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Using In Vitro Pull-Down and In-Cell Overexpression Assays to Study Protein Interactions with Arrestin
Nicole A Perry1, Xuanzhi Zhan2, Eugenia V Gurevich1
1Department of Pharmacology, Vanderbilt University, Nashville, TN, USA.
None:
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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