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Ubiquitination of GPCRs

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 746)

Abstract

In this chapter, we describe a method for detecting the ubiquitination status of G protein-coupled receptors (GPCRs). This involves co-expression of a GPCR with an epitope-tagged ubiquitin construct in a ­heterologous mammalian expression system. Stimulus-dependent modification of the GPCR by ­ubiquitin is detected by immunoprecipitation and subsequent immunoblotting to detect incorporation of the epitope-tagged ubiquitin. We describe here a well-established protocol to detect ubiquitination of the chemokine receptor CXCR4, which can be easily applied to detect the ubiquitination status of other GPCRs.

Key words

Ubiquitin G protein-coupled receptor Agonist Immunoblot Lysosome Sorting Degradation CXCR4 Immunoprecipitation De-ubiquitination 
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Notes

Acknowledgments

Work was supported by NIH grants GM075159 and DA026040 to A.M.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Molecular Pharmacology and Therapeutics, Stritch School of MedicineLoyola University ChicagoMaywoodUSA

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