Ubiquitination of G-Protein-Coupled Receptors
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 WordsAgonist CXCR4 degradation G-protein-coupled receptor immunoblot immunoprecipitation lysosome sorting ubiquitin