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Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide

Nature volume 497, pages 137–141 (2013)Cite this article

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Abstract

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins1. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors2, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization3. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways4. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1–V2Rpp–Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the ‘lariat loop’ implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

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Figure 1: Fab30 specifically recognizes and stabilizes an active state of β-arrestin-1.
Figure 2: Conformational changes associated with β-arrestin-1 activation.
Figure 3: V2Rpp interactions with β-arrestin-1.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for the b-arrestin-1–V2Rpp– Fab30 complex are deposited in the Protein Data Bank under accession code 4JQI.

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Acknowledgements

We thank D. Capel for technical assistance and V. Ronk, D. Addison and Q. Lennon for administrative and secretarial support. We thank S. Ahn and L. Wingler for critical reading of the manuscript. We acknowledge support from the Stanford Medical Scientist Training Program and the American Heart Association (A.M.), from the National Science Foundation (A.C.K.), from the National Institutes of Health Grants NS028471 (B.K.K.), HL16037 and HL70631 (R.J.L.), GM072688 and GM087519 (A.A.K. and S.K.), HL 075443 (K.X.) and from the Mathers Foundation (B.K.K. and W.I.W.). R.I.R is supported by a post-doctoral fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–CAPES. R.J.L. is an investigator with the Howard Hughes Medical Institute.

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  1. Arun K. Shukla, Aashish Manglik and Andrew C. Kruse: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Arun K. Shukla, Kunhong Xiao, Rosana I. Reis, Wei-Chou Tseng, Dean P. Staus, Li-Yin Huang, Prachi Tripathi-Shukla & Robert J. Lefkowitz

  2. Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, 94305, California, USA

    Aashish Manglik, Andrew C. Kruse, Daniel Hilger, William I. Weis & Brian K. Kobilka

  3. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, 60637, Illinois, USA

    Serdar Uysal, Marcin Paduch, Akiko Koide, Shohei Koide & Anthony A. Kossiakoff

  4. Department of Structural Biology, Stanford University School of Medicine, Stanford, 94305, California, USA

    William I. Weis

  5. Howard Hughes Medical Institute. Duke University Medical Center, Durham, 27710, North Carolina, USA

    Robert J. Lefkowitz

  6. Department of Biochemistry, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Robert J. Lefkowitz

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Contributions

A.K.S. conceived the project, designed the Fab selection strategy, selected and characterized Fab30, established and optimized complex formation and purification conditions, prepared protein for crystallization trials and supervised the experiments related to the biochemical characterization of the complex. A.M. purified the complex, performed crystallography trials and grew crystals. A.M. and A.C.K. collected and processed diffraction data, and solved and refined the structure with supervision from W.I.W. R.I.R. assisted with advanced Fab characterization and optimized complex formation. W.-C.T. assisted with Fab selection and preliminary characterization. K.X. performed and analysed the crosslinking experiments. D.P.S. performed and analysed radioligand binding experiments. L.-Y.H. assisted with functional characterization of the complex. P.T.-S. expressed and purified the receptor. S.U., M.P., A.K., S.K. and A.A.K. generated and provided the phage display library and the screening protocol and helped with the initial phase of Fab selection. D.H. performed the comparison of the structural model with EPR data. A.K.S., A.M. and A.C.K. made figures. A.K.S., A.M., A.C.K., B.K.K. and R.J.L. wrote the manuscript. B.K.K. and R.J.L. supervised the overall research.

Corresponding authors

Correspondence to Brian K. Kobilka or Robert J. Lefkowitz.

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The authors declare no competing financial interests.

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Shukla, A., Manglik, A., Kruse, A. et al. Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature 497, 137–141 (2013). https://doi.org/10.1038/nature12120

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