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

Perry NA, Zhan X, Gurevich EV, Iverson TM, Gurevich VV
Methods Mol Biol. 2019 1957: 107-120

PMID: 30919350 · DOI:10.1007/978-1-4939-9158-7_7

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.

MeSH Terms (12)

Animals Arrestin Biological Assay Cercopithecus aethiops COS Cells HEK293 Cells Humans Immobilized Proteins Mice Protein Binding Protein Interaction Mapping Recombinant Fusion Proteins

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