Filor, Jenny C.

Hoffmann, Carsten

Matthees, Edda S. F.

English

Digitale Bibliothek Thüringen

RESEARCH HIGHLIGHT OPENNovel atypical G protein-coupled receptor (GPCR)-arrestincomplexes: a structural snapshot of the barcode hypothesisJenny C. Filor1, Edda S. F. Matthees 1 and Carsten Hoffmann 1✉Signal Transduction and Targeted Therapy          (2025) 10:251 ; https://doi.org/10.1038/s41392-025-02338-0In a recent study published in Nature by Chen et al., six novel cryo-EMstructures of atypical chemokine receptor 3 (ACKR3) complexes withArrestin2 (Arr2, also known as β-arrestin1) and Arrestin3 (Arr3, alsoknown as β-arrestin2) were resolved using a novel nanobody, Fab7,which stabilizes active arrestin independent of the isoform, interact-ing receptor or its phosphorylation pattern.1 This work providescritical insights into G protein-coupled receptor (GPCR)–arrestininteractions under specific GPCR kinase (GRK) phosphorylationconditions, allowing an unprecedented direct comparison of thesedynamic signaling complexes.For the last decade, a central objective in structural biology hasbeen to elucidate GPCR–arrestin complexes to better understandspecific receptor regulation. One main challenge is to stabilizethese naturally dynamic interactions for their structural illumina-tion. The research teams of Tracy M. Handel and John J.G. Tesmermajorly advanced this endeavor with the development of theirnovel nanobody Fab7 as a powerful tool for future structuralinvestigations. Crucially, this enabled the direct comparison ofGRK2- or GRK5-specific receptor phosphorylation and its effect onGPCR–arrestin complex formation.1GRK-specific phosphorylation patterns, their impact onreceptor–arrestin complexes and the associated biology are oftensummarized as the “barcode hypothesis”. More specifically, it statesthat unique phosphorylation patterns at the receptor C-terminus orintracellular loops, induced by the availability or activation ofspecific GRKs, may result in distinct receptor–arrestin complexformations and defines the arrestin-mediated functional outcomes.2Now, Chen et al. provide long-awaited structural evidence for thisconcept.1 The comparison of different phosphorylation patterns byGRK5 (proximal) or GRK2 (distal) elucidate new details of theACKR3–arrestin complex formations and reveal an additional degreeof flexibility to arrestin biology (Fig. 1a). The authors demonstratethat the relative distance of the phosphorylated cluster on thereceptor C-terminus in relation to the transmembrane (TM) core hasa great influence on the orientation and stability of the resultingcomplex. The utilized ACKR3 is atypical in the sense that it does notactivate G proteins but exhibits phosphorylation-dependent inter-actions with both Arr2 and Arr3. Cellular assays indicate that theACKR3 is mainly regulated by GRK5,3 which localizes to the plasmamembrane independently of active free G protein βγ-subunits asopposed to the cytosolic GRK2/3 subfamily.4 However, ACKR3 canalso be phosphorylated by GRK2, following the release of Gβγsubunits by co-activation of the CXCR4 receptor with the sharedligand, if Gβγ dimers are abundantly expressed.3 This cross-regulatory mechanism suggests a scenario where ACKR3 can actas a sensor for CXCR4 activity and influence the efficiency ofchemokine scavenging.1The high-resolution cryo-EM structure of the GRK5-phosphorylatedACKR3–Arr2 complex (Fig. 1a, PDB: 9E82) reveals interaction details ofboth the phosphorylated C-terminal region (T338–T341) and the TMcore of the receptor with arrestins. Under these conditions, Arr2 isdrawn into a more tightly interacting complex with the receptor(Fig. 1a). In contrast, the more distal GRK2-phosphorylation pattern(PDB: 8Tll) at T352-S355 led to more flexibility of the overall complexand a more loosely associated arrestin (Fig. 1b). In both structures,the arrestin finger loop region (FLR) was shown to interact with thesurrounding membrane rather than inserting into the active cavity ofthe receptor. Notably, the exact FLR position could not be resolved inthe GRK2-phosphorylated state, highlighting its high conformationalheterogeneity (Fig. 1c). Using fluorescence quenching assays, Chen etal. collected additional evidence, further supporting that the FLR canindeed interact with the micelle membrane for both isoforms in away that resembles the glucagon receptor–arrestin complex.1,2Interestingly, mutation of FLR revealed that for the GRK5-phosphorylated state, the binding affinity of Arr2 is less dependenton this specific interaction site. In addition, Chen et al. were able todistinguish arrestin isoform-specific interaction interfaces also at theC-edge region, after interaction with the same phosphorylationpattern of one GPCR (Fig. 1d). This region marks an importantdifference between the arrestin isoforms, as it is naturally shorter inArr3, potentially adding to its increased flexibility and heterogeneityof formed receptor complexes.In this study, the authors elegantly expanded the definition ofarrestin binding requirements to the ACKR3 in relation to otherobserved phosphorylation motifs.1,5 By using an additional 12amino acid Gly-linker to extend the distance between the GRK5-phosphorylated cluster and the receptor TM region, the resultingACKR3–arrestin complex was shifted towards the GRK2-specificphenotype although phosphorylated via GRK5. Based on the GRK2or GRK5-phosphorylated residues in the ACKR3 and the minorinfluence of the particular sequence but rather the localization ofthe interacting phospho-peptide, Chen et al. defined the ACKR3-specific phosphorylation barcode as X(pT)XX(pS/T)XΦ (where Xrepresents any amino acid, while Φ symbolizes hydrophobicresidues).1 In clear and focused analyses, they compared theirfindings to previous identified phosphorylation motifs withspecific amino acid patterns and their occurrence in otherGPCRs.1,5 Finally, the authors were able to integrate these resultsinto a refined consensus sequence for arrestin interactions:requiring two closely located phosphorylated (or negativelyReceived: 17 June 2025 Revised: 6 July 2025 Accepted: 7 July 20251Institut für Molekulare Zellbiologie, CMB – Center for Molecular Biomedicine; Universitätsklinikum Jena; Friedrich-Schiller-Universität Jena, Jena, GermanyCorrespondence: Carsten Hoffmann (carsten.hoffmann@med.uni-jena.de)These authors contributed equally: Jenny C. Filor, Edda S. F. Mattheeswww.nature.com/sigtransSignal Transduction and Targeted Therapy© The Author(s) 20251234567890();,:charged) residues in combination with a hydrophobic anchor orthird position phosphorylation (PXXPΦ).1Chen et al. provide structural evidence to understand theimpact of GRK-mediated phosphorylation patterns on arrestin-dependent outcomes.1 The formation of GPCR–arrestin complexesis determined by the proximity of these sites to the receptor’s TMcore in addition to the specific phosphorylation “barcode”. Thiswill also affect the additionally utilized stabilizing interactioninterfaces, which might differ between the arrestin isoforms.Hence, it should be considered to broaden the “barcodehypothesis” to include a more holistic view of the structuraldeterminants of GPCR–arrestin complexes. As the authors high-light, this insight is essential for understanding the pathophysio-logical regulation of GPCRs and advancing the rational design ofarrestin-biased ligands, offering a more nuanced view of biasedsignaling that incorporates GRK-driven modulation.AUTHOR CONTRIBUTIONSC.H., J.C.F., and E.S.F.M. conceptualized the project; E.S.F.M. designed the figure withconstructive feedback by J.C.F. and C.H.; J.C.F., E.S.F.M., and C.H. wrote the manuscript.All authors have read and approved the article.FUNDINGOpen Access funding enabled and organized by Projekt DEAL.ADDITIONAL INFORMATIONCompeting interests: The authors declare no competing interests.Publisher’s note Springer Nature remains neutral with regard to jurisdictional claimsin published maps and institutional affiliations.REFERENCES1. Chen, Q. et al. Effect of phosphorylation barcodes on arrestin binding to a che-mokine receptor. Nature 643, 280–287 (2025).2. Underwood, O., Haider, R. S., Sanchez, J. & Canals, M. Arrestin-centred interactionsat the membrane and their conformational determinants. Br. J. Pharm. 182,3135–3150 (2025).3. Schafer, C. T., Chen, Q., Tesmer, J. J. G. & Handel, T. M. Atypical ChemokineReceptor 3 “Senses” CXC Chemokine Receptor 4 Activation Through GPCR KinasePhosphorylation. Mol. Pharm. 104, 174–186 (2023).4. Matthees, E. S. F. et al. GRK specificity and Gbetagamma dependency determinesthe potential of a GPCR for arrestin-biased agonism. Commun. Biol. 7, 802 (2024).5. Maharana, J. et al. Molecular insights into atypical modes of beta-arrestin inter-action with seven transmembrane receptors. Science 383, 101–108 (2024).Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in anymedium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons licence, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons licence, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons licence and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directlyfrom the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2025Fig. 1 The dynamic conformational landscape of arrestins upon complex formation with ACKR3. a Cryo-EM structure of GRK5-phosphorylatedACKR3 with Arr2 (PDB: 9E82). The utilized ligand CXCL12LRHQ is shown in pink and captured membrane lipids are indicated in yellow. Theresolved Arr2 structure with the GRK2-phosphorylated ACKR3 C-terminus (orange, PDB: 8TII) was positioned to symbolize the register shift ofinteracting with the more distal phosphorylation cluster, leading to a more loosely associated complex and hence yielding a higher flexibility.b Differential C-terminal cluster phosphorylation by GRK5 (proximal, T338-T341) and GRK2 (distal, T352-S355), interacting with Arr2 (light graybox). c Close-up of Arr2 finger loop region (FLR), inserting into the micelle membrane upon GRK5-mediated phosphorylation, which could notbe fully resolved in complex with the GRK2-phosphorylated ACKR3 C-terminus. d Close-up of the C-edge region of Arr2 (PDB: 9E82) and Arr3(PDB: 8TIL) upon interacting with the GRK5-phosphorylated ACKR3Novel atypical G protein-coupled receptor (GPCR)-arrestin complexes: a. . .Filor et al.2Signal Transduction and Targeted Therapy          (2025) 10:251 

Novel atypical G protein-coupled receptor (GPCR)-arrestin complexes: a structural snapshot of the barcode hypothesis

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Novel atypical G protein-coupled receptor (GPCR)-arrestin complexes: a structural snapshot of the barcode hypothesis

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