Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
Parichatikanond, W; Kyaw, ETH; Madreiter-Sokolowski, CT; Mangmool, S.
BRET-based assay to specifically monitor beta(2)AR/GRK2 interaction and beta-arrestin2 conformational change upon beta AR stimulation
METHOD CELL BIOL. 2021; 166: 67-81.
Doi: 10.1016/bs.mcb.2021.06.005
Web of Science
PubMed
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FullText_MUG
- Führende Autor*innen der Med Uni Graz
- Parichatikanond Warisara
- Co-Autor*innen der Med Uni Graz
- Madreiter-Sokolowski Corina
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- Abstract:
- The beta-adrenergic receptors (beta ARs) are members of G protein-coupled receptor (GPCR) family and have been one of themost important GPCRs for studying receptor endocytosis and signaling pathway. Agonist binding of beta ARs leads to an activation of G proteins and their canonical effectors. In a parallel way, beta AR stimulation triggers the termination of its signals by receptor desensitization. This termination process is initiated by G protein-coupled receptor kinase (GRK)-induced beta AR phosphorylation that promotes the recruitment of beta-arrestins to phosphorylated beta AR. The uncoupled beta ARs which formed a complex with GRK and beta-arrestin subsequently internalize into the cytosol. In addition, GRKs and beta-arrestins also act as scaffolding proteins and signal transducers in their own functions to modulate various downstream effectors. Upon translocation to the beta AR, beta-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 beta AR with both GRK and beta-arrestin. The assay described here allows the measurement of the BRET signal for detecting the interaction of beta 2AR with GRK2 and the conformational change of beta-arrestin2 following beta AR stimulation.