data by speculating that the complex bArr1-Galpha is transient low-affinity “cannot be measured by MST and minimal time-averaged protection below HDX sensitivity”. That makes the latter techniques unsuitable for the task negating the data thus obtained.\nWhich Galpha and in which form bArr1 interacts with also remains undefined, for the data from different techniques do not match.", "reviewer_2": "Happy with the responses.", "reviewer_3": "The authors have adequately addressed all my comments."}, "author_response": "We thank the reviewer for the careful re-evaluation of our revised manuscript. We recognize that our earlier use of the term “pre-active state” may have been misleading. Our intent was to convey that β-arrestins can occupy multiple, receptor- and context-dependent active conformations rather than a single binary “basal vs. active” state. Numerous studies (cited in the manuscript) have shown that arrestin conformations depend on receptor identity, ligand, and phosphorylation pattern, yielding diverse active conformers. We have revised the text to avoid implying a singular “pre-active” entity and instead refer to “active conformational ensemble state”.\n1) Why βarr1_ΔC shows binding while basal or V2Rpp-bound βarr1 do not (MST) and why V2Rpp-induced C-tail release is not sufficient for MST binding:\nV2Rpp-bound βarr1 represents one specific receptor-tail–stabilized conformer (tail engagement without receptor-core engagement). V2Rpp occupancy at phosphate-binding pockets can restrict conformational heterogeneity, making it suboptimal for Gα engagement under equilibrium MST conditions.\nIn contrast, βarr1_ΔC is relieved of C-tail autoinhibition yet not locked by a receptor phosphopeptide, samples a broader ensemble that includes Gα-competent sub-states detectable by MST. We therefore refer to βarr1_ΔC as “active conformational ensemble state”, not a unique “pre-active” state.\nPage 4:\nTruncation of the C-terminus including βXX transforms arrestins into “active conformational ensemble state” by mimicking βXX release but without occupying the phosphorylated receptor-binding pockets. This active conformational ensemble state is relieved from autoinhibition but not locked into a specific active conformation and therefore has been widely used to study the mechanism of arrestin activation19,25-27.\nIn this study, we used full-length βarr1 to represent the basal state, C-terminally truncated βarr1 (βarr1_ΔC) to represent the active conformational ensemble state rather than a single active state, and V2Rpp-bound βarr1 (V2Rpp, a phosphopeptide from the vasopressin type-2 receptor) as one specific receptor-tail–stabilized active conformer13,19,28.\nPage 5:\nThese results indicate that Gαs and Gαi1, whether in their inactive or active states, preferentially interact with the active conformational ensemble state βarr1 (βarr1_ΔC) but not with basal or V2Rpp-specific active states. V2Rpp occupancy at phosphate-binding pockets may favor a particular active-like conformer that does not engage Gα efficiently. On the contrary, C-tail truncation permits interconversion among multiple active-like sub-states, increasing the probability of populating a Gα-compatible conformation.\n2) Physiological plausibility of “active without receptor” arrestin:\nReports show that β-arrestins can remain signaling-competent after receptor dissociation (e.g., signaling from endocytic structures) (β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nature Cell Biology, 2016). This supports the notion that arrestin active-like ensembles can exist independent of a phosphorylated receptor tail ligand, consistent with our use of βarr1_ΔC to access such ensembles.\nPage 10:\nIn addition, β-arrestins can remain signaling-competent after receptor dissociation, sustaining an active-like ensemble that can engage other downstream signaling molecules39,40. This receptor-free, signaling-competent ensemble likely resembles βarr1_ΔC, providing a basis for its interaction with Gαs in cells.\nPage 12:\nMoreover, arrestins can remain active or signaling-competent state even after dissociation from the receptor39,40, potentially corresponding to the βXX-released “active conformational ensemble state”. It is possible that Gαs or Gαi1 can engage with βarr1 in this active conformational ensemble state within cells, promoting distinct conformational rearrangements, as captured by HDX-MS (Fig. 2c and 2d), which may underlie divergent downstream signaling profiles. This hypothesis also aligns with the \"barcode\" theory of arrestins, which posits that discrete arrestin conformations can encode specific downstream signaling outcomes46.\n3) Reconciling the data from in vitro system with those from the cell-based system\nThe apparent inconsistency arises from different systems. For example, MST measures stable equilibrium binding, and HDX-MS reports time-averaged backbone protection. Both MST and HDX-MS use purified protein systems, which lack cellular factors. In cells, additional factors (receptor-core engagement, membrane lipids such as PIP₂, scaffolds) can shift arrestin conformational ensembles, enabling transient or context-dependent Gα interactions that may not appear in the purified proteins. Our PLA and BRET data are therefore complementary to the biophysical assays, not contradictory, and support functional interaction in a cellular context. We explicitly discuss this in the manuscript.\nPage 9:\nCo-incubation with V2Rpp increased bimane fluorescence, reflecting βXX release upon V2Rpp-binding (Fig. 5b, gray solid bars). Conversely, GTPγS-bound Gαi1 or Gαs alone did not alter bimane fluorescence (Fig. 5b, green and red empty bars), suggesting that neither Gα directly activates βarr1 in the absence of receptor input in this in vitro system.\nPage 10:\nNotably, our MST with purified proteins showed that Gαs does not interact with V2Rpp-bound βarr1 (Fig. 1h,i), suggesting a discrepancy between in vitro and cellular observations. This difference can likely be attributed to the distinct conformational states of βarr1 induced in each system. In cells, receptor activation leads to dual engagement of βarr1 through both the phosphorylated C-terminal tail and the receptor core38, whereas V2Rpp alone promotes only tail engagement, which may not fully stabilize the Gαs-interacting conformation. In addition, β-arrestins can remain signaling-competent after receptor dissociation, sustaining an active-like ensemble that can engage other downstream signaling molecules39,40. This receptor-free, signaling-competent ensemble likely resembles βarr1_ΔC, providing a basis for its interaction with Gαs in cells.\nAs a next step, we investigated whether the interaction affects the activation status of βarr1 using an intramolecular BRET sensor (Fig. 6d). In this sensor, the basal (βXX–engaged) conformation yields a higher BRET signal, whereas βXX release reduces BRET. Gαs overexpression significantly decreased the BRET signal, indicating that Gαs alone can promote βarr1 activation (Fig. 6e). As expected, AVP reduced the BRET signal as V2R phosphorylated C-tail engages at the N-domain and replacing βXX (Fig. 6e). Co-expression of Gαs potentiated this AVP-induced βXX release (Fig. 6e).\nBy contrast, in our bimane fluorescence quenching assay with purified proteins, Gαs alone did not trigger βXX release (Fig. 5b). This discrepancy likely reflects cell-specific factors that prime βarr1 in vivo. For example, overexpressed V2R can exhibit basal activity, generating a subpopulation of phosphorylated receptors that biases βarr1 toward activation. In addition, plasma-membrane lipids such as phosphatidylinositol 4,5-bisphosphate (PIP ) can allosterically favor pre-active arrestin ensembles even in the absence of receptor engagement41-44. Thus, cellular βarr1 likely exists as an ensemble of basal and active-like states, with Gαs overexpression shifting the equilibrium toward βXX release.\nIn summary, convergent evidence from cell-based and in vitro assays supports a model in which Gαs preferentially engages the active conformational ensemble state of βarr1 and promotes or stabilizes the βXX–released conformation (Fig. 6g) while the precise βarr1 sub-state favored by Gαs remains to be defined.\nPage 11:\nWhen GTPγS-bound Gαs was co-incubated, full-length βarr1 either with or without V2Rpp did not show HDX changes. Similarly, we could not detect HDX changes in Gαs. The lack of Gαs-induced HDX changes in full-length βarr1 either with or without V2Rpp can be explained by the lack of stable binding between Gαs and the basal or V2Rpp-bound state βarr1 (Fig. 1d,e,h,i).\nNevertheless, Gαs facilitated βXX release from V2Rpp-bound state in in vitro (Fig. 5b) and both basal and V2R-activated βarr1 in cells (Fig. 6e,f). We interpret this as a transient, low-occupancy allosteric effect. Gαs briefly engages βarr1 when the protein samples active conformational ensembles, thereby promoting βXX release, but dissociates as βarr1 transitions to subsequent conformations (Fig. 6g). In the in vitro setting, V2Rpp may populate only a small fraction of such Gαs-competent ensembles, yielding functional potentiation without sufficient complex lifetime or abundance for detection by MST or HDX-MS. In cells, receptor activity and membrane factors likely enrich these active-like ensembles even within subpopulations of basal or V2R-activated βarr1, enabling the observed effect.\nWhich Galpha and in which form bArr1 interacts with also remains undefined, for the data from different techniques do not match.\nResponse:\nWe agree that the precise βarr1 sub-state preferred by each Gα remains to be defined. We have been careful to state this limitation and emphasize it in the Results and revised Discussion.\nPage 10:\nIn summary, convergent evidence from cell-based and in vitro assays supports a model in which Gαs preferentially engages the active conformational ensemble state of βarr1 and promotes or stabilizes the βXX–released conformation (Fig. 6g) while the precise βarr1 sub-state favored by Gαs remains to be defined.\nPage 13:\nTherefore, more sophisticated analysis is needed for understanding the complex structures; high-resolution understanding of stabilized sub-states will be required to define the precise βarr1 sub-states and Gα-selective interfaces.\nWe hope the revisions satisfactorily address the reviewer’s concerns: βarr1_ΔC represents an active conformational ensemble that includes Gα-competent sub-states; V2Rpp-bound βarr1 represents a specific receptor-tail–stabilized active conformer that is not necessarily Gα-competent; and the cellular context enriches these ensembles and permits transient allosteric coupling that is not captured as persistent complexes by MST/HDX. Accordingly, the in vitro and cellular datasets are complementary, not contradictory."}, {"round": 3, "reviewer_comments": {"reviewer_1": "In the revised version of the manuscript by Duan et al, the authors attempt to reconcile the contradictory data by suggesting specific conformational differences between βarr1_ΔC and full-length receptor-bound arrestin.\nThey argue that while the full-length arrestin represents basal state, βarr1_ΔC “represent the active conformational ensemble state rather than a single active state” and “C-tail truncation permits interconversion among multiple active-like sub-states, increasing the probability of populating a Gα-compatible conformation”.\nThis is possible but no experimental proof is offered to support this notion. βarr1_ΔC is an artificial construct, and it is unclear why it is taken to represent multiple active-like states as opposed to the state(s) that never occur in real life. There are other destabilized βarr1 mutants with released C-tail that could be examined to lend some experimental support to the idea.\n“Arrestin active-like ensembles can exist independent of a phosphorylated receptor tail ligand, consistent with our use of βarr1_ΔC to access such ensembles”. The conclusion is not quite correct, for b-arrestin signaling from clathrin-coated pits requires clathrin – and clathrin binding site on arrestin C-tail – which βarr1_ΔC does not possess.\n“convergent evidence from cell-based and in vitro assays supports a model in which Gαs preferentially engages the active conformational ensemble state of βarr1 and promotes or stabilizes the βXX–released conformation (Fig. 6g”) - If the in-cell data could be interpreted this way, the in vitro experiments show that both Gs and Gi in active or inactive form interact with βarr1_ΔC, although not with full length arrestin. Why then Gi shown no interaction at all in cells upon the release of C-tail (Fig. 5)?\nIn this revision, the manuscript remained essentially the same, with some changes in wording.", "reviewer_2": "Happy with the responses.", "reviewer_3": "The authors have adequately addressed all my comments."}, "author_response": "Response:\nWe appreciate the reviewer’s careful reading and would like to clarify our intent. We do not mean to suggest that C-tail truncation specifically biases arrestin toward a Gα-compatible conformation. Rather, our interpretation is that βarr1_ΔC samples a broader ensemble of active-like conformations, of which a Gα-compatible conformation represents one possible sub-state. Our conclusions are therefore framed in ensemble-based terms, not as evidence for a uniquely stabilized Gα-preferring structure.\nThis is possible but no experimental proof is offered to support this notion. βarr1_ΔC is an artificial construct, and it is unclear why it is taken to represent multiple active-like states as opposed to the state(s) that never occur in real life. There are other destabilized βarr1 mutants with released C-tail that could be examined to lend some experimental support to the idea.\nResponse:\nWe agree that βarr1_ΔC is an artificial construct. Nonetheless, C-terminal truncation has been extensively used in the arrestin field as an operational strategy to relieve autoinhibition and access C-tail–released conformations. Importantly, evidence from cellular studies indicates that arrestins can adopt active or signaling-competent conformations even in the absence of sustained engagement with a phosphorylated receptor tail, supporting the physiological plausibility of receptor-free, C-tail–released arrestin states.\nWhile alternative destabilizing mutations (e.g., polar core disruption) can also promote C-tail release, such perturbations generally induce more limited conformational changes compared with C-tail truncation. We therefore used βarr1_ΔC as a practical means to access a broad active-like conformational ensemble, rather than to claim that it precisely recapitulates a single physiological structure.\n“Arrestin active-like ensembles can exist independent of a phosphorylated receptor tail ligand, consistent with our use of βarr1_ΔC to access such ensembles”. The conclusion is not quite correct, for b-arrestin signaling from clathrin-coated pits requires clathrin – and clathrin binding site on arrestin C-tail – which βarr1_ΔC does not possess.\nResponse:\nWe appreciate this important distinction. Our statement was not intended to imply that βarr1_ΔC recapitulates clathrin-dependent endocytic signaling. Rather, our focus is on the conformational state of the arrestin core and its capacity to engage Gα proteins. Clathrin binding was not examined in this study, nor is it required for the Gα–arrestin interactions investigated here.\n“convergent evidence from cell-based and in vitro assays supports a model in which Gαs preferentially engages the active conformational ensemble state of βarr1 and promotes or stabilizes the βXX–released conformation (Fig. 6g”) - If the in-cell data could be interpreted this way, the in vitro experiments show that both Gs and Gi in active or inactive form interact with βarr1_ΔC, although not with full length arrestin. Why then Gi shown no interaction at all in cells upon the release of C-tail (Fig. 5)?\nResponse:\nFigure 5 reports βXX (C-tail) release, not direct Gα–βarr1 interaction.\nIn this revision, the manuscript remained essentially the same, with some changes in wording.\nResponse:\nWe respectfully disagree. In addition to substantial revisions in terminology (e.g., replacing “pre-active” with “active conformational ensemble”), we have significantly expanded the conceptual framework, clarified the interpretation of in vitro versus cellular assays, and revised multiple sections of the Results and Discussion to directly address the reviewer’s concerns."}]

Decision letter