BRET-based assay to specifically monitor β<inf>2</inf>AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation

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 w...

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Main Authors: Warisara Parichatikanond, Ei Thet Htar Kyaw, Corina T. Madreiter-Sokolowski, Supachoke Mangmool
Other Authors: Mahidol University
Format: Book Chapter
Published: 2022
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Online Access:https://repository.li.mahidol.ac.th/handle/123456789/76361
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spelling th-mahidol.763612022-08-04T15:14:09Z BRET-based assay to specifically monitor β<inf>2</inf>AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation Warisara Parichatikanond Ei Thet Htar Kyaw Corina T. Madreiter-Sokolowski Supachoke Mangmool Mahidol University Medizinische Universität Graz Biochemistry, Genetics and Molecular Biology 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. 2022-08-04T08:14:09Z 2022-08-04T08:14:09Z 2021-01-01 Book Chapter Methods in Cell Biology. Vol.166, (2021), 67-81 10.1016/bs.mcb.2021.06.005 0091679X 2-s2.0-85109437462 https://repository.li.mahidol.ac.th/handle/123456789/76361 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85109437462&origin=inward
institution Mahidol University
building Mahidol University Library
continent Asia
country Thailand
Thailand
content_provider Mahidol University Library
collection Mahidol University Institutional Repository
topic Biochemistry, Genetics and Molecular Biology
spellingShingle Biochemistry, Genetics and Molecular Biology
Warisara Parichatikanond
Ei Thet Htar Kyaw
Corina T. Madreiter-Sokolowski
Supachoke Mangmool
BRET-based assay to specifically monitor β<inf>2</inf>AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation
description 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.
author2 Mahidol University
author_facet Mahidol University
Warisara Parichatikanond
Ei Thet Htar Kyaw
Corina T. Madreiter-Sokolowski
Supachoke Mangmool
format Book Chapter
author Warisara Parichatikanond
Ei Thet Htar Kyaw
Corina T. Madreiter-Sokolowski
Supachoke Mangmool
author_sort Warisara Parichatikanond
title BRET-based assay to specifically monitor β<inf>2</inf>AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation
title_short BRET-based assay to specifically monitor β<inf>2</inf>AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation
title_full BRET-based assay to specifically monitor β<inf>2</inf>AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation
title_fullStr BRET-based assay to specifically monitor β<inf>2</inf>AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation
title_full_unstemmed BRET-based assay to specifically monitor β<inf>2</inf>AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation
title_sort bret-based assay to specifically monitor β<inf>2</inf>ar/grk2 interaction and β-arrestin2 conformational change upon βar stimulation
publishDate 2022
url https://repository.li.mahidol.ac.th/handle/123456789/76361
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