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Real-time monitoring of ubiquitination in living cells by BRET

Nature Methods volume 1, pages 203–208 (2004)Cite this article

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 β-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 β-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.

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Figure 1: Schematic representation of the β-arrestin ubiquitination BRET2 assay.
Figure 2: Specific BRET2 signal between Rluc–β-arrestin and GFP2-Ubi.
Figure 3: Detection of GPCR-induced β-arrestin ubiquitination.
Figure 4: Concomitant detection of β-arrestin ubiquitination and its recruitment to the activated receptor.
Figure 5: Real-time assessment of receptor stimulated β-arrestin ubiquitination.

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Acknowledgements

The authors are grateful to A. Vallée-Bélisle and S. Michnick for providing the plasmid pQE32-Ubi and to S. Marullo for providing the pCMV-Myc–tagged β-arrestin 2 plasmid. This work was supported by grants from the Canadian Institute for Health Research (M.B.) and the Natural Science and Engineering Research Council of Canada (M.A.). J.P. holds a fellowship from the European Molecular Biology Organization, M.A. holds a scholarship from the Fond de Recherche en Santé du Québec and M.B. is a Canada Research Chair in Signal Transduction and Molecular Pharmacology.

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  1. Julie Perroy and Stephanie Pontier: These authors contributed equally to this work.

Authors and Affiliations

  1. Département de Biochimie and Groupe de Recherche sur le Système Nerveux Autonome, Université de Montréal, C.P. 6128 Succursale Centre-Ville Montréal, Québec, H3C 3J7, Canada

    Julie Perroy, Stephanie Pontier, Pascale G Charest, Muriel Aubry & Michel Bouvier

  2. Laboratory of Functional Genomics, UPR CNRS 2580 CCIPE, 141 rue de la Cardonille, Montpellier, 34094 Cedex 05, France

    Julie Perroy

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  1. Julie Perroy
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Correspondence to Michel Bouvier.

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Supplementary information

Supplementary Fig. 1

Detection of receptor ubiquitination following short-term agonist stimulation. (PDF 35 kb)

Supplementary Fig. 2

Comparison of ubiquitination BRET signals obtained with either GFP2-Ubi or GFP2-UbiWT constructs. (PDF 47 kb)

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Perroy, J., Pontier, S., Charest, P. et al. Real-time monitoring of ubiquitination in living cells by BRET. Nat Methods 1, 203–208 (2004). https://doi.org/10.1038/nmeth722

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