Using In Vitro Pull-Down and In-Cell Overexpression Assays to Study Protein Interactions with Arrestin
AUTHORS
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
- PMID: 30919350[PubMed].
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.