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Detection of β-Arrestin-Mediated G Protein-Coupled Receptor Ubiquitination Using BRET

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1957)

Abstract

Ubiquitination of G protein-coupled receptors (GPCRs) is an important dynamic posttranslational modification that has been linked to the intracellular trafficking of internalized GPCRs to lysosomes. Ubiquitination of GPCRs is mediated by specific E3 ubiquitin ligases that are scaffolded by the adaptor proteins called β-arrestins. Traditionally, detection of GPCR ubiquitination is achieved by using ubiquitin antibodies to Western blot immunoprecipitates of detergent-solubilized GPCRs expressed in heterologous cells. However, studies have also shown that bioluminescence resonance energy transfer (BRET)-based techniques can reveal ubiquitination of GPCRs in intact cells and in real time. This chapter describes a step-by-step protocol to evaluate ubiquitination of GPCRs using the BRET methodology.

Key words

GPCR Arrestin Ubiquitination BRET Ubiquitin Titration Adrenergic receptor 
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Notes

Acknowledgments

We thank Drs. Michel Bouvier, Marc Caron, and Stéphane Laporte for providing plasmid constructs. We thank Dr. Asuka Inoue for providing β-arrestin1/β-arrestin2 double-knockout HEK-293T cells.

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Cell BiologyDuke University Medical CenterDurhamUSA
  2. 2.Department of Medicine (Cardiology)Duke University Medical CenterDurhamUSA
  3. 3.College of Medicine, Member of QU HealthQatar UniversityDohaQatar

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