Methods to study endocytic trafficking of the EGF receptor.

Endocytosis and postendocytic sorting of epidermal growth factor (EGF) receptor (EGFR) are the major regulators of EGFR signaling. EGFR endocytosis and ubiquitin-dependent lysosomal targeting are also considered to be the prototypic experimental system for studying the molecular mechanisms of stimulus-induced and constitutive endocytic trafficking. Therefore, elucidation of the mechanisms of EGFR endocytosis and its regulation of the signaling network is essential not only for better understanding of the EGFR biology but also for defining general regulatory principles in the endocytosis system. Comprehensive analysis of these mechanisms requires quantitative and physiologically relevant methodological approaches for measuring the rates of EGFR internalization, degradation, and recycling. Basic experimental protocols described in this chapter cover a combination of single-cell microscopy and biochemical methods that are used to follow EGF-induced endocytosis of EGFR in real time, measure the kinetic rate parameters of EGFR internalization and recycling, and analyze EGF-dependent ubiquitination and degradation of EGFR.