Assays to Monitor Degradation of the EGF Receptor

Recently the life cycles of receptor tyrosine kinases (RTKs) have become a focus of signal transduction research. Ligand-induced ubiquitination of RTKs followed by their internalization and degradation has, in particular, been extensively studied. This chapter describes the basic methods used to measure ubiquitination and degradation of RTKs using the example of the epidermal growth factor receptor (EGFR). Common sources for endogenous and recombinant EGFR as well as cell lines used to conduct receptor downregulation assays are described. Monitoring of ubiquitination and degradation of the EGFR subsequent to stimulation with the receptor ligand EGF is described. Finally, protocols to quantitatively measure degradation of the EGFR by pulse chase experiments or using radiolabeled ligands such as ¹²⁵ I-EGF are presented.