Real-time monitoring of ubiquitination in living cells by BRET
- PMID: 15782195
- DOI: 10.1038/nmeth722
Real-time monitoring of ubiquitination in living cells by BRET
Abstract
Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiological environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioluminescence resonance energy transfer (BRET), we measured the ubiquitination of beta-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addition to allowing the detection of basal and GPCR-regulated ubiquitination of beta-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.
Comment in
-
Resonating to the music of ubiquitination.Nat Methods. 2004 Dec;1(3):191-3. doi: 10.1038/nmeth1204-191. Nat Methods. 2004. PMID: 15782194 No abstract available.
Similar articles
-
Resonating to the music of ubiquitination.Nat Methods. 2004 Dec;1(3):191-3. doi: 10.1038/nmeth1204-191. Nat Methods. 2004. PMID: 15782194 No abstract available.
-
Improved donor/acceptor BRET couples for monitoring beta-arrestin recruitment to G protein-coupled receptors.Biotechnol J. 2009 Sep;4(9):1337-44. doi: 10.1002/biot.200900016. Biotechnol J. 2009. PMID: 19557797
-
Detection of GPCR/beta-arrestin interactions in live cells using bioluminescence resonance energy transfer technology.Methods Mol Biol. 2009;552:305-17. doi: 10.1007/978-1-60327-317-6_22. Methods Mol Biol. 2009. PMID: 19513659
-
Monitoring interactions between G-protein-coupled receptors and beta-arrestins.Biochem Soc Trans. 2007 Aug;35(Pt 4):764-6. doi: 10.1042/BST0350764. Biochem Soc Trans. 2007. PMID: 17635143 Review.
-
New technologies: bioluminescence resonance energy transfer (BRET) for the detection of real time interactions involving G-protein coupled receptors.Pituitary. 2003;6(3):141-51. doi: 10.1023/b:pitu.0000011175.41760.5d. Pituitary. 2003. PMID: 14974443 Review.
Cited by
-
BRET biosensors to study GPCR biology, pharmacology, and signal transduction.Front Endocrinol (Lausanne). 2012 Aug 29;3:105. doi: 10.3389/fendo.2012.00105. eCollection 2012. Front Endocrinol (Lausanne). 2012. PMID: 22952466 Free PMC article.
-
Visualizing dynamic activities of signaling enzymes using genetically encodable FRET-based biosensors from designs to applications.Methods Enzymol. 2012;504:317-40. doi: 10.1016/B978-0-12-391857-4.00016-1. Methods Enzymol. 2012. PMID: 22264542 Free PMC article. Review.
-
Development and Applications of Bioluminescent and Chemiluminescent Reporters and Biosensors.Annu Rev Anal Chem (Palo Alto Calif). 2019 Jun 12;12(1):129-150. doi: 10.1146/annurev-anchem-061318-115027. Epub 2019 Feb 20. Annu Rev Anal Chem (Palo Alto Calif). 2019. PMID: 30786216 Free PMC article. Review.
-
Evolution of BRET Biosensors from Live Cell to Tissue-Scale In vivo Imaging.Front Endocrinol (Lausanne). 2013 Sep 23;4:131. doi: 10.3389/fendo.2013.00131. Front Endocrinol (Lausanne). 2013. PMID: 24065957 Free PMC article. Review.
-
Resistance to inhibitors of cholinesterase (Ric)-8A and Gαi contribute to cytokinesis abscission by controlling vacuolar protein-sorting (Vps)34 activity.PLoS One. 2014 Jan 21;9(1):e86680. doi: 10.1371/journal.pone.0086680. eCollection 2014. PLoS One. 2014. PMID: 24466196 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
