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A Mass Spectrometry-Based Structural Assay for Activation-Dependent Conformational Changes in β-Arrestins

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Beta-Arrestins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1957))

Abstract

β-Arrestins are key regulation proteins for G protein-coupled receptors (GPCRs) signaling. Experimental evidence suggests that β-arrestins undergo conformational changes concomitant with binding to activated, phosphorylated GPCRs. We developed a mass spectrometry-based structural proteomic assay to monitor conformational changes associated with the activation of β-arrestins. This assay utilizes synthesized phosphopeptides mimicking phosphorylated C-terminal tails of GPCRs to activate β-arrestins. The activation-dependent conformational changes of β-arrestins are revealed using limited proteolysis coupled with both SDS-PAGE and mass spectrometry analysis. As an in vitro β-arrestin activation assay, this mass spectrometry-based structural method can be adapted as a simple but useful tool to study the nature and extent of conformational changes of β-arrestins downstream of different receptors as well as β-arrestin conformations associated with different functions, such as desensitization, internalization, and signaling.

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Acknowledgment

This work was supported, in part, by US National Institutes of Health grant no HL-075443 Proteomics Core support to K.X. This publication was also made possible by seed funding support to K.X. from the Department of Pharmacology and Chemical Biology, the University of Pittsburgh and Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania, and the Institute for Transfusion Medicine.

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Authors and Affiliations

  1. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA

    Yang Zhao & Kunhong Xiao

  2. Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA

    Kunhong Xiao

  3. Biomedical Mass Spectrometry Center, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Kunhong Xiao

Authors
  1. Yang Zhao
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  2. Kunhong Xiao
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Corresponding author

Correspondence to Kunhong Xiao .

Editor information

Editors and Affiliations

  1. Institut Cochin, INSERM U1016, Paris, France

    Mark G. H. Scott

  2. Department of Medicine, Research Institute of the McGill, University Health Center (RI-MUHC), McGill University, Montreal, QC, Canada

    Stéphane A. Laporte

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Zhao, Y., Xiao, K. (2019). A Mass Spectrometry-Based Structural Assay for Activation-Dependent Conformational Changes in β-Arrestins. In: Scott, M., Laporte, S. (eds) Beta-Arrestins. Methods in Molecular Biology, vol 1957. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9158-7_18

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  • DOI: https://doi.org/10.1007/978-1-4939-9158-7_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9157-0

  • Online ISBN: 978-1-4939-9158-7

  • eBook Packages: Springer Protocols

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