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


Perry NANicole A , Zhan XXuanzhi , Gurevich EVEugenia V , Iverson TMT M , Gurevich VVVsevolod V . Methods in molecular biology (Clifton, N.J.). 2019 3 ; 1957(). 107-120


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.