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Using In Vitro Pull-Down and In-Cell Overexpression Assays to Study Protein Interactions with Arrestin

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Beta-Arrestins

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

Abstract

Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cell-based functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.

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Acknowledgments

This work was supported by NIH R01 grants EY011500, GM109955 and GM077561 (the latter two were merged into R35 GM122491) (VVG), NS065868 and DA030103 (EVG), GM120569 (TMI), R21grant DA DA043680 (TMI/VVG), and an AHA Predoctoral Fellowship 16PRE30180007 (NAP).

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Authors and Affiliations

  1. Department of Pharmacology, Vanderbilt University, Nashville, TN, USA

    Nicole A. Perry, Eugenia V. Gurevich, T. M. Iverson & Vsevolod V. Gurevich

  2. Department of Chemistry, Tennessee Technological University, Cookeville, TN, USA

    Xuanzhi Zhan

  3. Department of Biochemistry, Vanderbilt University, Nashville, TN, USA

    T. M. Iverson

  4. Center for Structural Biology, Vanderbilt University, Nashville, TN, USA

    T. M. Iverson

  5. Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA

    T. M. Iverson

Authors
  1. Nicole A. Perry
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  2. Xuanzhi Zhan
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  3. Eugenia V. Gurevich
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  4. T. M. Iverson
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  5. Vsevolod V. Gurevich
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Corresponding author

Correspondence to Vsevolod V. Gurevich .

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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Perry, N.A., Zhan, X., Gurevich, E.V., Iverson, T.M., Gurevich, V.V. (2019). Using In Vitro Pull-Down and In-Cell Overexpression Assays to Study Protein Interactions with Arrestin. 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_7

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  • DOI: https://doi.org/10.1007/978-1-4939-9158-7_7

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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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