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 a nalyzed in cells conditionally defective for clathrin-dependent endocy tosis by overexpression of mutant dynamin (K44A). EGF-induced autophos phorylation of the EGFR on ice was strongly reduced in cells overexpre ssing mutant dynamin, and consistently, binding analyses showed that h igh-affinity EGFRs were lost. In the absence of mutant dynamin the cel ls displayed both high-and low-affinity EGFR. At 4 degrees C EGF-EGFR localized mainly outside coated pits regardless of expression of mutan t dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37 degrees C, Thus, expression of mutan t dynamin disrupts high-affinity binding of EGF, but not ligand-induce d recruitment of EGFR to clathrin-coated pits.