Protocol

Receptor Signal Transduction Protocols

Volume 259 of the series Methods in Molecular Biology pp 299-305

Ubiquitination of G-Protein-Coupled Receptors

  • Adriano MarcheseAffiliated withDepartment of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University
  • , Jeffrey L. BenovicAffiliated withDepartment of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University

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Abstract

In this chapter we describe methods for detecting the ubiquitination state of G-protein-coupled receptors (GPCRs). This involves coexpression of a GPCR with an epitope-tagged ubiquitin construct in a heterologous expression system. Modification by ubiquitin of the GPCR resulting from agonist activation is detected by immunoprecipation and subsequent immunoblotting for the epitope-tagged ubiquitin. We use here the chemokine receptor CXCR4 as the model receptor; however, this could be easily modified to detect the ubiquitination state of any GPCR.

Key Words

Agonist CXCR4 degradation G-protein-coupled receptor immunoblot immunoprecipitation lysosome sorting ubiquitin