Abstract
Beta-arrestins (βarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how βarrs communicate with their downstream effectors. Here, we delineate structural mechanisms underlying βarr-mediated signal transduction. Using cryo-electron microscopy, we elucidate how βarr1 recruits and activates the non-receptor tyrosine kinase Src, a well-established signaling partner of βarrs. βarr1 engages Src SH3 through two distinct sites, each employing a different recognition mechanism: a polyproline motif in the N-domain and a non-proline-based interaction in the central crest region. At both sites βarr1 interacts with the aromatic surface of SH3, disrupting the autoinhibited conformation of Src and directly triggering its allosteric activation. This structural evidence establishes βarr1 as an active regulatory protein rather than a passive scaffold and suggests a potentially general mechanism for βarr-mediated signaling across diverse effectors.
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Data availability
The cryo-EM maps have been deposited in the EMDB under accession codes EMD-45977 (SH3–βarr1-CC complex), EMD-45982 (SH3–βarr1-N complex), and EMD-44881 (Src–βarr1-CC complex). The atomic coordinates have been deposited in the Protein Data Bank under accession codes 9CX3 (SH3–βarr1-CC complex); 9CX9 (SH3–βarr1-N complex); 9BT8 (Src–βarr1-CC complex). The HDX-MS data have been deposited to the ProteomeXchange Consortium [http://proteomecentral.proteomexchange.org] via the MassIVE repository [https://massive.ucsd.edu/] with the dataset identifier PXD073493. CXMS data have been deposited to the ProteomeXchange Consortium [http://proteomecentral.proteomexchange.org] via the PRIDE partner repository74 with the dataset identifier PXD073058. All other data generated or analyzed in this study are included in the article and its Supplementary Information. Source data are provided within the Source Data File. The manuscript refers to the following previously published PDB accession codes: 4JQI (βarr1–V2Rpp); 2PTK (Src); 1G4M (βarr1); 1FMK (Src); 6TKO (β1V2R–βarr1); 6U1N (M2V2R–βarr1); 6UP7 (NTSR1–βarr1); 4U5W (Hck–Nef); 2KNB (endophilin A1 SH3–parkin Ubl); 3A98 (DOCK2 SH3–ELMO 1); 1JT4 (Sla1 SH3-ubiquitin); 1Y57 (Src). Source data are provided with this paper.
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Acknowledgements
We acknowledge the use of cryo-EM microscopes at the Shared Materials Instrumentation Facility (Duke University) and thank Nilakshee Bhattacharya for assistance with microscope operation. We are grateful to Liyin Huang for their helpful discussions throughout this work and to Yangyang Li for administrative assistance. R.J.L. is an Investigator of the Howard Hughes Medical Institute. This work was supported, in part, by US National Institutes of Health (National Heart, Lung, and Blood Institute: R01 HL16037 to R.J.L.; National Institute of General Medical Sciences: R35GM133598 to A.G.). N.P. is supported by postdoctoral fellowships from Human Frontier Science Program (LT000174/2018) and European Molecular Biology Organization (ALTF 1071-2017). K.X. is supported by the Moonshot Biomarker Program of Allegheny Health Network Cancer Institute and Highmark Health, the Prostate Cancer Foundation Challenge Award (2023CHAL4223), the PA State Formula Grant (SAP #: 4100095527), and The Pittsburgh Foundation (cc#45126409).
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N.P. and R.J.L. conceived the study. N.P., R.R.A., J.K., A.W.K., B.P., K.X., and S.A. designed the experiments. N.P., B.N.T., L.L., D.K.B., A.W.K., B.P., K.X., R.O., and X.Z. performed biochemical experiments. N.P., B.N.T., J.K. and S.A. performed cellular functional assays. N.P., B.N.T., and D.K.B. performed structural biology experiments. N.P. and H.B. built and refined structural models. R.R.A. and S.L. performed HDX-MS experiments. N.P., B.N.T., H.B., D.K.B., R.R.A., J.K., A.W.K., B.P., and K.X. analyzed the data. N.P. wrote the manuscript with input from all authors. A.G. and R.J.L. supervised the work.
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Pakharukova, N., Thomas, B.N., Bansia, H. et al. Mechanism of beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69884-1
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DOI: https://doi.org/10.1038/s41467-026-69884-1
