BRET-based assay to specifically monitor ß2AR/GRK2 interaction and ß-arrestin2 conformational change upon ßAR stimulation.
Methods Cell Biol
; 166: 67-81, 2021.
Article
em En
| MEDLINE
| ID: mdl-34752340
The ß-adrenergic receptors (ßARs) are members of G protein-coupled receptor (GPCR) family and have been one of the most important GPCRs for studying receptor endocytosis and signaling pathway. Agonist binding of ßARs leads to an activation of G proteins and their canonical effectors. In a parallel way, ßAR stimulation triggers the termination of its signals by receptor desensitization. This termination process is initiated by G protein-coupled receptor kinase (GRK)-induced ßAR phosphorylation that promotes the recruitment of ß-arrestins to phosphorylated ßAR. The uncoupled ßARs which formed a complex with GRK and ß-arrestin subsequently internalize into the cytosol. In addition, GRKs and ß-arrestins also act as scaffolding proteins and signal transducers in their own functions to modulate various downstream effectors. Upon translocation to the ßAR, ß-arrestin is believed to undergo an important conformational change in the structure that is necessary for its signal transduction. The bioluminescence resonance energy transfer (BRET) technique involves the fusion of donor (luciferase) and acceptor (fluorescent) molecules to the interested proteins. Co-expression of these fusion proteins enables direct detection of their interactions in living cells. Here we describe the use of our established BRET technique to track the interaction of ßAR with both GRK and ß-arrestin. The assay described here allows the measurement of the BRET signal for detecting the interaction of ß2AR with GRK2 and the conformational change of ß-arrestin2 following ßAR stimulation.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Beta-Arrestina 2
Idioma:
En
Revista:
Methods Cell Biol
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Tailândia