A high-resolution analysis of arrestin2 interactions responsible for CCR5 endocytosis
Curation statements for this article:-
Curated by eLife
eLife Assessment
The authors investigate arrestin2-mediated CCR5 endocytosis in the context of clathrin and AP2 contributions. Using an extensive set of NMR experiments, and supported by microscopy and other biophysical assays, the authors provide convincing data on the roles of AP2 and clathrin in CCR5 endocytosis. This important work will appeal to an audience beyond those studying chemokine receptors, including those studying GPCR regulation and trafficking. The distinct role of AP2 and not clathrin will be of particular interest to those studying GPCR internalization mechanisms.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Abstract
Clathrin-mediated endocytosis (CME) is crucial for regulating G protein-coupled receptors (GPCRs) via phosphorylation-dependent arrestin interactions. Despite detailed structural knowledge on the arrestin interactions with phosphorylated tails of GPCRs, the interplay between receptor phosphorylation and arrestin coupling to the CME machinery is not well understood, in particular due to the weakness and dynamics of the individual molecular interactions. Here we have characterized the interactions of arrestin2, which is activated by the phosphorylated C-terminus of the human chemokine receptor 5 (CCR5), with the main protein constituents of CME, namely clathrin and AP2 proteins by solution NMR spectroscopy, biochemical and cellular assays. The NMR analysis revealed that arrestin2 interacts weakly with clathrin through a single binding site, independent of arrestin2 activation. In contrast, the arrestin2-AP2 interaction is stronger, requires arrestin2 activation by the CCR5 phospho-tail, and depends quantitatively on its degree of phosphorylation. These in vitro results are corroborated by cellular assays, which show that the chemokine-induced formation of a long-lived CCR5-arrestin2 internalization complex depends strongly on the interaction of arrestin2 with AP2, but not with clathrin. Taken together, these findings provide quantitative, atom-scale insights on the first steps of CCR5 endocytosis.
Article activity feed
-
-
-
eLife Assessment
The authors investigate arrestin2-mediated CCR5 endocytosis in the context of clathrin and AP2 contributions. Using an extensive set of NMR experiments, and supported by microscopy and other biophysical assays, the authors provide convincing data on the roles of AP2 and clathrin in CCR5 endocytosis. This important work will appeal to an audience beyond those studying chemokine receptors, including those studying GPCR regulation and trafficking. The distinct role of AP2 and not clathrin will be of particular interest to those studying GPCR internalization mechanisms.
-
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 …
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-fits-all model for GPCR-arrestin signaling and instead support a nuanced, receptor-specific view, with one example summarized effectively in the mechanistic figure.
-
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 …
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.
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?
(2) I don't understand how methyl TROSY spectra of arrestin2 with phosphopeptide could look so broadened unless there are sample stability problems.
(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?
(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?
(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.
-
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 …
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).
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.
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 receptor may be related to the position of the tags.
-
-