Quantitative analysis of endocytosis and turnover of epidermal growth factor (EGF) and EGF receptor.
Binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) initiates signal transduction, ultimately leading to altered gene expression. Ligand-activated EGFR is also rapidly internalized and then targeted to lysosomes for degradation or recycled back to the plasma membrane. Endocytosis is a major regulator of EGFR signaling. Therefore, elucidation of the mechanisms of EGFR endocytosis is essential for a better understanding of EGFR biology. In order to achieve a comprehensive analysis of these mechanisms, reliable methods for measuring the rates of EGFR protein turnover and the rate parameters for individual steps of EGFR endocytic trafficking must be employed. The protocols in this unit describe methodologies to measure the rates of EGFR synthesis and degradation, to monitor EGF-induced down-regulation of surface EGFR, to measure the kinetic rate parameters of internalization, recycling, and degradation of radiolabeled EGF, and to perform radioiodination of EGF by the chloramine T method.
Sorkin
Alexander
A
Department of Pharmacology, University of Colorado Denver Medical School, Aurora, Colorado, USA.
Duex
Jason E
JE
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D015536:Down-Regulation; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000378:metabolism*; D007457:Iodine Radioisotopes; D051379:Mice; D041681:NIH 3T3 Cells; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D013194:Staining and Labeling; D034421:Sus scrofa
D007457:Iodine Radioisotopes; D004815:Epidermal Growth Factor; D066246:ErbB Receptors