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 microscale thermophoresis (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.
Similar content being viewed by others
Data availability
Data supporting the findings of this manuscript are available in the Source Data File. A reporting summary for this Article is available as a Supplementary Information file. Data for sequence analysis in Supplementary Fig. 4 are available from the GPCR database (gpcrdb.org). HDX-MS data have been deposited to ProteomeXchange Consortium via PRIDE partner repository with the set identifier PXD065019. The following accession codes are used in the study: 1G4M, 4JQI, 1GP2, 6EG8, 3SN6. Further information and requests for reagents should be directed to and fulfilled by the corresponding author, Ka Young Chung (kychung2@skku.edu). Source data files are provided in this paper. Source data are provided with this paper.
References
Shukla, A. K., Xiao, K. & Lefkowitz, R. J. Emerging paradigms of beta-arrestin-dependent seven transmembrane receptor signaling. Trends Biochem. Sci. 36, 457–469 (2011).
Hilger, D., Masureel, M. & Kobilka, B. K. Structure and dynamics of GPCR signaling complexes. Nat. Struct. Mol. Biol. 25, 4–12 (2018).
Weis, W. I. & Kobilka, B. K. The molecular basis of G protein-coupled receptor activation. Annu. Rev. Biochem. 87, 897–919 (2018).
Syrovatkina, V., Alegre, K. O., Dey, R. & Huang, X. Y. Regulation, signaling, and physiological functions of G-proteins. J. Mol. Biol. 428, 3850–3868 (2016).
Mahoney, J. P. & Sunahara, R. K. Mechanistic insights into GPCR-G protein interactions. Curr. Opin. Struct. Biol. 41, 247–254 (2016).
Milligan, G. & Kostenis, E. Heterotrimeric G-proteins: a short history. Br. J. Pharmacol. 147, S46–S55 (2006).
Wilden, U., Hall, S. W. & Kuhn, H. Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. Proc. Natl. Acad. Sci. USA 83, 1174–1178 (1986).
Benovic, J. L. et al. Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein). Proc. Natl. Acad. Sci. USA 84, 8879–8882 (1987).
Smith, J. S. & Rajagopal, S. The beta-arrestins: multifunctional regulators of G protein-coupled receptors. J. Biol. Chem. 291, 8969–8977 (2016).
Lohse, M. J., Benovic, J. L., Codina, J., Caron, M. G. & Lefkowitz, R. J. beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science 248, 1547–1550 (1990).
DeFea, K. A. Beta-arrestins as regulators of signal termination and transduction: how do they determine what to scaffold? Cell. Signal. 23, 621–629 (2011).
Attramadal, H. et al. Beta-arrestin2, a novel member of the arrestin/beta-arrestin gene family. J. Biol. Chem. 267, 17882–17890 (1992).
Daly, C. et al. beta-Arrestin-dependent and -independent endosomal G protein activation by the vasopressin type 2 receptor. eLife https://doi.org/10.7554/eLife.87754 (2023).
Smith, J. S. et al. Noncanonical scaffolding of G(alphai) and beta-arrestin by G protein-coupled receptors. Science https://doi.org/10.1126/science.aay1833 (2021).
Thomsen, A. R. B. et al. GPCR-G protein-beta-arrestin super-complex mediates sustained G protein signaling. Cell 166, 907–919 (2016).
Li, B., Wang, C., Zhou, Z., Zhao, J. & Pei, G. beta-Arrestin-1 directly interacts with Galphas and regulates its function. FEBS Lett. 587, 410–416 (2013).
Granzin, J. et al. X-ray crystal structure of arrestin from bovine rod outer segments. Nature 391, 918–921 (1998).
Hirsch, J. A., Schubert, C., Gurevich, V. V. & Sigler, P. B. The 2.8 A crystal structure of visual arrestin: a model for arrestin’s regulation. Cell 97, 257–269 (1999).
Shukla, A. K. et al. Structure of active beta-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature 497, 137–141 (2013).
Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561–567 (2015).
Liu, Q. et al. DeSiphering receptor core-induced and ligand-dependent conformational changes in arrestin via genetic encoded trimethylsilyl 1H-NMR probe. Nat. Commun. 11, 4857 (2020).
Zhai, R. et al. Distinct activation mechanisms of beta-arrestin-1 revealed by (19)F NMR spectroscopy. Nat. Commun. 14, 7865 (2023).
Maharana, J. et al. Molecular insights into atypical modes of beta-arrestin interaction with seven transmembrane receptors. Science 383, 101–108 (2024).
He, Q. T. et al. Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2. Nat. Commun. 12, 2396 (2021).
Pulvermuller, A. et al. Functional differences in the interaction of arrestin and its splice variant, p44, with rhodopsin. Biochemistry 36, 9253–9260 (1997).
Kim, Y. J. et al. Crystal structure of pre-activated arrestin p44. Nature 497, 142–146 (2013).
Kim, D. K., Yun, Y., Kim, H. R., Seo, M. D. & Chung, K. Y. Different conformational dynamics of various active states of beta-arrestin1 analyzed by hydrogen/deuterium exchange mass spectrometry. J. Struct. Biol. 190, 250–259 (2015).
Latorraca, N. R. et al. How GPCR phosphorylation patterns orchestrate arrestin-mediated signaling. Cell 183, 1813–1825.e1818 (2020).
Hvidt, A. & Nielsen, S. O. in Advances in Protein Chemistry Vol. 21 (eds Anfinsen, C. B. et al.) 287–386 (Academic Press, 1966).
Wales, T. E. & Engen, J. R. Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom. Rev. 25, 158–170 (2006).
Qu, C. et al. Scaffolding mechanism of arrestin-2 in the cRaf/MEK1/ERK signaling cascade. Proc. Natl. Acad. Sci. USA https://doi.org/10.1073/pnas.2026491118 (2021).
Ham, D., Inoue, A., Xu, J., Du, Y. & Chung, K. Y. Molecular mechanism of muscarinic acetylcholine receptor M3 interaction with Gq. Commun. Biol. 7, 362 (2024).
Nehme, R. et al. Mini-G proteins: novel tools for studying GPCRs in their active conformation. PLoS ONE 12, e0175642 (2017).
McEwen, D. P., Gee, K. R., Kang, H. C. & Neubig, R. R. Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins. Anal. Biochem. 291, 109–117 (2001).
Tontson, L., Babina, A., Vosumaa, T., Kopanchuk, S. & Rinken, A. Characterization of heterotrimeric nucleotide-depleted Galpha(i)-proteins by Bodipy-FL-GTPgammaS fluorescence anisotropy. Arch. Biochem. Biophys. 524, 93–98 (2012).
Kim, K. & Chung, K. Y. Molecular mechanism of beta-arrestin-2 pre-activation by phosphatidylinositol 4,5-bisphosphate. EMBO Rep. https://doi.org/10.1038/s44319-024-00239-x (2024).
Jones Brunette, A. M. & Farrens, D. L. Distance mapping in proteins using fluorescence spectroscopy: tyrosine, like tryptophan, quenches bimane fluorescence in a distance-dependent manner. Biochemistry 53, 6290–6301 (2014).
Bous, J. et al. Structure of the vasopressin hormone-V2 receptor-beta-arrestin1 ternary complex. Sci. Adv. 8, eabo7761 (2022).
Ranjan, R., Gupta, P. & Shukla, A. K. GPCR Signaling: beta-arrestins Kiss and Remember. Curr. Biol. 26, R285–R288 (2016).
Eichel, K., Jullie, D. & von Zastrow, M. beta-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat. Cell Biol. 18, 303–310 (2016).
Eichel, K. et al. Catalytic activation of beta-arrestin by GPCRs. Nature 557, 381–386 (2018).
Grimes, J. et al. Plasma membrane preassociation drives beta-arrestin coupling to receptors and activation. Cell 186, 2238–2255.e2220 (2023).
Janetzko, J. et al. Membrane phosphoinositides regulate GPCR-beta-arrestin complex assembly and dynamics. Cell 185, 4560–4573.e4519 (2022).
Fisher, N. M. & von Zastrow, M. Opioid receptors reveal a discrete cellular mechanism of endosomal G protein activation. Proc. Natl. Acad. Sci. USA 122, e2420623122 (2025).
Nobles, K. N. et al. Distinct phosphorylation sites on the beta(2)-adrenergic receptor establish a barcode that encodes differential functions of beta-arrestin. Sci. Signal 4, ra51 (2011).
Ahn, D. et al. Galphas slow conformational transition upon GTP binding and a novel Galphas regulator. iScience 26, 106603 (2023).
Ham, D. et al. Conformational switch that induces GDP release from Gi. J. Struct. Biol. 213, 107694 (2021).
Ham, D., Ahn, D., Chung, C. & Chung, K. Y. Isolation and conformational analysis of the Galpha alpha-helical domain. Biochem. Biophys. Res. Commun. 685, 149153 (2023).
Du, Y. et al. Assembly of a GPCR-G protein complex. Cell 177, 1232–1242 e1211 (2019).
Kim, H. R. et al. Structural mechanism underlying primary and secondary coupling between GPCRs and the Gi/o family. Nat. Commun. 11, 3160 (2020).
Charest, P. G., Terrillon, S. & Bouvier, M. Monitoring agonist-promoted conformational changes of beta-arrestin in living cells by intramolecular BRET. EMBO Rep. 6, 334–340 (2005).
Acknowledgements
We thank Asuka Inoue (Kyoto University) for kindly providing the ΔGαs cell line. This study was supported by grants from the Ministry of Science and ICT (RS-2019-NR040057, RS-2021-NR059713, and RS-2025-23323103 to K.Y.C.). S.K. was supported by the Brain Korea 21 FOUR program “Education and Research Center for Fostering Pharmaceutical Researchers towards Leading Innovative Growth” at the School of Pharmacy, Sungkyunkwan University. Y.K. was supported by grants of the Korea Basic Science Institute (National research Facilities and Equipment Center), National Research Foundation of Korea (NRF) (RS-2024-00340059 and RS-2024-00398706), the Korea Health Technology R&D Project (the Korea Health Industry Development Institute, KHIDI) (RS-2024-00512909), and the Gwangju Institute of Science and Technology (GIST) research fund (Future-leading Specialized Research project, 2025). J.S was supported by the National Research Foundation of Korea (NRF) (RS-2023-00227950, RS-2024-00407331, RS-2024-00338426).
Author information
Authors and Affiliations
Contributions
L.D. prepared βarr1 and Gα proteins and performed MST, HDX-MS, BODIPY-FL–GTPγS assay, and β-arrestin βXX release assay for all samples. H.K. and J.S. performed the BRET assay. Y.S. and Y.K. performed the PLA assay. D.A. assisted in Gα purification and BODIPY-FL–GTPγS assay. S.K. and J.H. performed the structural analysis. K.Y.C. initiated and supervised the project. K.Y.C. and L.D. analyzed the data and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
L.D. and K.Y.C. have a patent pending for βarr C-tail release assay (application number: 10-2024-0091925). The remaining authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Duan, L., Kim, H., Suh, Y. et al. Functional and structural insights into interactions between β-Arrestin 1 and Gαs or Gαi1. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68690-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-68690-z
