A high-resolution analysis of arrestin2 interactions responsible for CCR5 endocytosis

Reviewer #1 (Public review):

Petrovic et al. investigate CCR5 endocytosis via arrestin2, with a particular focus on clathrin and AP2 contributions. The study is thorough and methodologically diverse. The NMR titration data clearly demonstrate chemical shift changes at the canonical clathrin-binding site (LIELD), present in both the 2S and 2L arrestin splice variants. To assess the effect of arrestin activation on clathrin binding, the authors compare: truncated arrestin (1-393), full-length arrestin, and 1-393 incubated with CCR5 phosphopeptides. All three bind clathrin comparably, whereas controls show no binding. These findings are consistent with prior crystal structures showing peptide-like binding of the LIELD motif, with disordered flanking regions. The manuscript also evaluates a non-canonical clathrin binding site specific to the 2L splice variant. Though this region has been shown to enhance beta2-adrenergic receptor binding, it appears not to affect CCR5 internalization.

Similar analyses applied to AP2 show a different result. AP2 binding is activation-dependent and influenced by the presence and level of phosphorylation of CCR5-derived phosphopeptides. These findings are reinforced by cellular internalization assays.

In sum, the results highlight splice-variant-dependent effects and phosphorylation-sensitive arrestin-partner interactions. The data argue against a (rapidly disappearing) one-size-fits-all model for GPCR-arrestin signaling and instead support a nuanced, receptor-specific view, with one example summarized effectively in the mechanistic figure.

Weaknesses:

Figure 1 shows regions alphaFold model that are intrinsically disordered without making it clear that this is not an expected stable position. The authors NMR titration data are n=1. Many figure panels require that readers pinch and zoom to see the data.

Reviewer #2 (Public review):

Summary:

Based on extensive live cell assays, SEC, and NMR studies of reconstituted complexes, these authors explore the roles of clathrin and the AP2 protein in facilitating clathrin mediated endocytosis via activated arrestin-2. NMR, SEC, proteolysis, and live cell tracking confirm a strong interaction between AP2 and activated arrestin using a phosphorylated C-terminus of CCR5. At the same time a weak interaction between clathrin and arrestin-2 is observed, irrespective of activation.

These results contrast with previous observations of class A GPCRs and the more direct participation by clathrin. The results are discussed in terms of the importance of short and long phosphorylated bar codes in class A and class B endocytosis.

Strengths:

The 15N,1H and 13C,methyl TROSY NMR and assignments represent a monumental amount of work on arrestin-2, clathrin, and AP2. Weak NMR interactions between arrestin-2 and clathrin are observed irrespective of activation of arrestin. A second interface, proposed by crystallography, was suggested to be a possible crystal artifact. NMR establishes realistic information on the clathrin and AP2 affinities to activated arrestin with both kD and description of the interfaces.

Weaknesses:

This reviewer has identified only minor weaknesses with the study.

(1) I don't observe two overlapping spectra of Arrestin2 (1-393) +/- CLTC NTD in Supp Figure 1

(2) Arrestin-2 1-418 resonances all but disappear with CCR5pp6 addition. Are they recovered with Ap2Beta2 addition and is this what is shown in Supp Fig 2D

(3) I don't understand how methyl TROSY spectra of arrestin2 with phosphopeptide could look so broadened unless there are sample stability problems?

(4) At one point the authors added excess fully phosphorylated CCR5 phosphopeptide (CCR5pp6). Does the phosphopeptide rescue resolution of arrestin2 (NH or methyl) to the point where interaction dynamics with clathrin (CLTC NTD) are now more evident on the arrestin2 surface?

(5) Once phosphopeptide activates arrestin-2 and AP2 binds can phosphopeptide be exchanged off? In this case, would it be possible for the activated arrestin-2 AP2 complex to re-engage a new (phosphorylated) receptor?

(6) I'd be tempted to move the discussion of class A and class B GPCRs and their presumed differences to the intro and then motivate the paper with specific questions.

(7) Did the authors ever try SEC measurements of arrestin-2 + AP2beta2+CCR5pp6 with and without PIP2, and with and without clathrin (CLTC NTD? The question becomes what the active complex is and how PIP2 modulates this cascade of complexation events in class B receptors.

Reviewer #3 (Public review):

Summary:

Overall, this is a well-done study, and the conclusions are largely supported by the data, which will be of interest to the field.

Strengths:

Strengths of this study include experiments with solution NMR that can resolve high-resolution interactions of the highly flexible C-terminal tail of arr2 with clathrin and AP2. Although mainly confirmatory in defining the arr2 CBL 376LIELD380 as the clathrin binding site, the use of the NMR is of high interest (Fig. 1). The 15N-labeled CLTC-NTD experiment with arr2 titrations reveals a span from 39-108 that mediates an arr2 interaction, which corroborates previous crystal data, but does not reveal a second area in CLTC-NTD that in previous crystal structures was observed to interact with arr2.

SEC and NMR data suggest that full-length arr2 (1-418) binding with 2-adaptin subunit of AP2 is enhanced in the presence of CCR5 phospho-peptides (Fig. 3). The pp6 peptide shows the highest degree of arr2 activation, and 2-adaptin binding, compared to less phosphorylated peptide or not phosphorylated at all. It is interesting that the arr2 interaction with CLTC NTD and pp6 cannot be detected using the SEC approach, further suggesting that clathrin binding is not dependent on arrestin activation. Overall, the data suggest that receptor activation promotes arrestin binding to AP2, not clathrin, suggesting the AP2 interaction is necessary for CCR5 endocytosis.

To validate the solid biophysical data, the authors pursue validation experiments in a HeLa cell model by confocal microscopy. This requires transient transfection of tagged receptor (CCR5-Flag) and arr2 (arr2-YFP). CCR5 displays a "class B"-like behavior in that arr2 is rapidly recruited to the receptor at the plasma membrane upon agonist activation, which forms a stable complex that internalizes onto endosomes (Fig. 4). The data suggest that complex internalization is dependent on AP2 binding not clathrin (Fig. 5).

The addition of the antagonist experiment/data adds rigor to the study.

Overall, this is a solid study that will be of interest to the field.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Petrovic et al. investigate CCR5 endocytosis via arrestin2, with a particular focus on clathrin and AP2 contributions. The study is thorough and methodologically diverse. The NMR titration data are particularly compelling, clearly demonstrating chemical shift changes at the canonical clathrin-binding site (LIELD), present in both the 2S and 2L arrestin splice variants.

To assess the effect of arrestin activation on clathrin binding, the authors compare: truncated arrestin (1-393), full-length arrestin, and 1-393 incubated with CCR5 phosphopeptides. All three bind clathrin comparably, whereas controls show no binding. These findings are consistent with prior crystal structures showing peptide-like binding of the LIELD motif, with disordered flanking regions. The manuscript also evaluates a non-canonical clathrin binding site specific to the 2L splice variant. Though this region has been shown to enhance beta2-adrenergic receptor binding, it appears not to affect CCR5 internalization.

Similar analyses applied to AP2 show a different result. AP2 binding is activation-dependent and influenced by the presence and level of phosphorylation of CCR5-derived phosphopeptides. These findings are reinforced by cellular internalization assays.

In sum, the results highlight splice-variant-dependent effects and phosphorylation-sensitive arrestin-partner interactions. The data argue against a (rapidly disappearing) one-size-fitsall model for GPCR-arrestin signaling and instead support a nuanced, receptor-specific view, with one example summarized effectively in the mechanistic figure.

We thank the referee for this positive assessment of our manuscript. Indeed, by stepping away from the common receptor models for understanding internalization (b2AR and V2R), we revealed the phosphorylation level of the receptor as a key factor in driving the sequestration of the receptor from the plasma membrane. We hope that the proposed mechanistic model will aid further studies to obtain an even more detailed understanding of forces driving receptor internalization.

Reviewer #2 (Public review):

Summary:

Based on extensive live cell assays, SEC, and NMR studies of reconstituted complexes, these authors explore the roles of clathrin and the AP2 protein in facilitating clathrin-mediated endocytosis via activated arrestin-2. NMR, SEC, proteolysis, and live cell tracking confirm a strong interaction between AP2 and activated arrestin using a phosphorylated C-terminus of CCR5. At the same time, a weak interaction between clathrin and arrestin-2 is observed, irrespective of activation.

These results contrast with previous observations of class A GPCRs and the more direct participation by clathrin. The results are discussed in terms of the importance of short and long phosphorylated bar codes in class A and class B endocytosis.

Strengths:

The 15N,1H, and 13C, methyl TROSY NMR and assignments represent a monumental amount of work on arrestin-2, clathrin, and AP2. Weak NMR interactions between arrestin-2 and clathrin are observed irrespective of the activation of arrestin. A second interface, proposed by crystallography, was suggested to be a possible crystal artifact. NMR establishes realistic information on the clathrin and AP2 affinities to activated arrestin, with both kD and description of the interfaces.

We sincerely thank the referee for this encouraging evaluation of our work and appreciate the recognition of the NMR efforts and insights into the arrestin–clathrin–AP2 interactions.

Weaknesses:

This reviewer has identified only minor weaknesses with the study.

(1) Arrestin-2 1-418 resonances all but disappear with CCR5pp6 addition. Are they recovered with Ap2Beta2 addition, and is this what is shown in Supplementary Figure 2D?

We believe the reviewer is referring to Figure 3 - figure supplement 1. In this figure, the panels E and F show resonances of arrestin2^1-418 (apo state shown with black outline) disappear upon the addition of CCR5pp6 (arrestin2^1-418•CCR5pp6 complex spectrum in red). The panels C and D show resonances of arrestin2^1-418 (apo state shown with black outline), which remain unchanged upon addition of AP2b2^701-937 (orange), indicating no complex formation. We also recorded a spectrum of the arrestin2^1-418 •CCR5pp6 complex under addition of AP2b2 ^701-937(not shown), but the arrestin2 resonances in the arrestin2¹⁴¹⁸ •CCR5pp6 complex were already too broad for further analysis. This had been already explained in the text.

“In agreement with the AP2b2 NMR observations, no interaction was observed in the arrestin2 methyl and backbone NMR spectra upon addition of AP2b2 in the absence of phosphopeptide (Figure 3-figure supplement 1C, D). However, the significant line broadening of the arrestin2 resonances upon phosphopeptide addition (Figure 3-figure supplement 1E, F) precluded a meaningful assessment of the effect of the AP2b2 addition on arrestin2 in the presence of phosphopeptide””.

(2) I don't understand how methyl TROSY spectra of arrestin2 with phosphopeptide could look so broadened unless there are sample stability problems.

We thank the referee for this comment. We would like to clarify that in general a broadened spectrum beyond what is expected from the rotational correlation time does not necessarily correlate with sample stability problems. It is rather evidence of conformational intermediate exchange on the micro- to millisecond time scale.

The displayed ¹H-¹⁵ N spectra of apo arrestin2 already suffer from line broadening due to such intrinsic mobility of the protein. These spectra were recorded with acquisition times of 50 ms (¹⁵N) and 55 ms (¹H) and resolution-enhanced by a 60˚-shifted sine-bell filter for ¹⁵N and a 60˚-shifted squared sine-bell filter for ¹H, respectively, which leads to the observed resolution with still reasonable sensitivity. The ¹H-¹⁵ resonances in Fig. 1b (arrestin2^1-393) look particularly narrow. However, this region contains a large number of flexible residues. The full spectrum, e.g. Figure 1-figure supplement 2, shows the entire situation with a clear variation of linewidths and intensities. The linewidth variation becomes stronger when omitting the resolution enhancement filters.

The addition of the CCR5pp6 phosphopeptide does not change protein stability, which we assessed by measuring the melting temperature of arrestin2^1-418 and arrestin2^1-418 •CCR5pp6 complex (Tm = 57°C in both cases). We believe that the explanation for the increased broadening of the arrestin2 resonances is that addition of the CCR5pp6, possibly due to the release of the arrestin2 strand b20, amplifies the mentioned intermediate timescale protein dynamics. This results in the disappearance of arrestin2 resonances.

We have now included the assessment of arrestin2^1-418 and arrestin2^1-418 •CCR5pp6 stability in the manuscript:

“The observed line broadening of arrestin2 in the presence of phosphopeptide must be a result of increased protein motions and is not caused by a decrease in protein stability, since the melting temperature of arrestin2 in the absence and presence of phosphopeptide are identical (56.9 ± 0.1 °C)”.

(3) At one point, the authors added an excess fully phosphorylated CCR5 phosphopeptide (CCR5pp6). Does the phosphopeptide rescue resolution of arrestin2 (NH or methyl) to the point where interaction dynamics with clathrin (CLTC NTD) are now more evident on the arrestin2 surface?

Unfortunately, when we titrate arrestin2 with CCR5pp6 (please see Isaikina & Petrovic et. al, Mol. Cell, 2023 for more details), the arrestin2 resonances undergo fast-to-intermediate exchange upon binding. In the presence of phosphopeptide excess, very few resonances remain, the majority of which are in the disordered region, including resonances from the clathrin-binding loop. Due to the peak overlap, we could not unambiguously assign arrestin2 resonances in the bound state, which precluded our assessment of the arrestin2-clathrin interaction in the presence of phosphopeptide. We have made this now clearer in the paragraph ‘The arrestin2-clathrin interaction is independent of arrestin2 activation’

“Due to significant line broadening and peak overlap of the arrestin2 resonances upon phosphopeptide addition, the influence of arrestin activation on the clathrin interaction could not be detected on either backbone or methyl resonances”.

(4) Once phosphopeptide activates arrestin-2 and AP2 binds, can phosphopeptide be exchanged off? In this case, would it be possible for the activated arrestin-2 AP2 complex to re-engage a new (phosphorylated) receptor?

This would be an interesting mechanism. In principle, this should be possible as long as the other (phosphorylated) receptor outcompetes the initial phosphopeptide with higher affinity towards the binding site. However, we do not have experiments to assess this process directly. Therefore, we rather wish not to further speculate.

(5) Did the authors ever try SEC measurements of arrestin-2 + AP2beta2+CCR5pp6 with and without PIP2, and with and without clathrin (CLTC NTD? The question becomes what the active complex is and how PIP2 modulates this cascade of complexation events in class B receptors.

We thank the referee for this question. Indeed, we tested whether PIP2 can stabilize the arrestin2•CCR5pp6•AP2 complex by SEC experiments. Unfortunately, the addition of PIP2 increased the formation of arrestin2 dimers and higher oligomers, presumably due to the presence of additional charges. The resolution of SEC experiments was not sufficient to distinguish arrestin2 in oligomeric form or in arrestin2•CCR5pp6•AP2 complex. We now mention this in the text:

“We also attempted to stabilize the arrestin2-AP2b2-phosphopetide complex through the addition of PIP2, which can stabilize arrestin complexes with the receptor (Janetzko et al., 2022). The addition of PIP2 increased the formation of arrestin2 dimers and higher oligomers, presumably due to the presence of additional charges. Unfortunately, the resolution of the SEC experiments was not sufficient to separate the arrestin2 oligomers from complexes with AP2b2”.

Reviewer #3 (Public review):

Summary:

Overall, this is a well-done study, and the conclusions are largely supported by the data, which will be of interest to the field.

Strengths:

(1) The strengths of this study include experiments with solution NMR that can resolve high-resolution interactions of the highly flexible C-terminal tail of arr2 with clathrin and AP2. Although mainly confirmatory in defining the arr2 CBL 376LIELD380 as the clathrin binding site, the use of the NMR is of high interest (Figure 1). The 15N-labeled CLTC-NTD experiment with arr2 titrations reveals a span from 39-108 that mediates an arr2 interaction, which corroborates previous crystal data, but does not reveal a second area in CLTC-NTD that in previous crystal structures was observed to interact with arr2.

(2) SEC and NMR data suggest that full-length arr2 (1-418) binding with the 2-adaptin subunit of AP2 is enhanced in the presence of CCR5 phospho-peptides (Figure 3). The pp6 peptide shows the highest degree of arr2 activation and 2-adaptin binding, compared to less phosphorylated peptides or not phosphorylated at all. It is interesting that the arr2 interaction with CLTC NTD and pp6 cannot be detected using the SEC approach, further suggesting that clathrin binding is not dependent on arrestin activation. Overall, the data suggest that receptor activation promotes arrestin binding to AP2, not clathrin, suggesting the AP2 interaction is necessary for CCR5 endocytosis.

(3) To validate the solid biophysical data, the authors pursue validation experiments in a HeLa cell model by confocal microscopy. This requires transient transfection of tagged receptor (CCR5-Flag) and arr2 (arr2-YFP). CCR5 displays a "class B"-like behavior in that arr2 is rapidly recruited to the receptor at the plasma membrane upon agonist activation, which forms a stable complex that internalizes into endosomes (Figure 4). The data suggest that complex internalization is dependent on AP2 binding, not clathrin (Figure 5).

We thank the referee for the careful and encouraging evaluation of our work. We appreciate the recognition of the solidity of our data and the support for our conclusions regarding the distinct roles of AP2 and clathrin in arrestin-mediated receptor internalization.

Weaknesses:

The interaction of truncated arr2 (1-393) was not impacted by CCR5 phospho-peptide pp6, suggesting the interaction with clathrin is not dependent on arrestin activation (Figure 2). This raises some questions.

We thank the referee for raising this concern, as we were also surprised by the discovery that the interaction does not depend on arrestin activation. However, the NMR data clearly show at atomic resolution that arrestin activation does not influence the interaction with clathrin in vitro. Evolutionary, the arrestin-clathrin interaction appears not to be conserved as the visual arrestin completely lacks a clathrin-binding motif. For that reason, we believe that the weak arrestin-clathrin interaction provides more of a supportive role during the internalization rather than the regulatory interaction with AP2, which requires and quantitatively depends on the arrestin2 activation. We have reflected on this in the Discussion:

“Although the generalization of this mechanism from CCR5 to other arr-class B receptors has to be explored further, it is indirectly corroborated in the visual rhodopsin-arrestin1 system. The arr-class B receptor rhodopsin (Isaikina et al., 2023) also undergoes CME (Moaven et al., 2013) with arrestin1 harboring the conserved AP2 binding motif, but missing the clathrinbinding motif (Figure 1-figure supplement 1A)”.

Overall, the data are solid, but for added rigor, can these experiments be repeated without tagged receptor and/or arr2? My concern stems from the fact that the stability of the interaction between arr2 and the receptor may be related to the position of the tags.

We thank the referee for this suggestion, which refers to the cellular experiments; the biophysical experiments were carried out without tags. To eliminate the possibility of tags contributing to receptor-arrestin2 binding in the cellular experiments, we also performed the experiments in the presence of CCR5 antagonist [5P12]CCL5 (Figure 4). These data show that in the case of inactive CCR5, arrestin2 is not recruited to CCR5, nor does it form internalization complexes, which would be the case if the tags were increasing the receptorarrestin interaction. In contrast, if the tags were decreasing the interaction, we would not expect such a strong internalization. As indicated below, we have also attempted to perform our cellular experiments using an N-terminally SNAP-tagged CCR5. Unfortunately, this construct did not express in HeLa cells indicating that SNAP-CCR5 was either toxic or degraded.

Reviewing Editor Comments:

Overall, the reviewers did not suggest much by way of additional experiments. They do suggest several aspects of the manuscript that would benefit from further clarification.

Reviewer #1 (Recommendations for the authors):

(1) The distinction between arrestin 2S and arrestin 2L as relates to the canonical and non-canonical clathrin binding sites would benefit from clarification, particularly because the second binding site depends on the splice variant. This is something that some readers may not be familiar with (particularly young ones that are hopefully part of the intended readership).

We thank the referee for this suggestion. We would like to emphasize that in our work, only the long arrestin2 splice variant was used, which contains both binding sites. We have now introduced the splice variants and their relation to the clathrin binding sites in the text.

In section ‘Localizing and quantifying the arrestin2-clathrin interaction by NMR spectroscopy’:

“Clathrin and arrestin interact in their basal state (Goodman et al., 1996), and a structure of a complex between arrestin2 and the clathrin heavy chain N-terminal domain (residues 1-363, named clathrin-N in the following) has been solved by X-ray crystallography (PDB:3GD1) in the absence of an arrestin2-activating phosphopeptide (Kang et al., 2009). This structure (Figure 1-figure supplement 1B) suggests a 2:1 binding model between arrestin2 and clathrinN. The first interaction (site I) is observed between the ³⁷⁶LIELD³⁸⁰ clathrin-binding motif of the arrestin2 CBL and the edge of the first two β-sheet blades of clathrin-N, whereas the second interaction (site II) occurs between arrestin2 residues ³³⁴LLGDLA³³⁹ and the 4th and 5th blade of clathrin-N. The latter arrestin interaction site is not present in the arrestin2 splice variant arrestin2S (for short) where an 8-amino acid insert (residues 334-341) between β-strands 18 and 19 is removed (Kang et al., 2009)”.

Section ‘The arrestin2-clathrin interaction is independent of arrestin2 activation’

“Figure 2A (left) shows the intensity changes (full spectra in Figure 2-figure supplement 1A) of the clathrin-N ¹H-¹⁵N TROSY resonances [assignments transferred from BMRB, ID:25403 (Zhuo et al., 2015)] upon addition of a one-molar equivalent of arrestin2^1-393. A significant intensity reduction due to line broadening is detected for clathrin-N residues 39-40, 48-50, 62-72, 83-90, 101-106, and 108. These residues form a clearly defined binding region at the edges of blade 1 and blade 2 of clathrin-N (Figure 2A, right), which corresponds to interaction site I in the 3GD1 crystal structure, involving the conserved arrestin2 ³⁷⁶LIELD³⁸⁰ motif. However, no significant signal attenuation was observed for clathrin-N residues in blade 4 and blade 5, which would correspond to the crystal interaction site II with arrestin2 residues ³³⁴LLGDLA³³⁹ that are absent in the arrestin2S splice variant. Thus only one arrestin2 binding site in clathrin-N is detected in solution, and site II of the crystal structure may be a result of crystal packing”.

(2) Acronym density is high throughout. While many are standard in the clathrin literature, this could hinder accessibility for readers with a GPCR or arrestin focus.

We agree with the referee. The acronyms were hard to avoid. The most non-obvious acronym seems ‘CLTC-NTD’ for the N-terminal domain of the clathrin heavy chain, which uses the non-obvious, but common gene name CLTC for the clathrin heavy chain. We have now replaced ‘CLTC-NTD’ by ‘clathrin-N’ and hope that this makes the text easier to follow.

(3) The NMR section, while impressive in scope, had writing that was more difficult to follow than the rest. I am curious what percentage of resonance could be assigned.

We apologize if the NMR sections of this manuscript were unclear. We attempted to provide a very detailed description of the experimental setup and the spectral results. Being experienced NMR spectroscopists, we have tried very hard to obtain good 3D triple resonance spectra for assignments, but their sensitivity is very low. We believe that this is due to the microsecond dynamics present in the system, which makes the heteronuclear transfers inefficient. So far, we have been able to assign ~30% of the visible arrestin2 resonances. We are still validating the assignments and are working on the analysis and an explanation for this arrestin2 behavior. Therefore, at this point, we want to refrain from stronger statements besides that considerable intrinsic microsecond dynamics is impeding the assignment process.

(4) It may be worth noting in the main text that truncated arrestins have slightly higher basal activation. I was curious why the truncated arrestin was not chosen for the AP2 NMR titrations. Presumably, an effect would be more likely to be seen.

While some truncated arrestin2 variants (comprising residues 1-382 or 1-360) indeed show higher basal activity than the full-length arrestin2, they typically completely lack the b20 strand (residues 386-390), which is crucial for the formation of a parallel b-sheet with strand b1, and whose release governs arrestin activation. Our truncated arrestin2 construct comprises residues 1-393 and contains strand b20. In our experience, no significant difference in basal activity, as assessed by Fab30 binding, was detected for arrestin2^1-393 and arrestin2^1-418 (Author response image 1).

Author response image 1.

SEC profiles showing arrestin2^1–393 (left) and arrestin2^1-418 (right) activation by the CCR5pp6 phosphopeptide as assayed by Fab30 binding. The active ternary arrestin2-phosphopeptide-Fab30 complex elutes at a lower volume than the inactive apo arrestin2 or the binary arrestin2-phosphopeptide complex. Both arrestin2 constructs are activated by the phosphopeptide to a similar level as assessed by the integrated SEC volumes.

We want to emphasize that we used full-length arrestin2^1-418 in order to assess the AP2 interaction, as the crystal structure of arrestin2 peptide-AP2 (PDB:2IV8) shows residues past the residue 393 involved in binding.

PDB codes are currently not accompanied by corresponding literature citations throughout. Please add these.

Thank you for this suggestion. In the manuscript, we were careful to provide the full literature citation the first time each PDB code is mentioned. To avoid redundancy and maintain clarity, we rather do not want to repeat the citations with every subsequent mentioning of the PDB code.

(5) The AlphaFold model could benefit from a more transparent discussion of prediction confidence and caveats. The younger crowd (part of the presumed intended readership) tends to be more certain that computational output is 'true'. Figure 1A shows long loops that are likely regions of low confidence in the prediction. Displaying expected disordered regions as transparent or color-coded would help highlight these as flexible rather than stable, especially for that same younger readership.

We need to explain that the AlphaFold model of arrestin2 was only used to visualize the clathrin-binding loop and the 344-loop of the arrestin2 C-domain, which are not detected in the available apo bovine (PDB:1G4M) and apo human (PDB:8AS4) arrestin2 crystal structures. However, the AlphaFold model of arrestin2 is basically identical to the crystal structures in the regions that are visible in the crystal structures. We have clarified this now in the caption to Figure 1.

“The model was used to visualize the clathrin-binding loop and the 344-loop of the arrestin2 C-domain, which are not detected in the available crystal structures of apo arrestin2 [bovine: PDB 1G4M (Han et al., 2001), human: PDB 8AS4 (Isaikina et al., 2023)]. In the other structured regions, the model is virtually identical to the crystal structures”.

(6) Several figure panels were difficult to interpret due to their small size. Especially microscopy insets, where I needed to simply trust that the authors were accurately describing the data. Enlarging panels is essential, and this may require separating them into different figures.

We appreciate the referee’s concern regarding figure readability. However, we want to indicate that all our figures are provided as either high-resolution pixel or scalable vector graphics, which allow for zooming in to very fine detail, either electronically or in print. This ensures that microscopy insets and other small panels can be examined clearly when viewed appropriately. We believe the current layout of the figures is necessary to be able to efficiently compare the data between different conditions.

Many figure panels had text size that was too small. Font inconsistencies across figures also stand out.

We apologize for this. We have now enlarged the font size in the figures and made the styles more consistent.

For Fig. 1F, consider adding individual data points and error bars.

Thank you for this suggestion. However, Figure 1F already contains the individual data points, with colored circles corresponding to the titration condition. As we did not have replicates of the titration, no error bars are shown. However, the close agreement of the theoretical fit with the individual measured data points stemming from different experiments shows that the statistical errors are indeed very small. We have estimated an overall error for the Kd (as indicated in panel F, right) by error propagation based on an estimate of the chemical shift error as obtained in the NMR software POKY (based on spectral noise).

Reviewer #2 (Recommendations for the authors):

(1) I don't observe two overlapping spectra of Arrestin2 (1393) +/- CLTC NTD in Supplementary Figure 1.

As explained above all the spectra are shown as scalable vector graphics. The overlapping spectra are visible when zoomed in.

(2) I'd be tempted to move the discussion of class A and class B GPCRs and their presumed differences to the intro and then motivate the paper with specific questions.

We appreciate the referee’s suggestion and had a similar idea previously. However, as we do not have data on other class-A or class-B receptors, we rather don’t want to motivate the entire manuscript by this question.

Reviewer #3 (Recommendations for the authors):

(1) What happens with full-length arr2 (1-418) when the phospho-peptide pp6 is added to the reaction? It's unclear to me that 1-418 would behave the same as 1-393 because the arr2 tail of 1-393 is likely sufficiently mobile to accommodate binding to CLTC NTD. I suggest attempting this experiment for added rigor.

We believe that there is a misunderstanding. The 1-393 and 1-418 constructs differ by the disordered C-terminal tail, which is not involved in the clathrin interaction with the arrestin2 376-380 (LIELD) residues. Accordingly, both 1-393 and 1-418 constructs show almost identical interactions with clathrin (Figure 2A and 2C). Moreover, the phospho-activated arrestin2^1-393 (Figure 2B) interacts identically with clathrin as inactive arrestin2^1-393 and inactive arrestin2^1-418. We believe that this comparison is sufficient for the conclusion that arrestin activation does not play a role in arrestin-clathrin binding.

(2) If the tags were moved to the N-terminus of the receptor and/or arr2, I wonder if the complex is as stable (Figure 4)?

We thank the referee for their suggestion. We have indeed attempted to perform our experiments using an N-terminally SNAP-tagged CCR5. Unfortunately, this construct did not express in the HeLa cells indicating that SNAP-CCR5 was either toxic or degraded. Unfortunately, as the lab is closing due to the retirement of the PI, we are not able to repeat these experiments with further differently positioned tags. We refer also to our answer above that the experiments with the antagonist [5P12]CCL5 present a certain control.

(3) A biochemical assay to measure receptor internalization, in addition to the cell biological approach (Figure 5), would add additional rigor to the study and conclusions.

We tried to measure internalization using a biochemical approach. We tried to pull-down CCR5 from HeLa cells and assess arrestin binding. Unfortunately, even using different buffer conditions, we found that CCR5 was aggregating once solubilized from membranes, preventing us from doing this analysis. We had a similar problem when we exogenously expressed CCR5 in insect cells for purification purposes. We have long experience with CCR5, and this receptor is very aggregation-prone due to extended charged surfaces, which interact with the chemokines.

As an alternative, and in support of the cellular immunofluorescence assays, we also attempted to obtain internalization data via FACS using a CCR5 surface antibody (CD195 Monoclonal Antibody eBioT21/8). CD195 recognizes the N-terminus of the receptor. Unfortunately, the presence of the chemokine ligand (~ 8 kDa) interferes with antibody binding, precluding the quantitative biochemical assessment of the arrestin2 mutants on the CCR5 internalization.

For these reasons, we were particularly careful to quantify CCR5 internalization from the immunofluorescence microscopy data using colocalization coefficients as well as puncta counting (Figure 4+5).