BRET-based assay to specifically monitor β2AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation.

Warisara Parichatikanond, Ei Thet Htar Kyaw, Corina T Madreiter-Sokolowski, Supachoke Mangmool

Methods in cell biology 2021

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The β-adrenergic receptors (βARs) 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 βARs leads to an activation of G proteins and their canonical effectors. In a parallel way, βAR stimulation triggers the termination of its signals by receptor desensitization. This termination process is initiated by G protein-coupled receptor kinase (GRK)-induced βAR phosphorylation that promotes the recruitment of β-arrestins to phosphorylated βAR. The uncoupled βARs which formed a complex with GRK and β-arrestin subsequently internalize into the cytosol. In addition, GRKs and β-arrestins also act as scaffolding proteins and signal transducers in their own functions to modulate various downstream effectors. Upon translocation to the βAR, β-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 βAR with both GRK and β-arrestin. The assay described here allows the measurement of the BRET signal for detecting the interaction of β2AR with GRK2 and the conformational change of β-arrestin2 following βAR stimulation. Copyright © 2021 Elsevier Inc. All rights reserved.

Citation

Warisara Parichatikanond, Ei Thet Htar Kyaw, Corina T Madreiter-Sokolowski, Supachoke Mangmool. BRET-based assay to specifically monitor β2AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation. Methods in cell biology. 2021;166:67-81