Detection of β-Arrestin-Mediated G Protein-Coupled Receptor Ubiquitination Using BRET.
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.
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Related Subject Headings
- beta-Arrestins
- Ubiquitination
- Ubiquitin
- Receptors, G-Protein-Coupled
- Protein Binding
- Humans
- HEK293 Cells
- Developmental Biology
- Data Analysis
- Bioluminescence Resonance Energy Transfer Techniques
Citation
Published In
DOI
EISSN
Publication Date
Volume
Start / End Page
Location
Related Subject Headings
- beta-Arrestins
- Ubiquitination
- Ubiquitin
- Receptors, G-Protein-Coupled
- Protein Binding
- Humans
- HEK293 Cells
- Developmental Biology
- Data Analysis
- Bioluminescence Resonance Energy Transfer Techniques