Alpha 2.0
Login Register
» Articles » PMID: 26260597

Monitoring G Protein Activation in Cells with BRET

Overview
Journal Methods Mol Biol
Specialty Molecular Biology
Date 2015 Aug 12
PMID 26260597
Citations 55
Authors
Ikuo Masuho
Kirill A Martemyanov
Nevin A Lambert
Affiliations
Soon will be listed here.
Abstract

Live-cell assays based on fluorescence and luminescence are now indispensable tools for the study of G protein signaling. Assays based on fluorescence and bioluminescence resonance energy transfer (FRET and BRET) have been particularly valuable for monitoring changes in second messengers, protein-protein interactions, and protein conformation. Here, we describe a BRET assay that monitors the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits from all four G protein subfamilies. This assay provides useful kinetic and pharmacological information with reasonably high throughput using a standard laboratory equipment.

Citing Articles

The structure and function of the ghrelin receptor coding for drug actions.

Shiimura Y, Im D, Tany R, Asada H, Kise R, Kurumiya E Nat Struct Mol Biol. 2025; .

PMID: 39833471 DOI: 10.1038/s41594-024-01481-6.

N-terminal fragment shedding contributes to signaling of the full-length adhesion receptor ADGRL3.

Perry-Hauser N, Du Rand J, Lee K, Shi L, Javitch J J Biol Chem. 2025; 301(2):108174.

PMID: 39798870 PMC: 11849108. DOI: 10.1016/j.jbc.2025.108174.

Essential strategies for the detection of constitutive and ligand-dependent Gi-directed activity of 7TM receptors using bioluminescence resonance energy transfer.

Endzhievskaya S, Chahal K, Resnick J, Khare E, Roy S, Handel T bioRxiv. 2024; .

PMID: 39713355 PMC: 11661105. DOI: 10.1101/2024.12.04.626681.

An activating calcium-sensing receptor variant with biased signaling reveals a critical residue for Gα11 coupling.

Benson M, Wyatt R, Levine M, Gorvin C J Bone Miner Res. 2024; 40(2):270-282.

PMID: 39658204 PMC: 11789390. DOI: 10.1093/jbmr/zjae199.

Protocol to investigate G protein-coupled receptor signaling kinetics and concentration-dependent responses using ONE-GO biosensors.

Janicot R, Garcia-Marcos M STAR Protoc. 2024; 5(4):103383.

PMID: 39395175 PMC: 11735995. DOI: 10.1016/j.xpro.2024.103383.

References
1.
Pierce K, Premont R, Lefkowitz R . Seven-transmembrane receptors. Nat Rev Mol Cell Biol. 2002; 3(9):639-50. DOI: 10.1038/nrm908. View
2.
Hynes T, Tang L, Mervine S, Sabo J, Yost E, Devreotes P . Visualization of G protein betagamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting. J Biol Chem. 2004; 279(29):30279-86. DOI: 10.1074/jbc.M401432200. View
3.
Hollins B, Kuravi S, Digby G, Lambert N . The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers. Cell Signal. 2009; 21(6):1015-21. PMC: 2668204. DOI: 10.1016/j.cellsig.2009.02.017. View
4.
Lambert N, Johnston C, Cappell S, Kuravi S, Kimple A, Willard F . Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity. Proc Natl Acad Sci U S A. 2010; 107(15):7066-71. PMC: 2872438. DOI: 10.1073/pnas.0912934107. View
5.
Lohse M, Nuber S, Hoffmann C . Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol Rev. 2012; 64(2):299-336. DOI: 10.1124/pr.110.004309. View