High-affinity binding of epidermal growth factor (EGF) to EGF receptor is disrupted by overexpression of mutant dynamin (K44A)

Activation of the epidermal growth factor receptor (EGFR) kinase was analyzed in cells conditionally defective for clathrin-dependent endocytosis by overexpression of mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on ice was strongly reduced in cells overexpressing mutant dynamin, and consistently, binding analyses showed that high-affinity EGFRs were lost. In the absence of mutant dynamin the cells displayed both high-and low-affinity EGFR. At 4 degrees C EGF-EGFR localized mainly outside coated pits regardless of expression of mutant dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37 degrees C, Thus, expression of mutant dynamin disrupts high-affinity binding of EGF, but not ligand-induced recruitment of EGFR to clathrin-coated pits.