Caveolae-dependent endocytosis has recently been proposed in the uptake of EGF receptor (EGFR) at high concentrations of ligand. Consistently, upon incubation of HEp2 and HeLa cells with methyl-beta-cyclodextrin, we observed a small inhibitory effect on endocytosis of ligated EGFR in HEp2 cells. However, immunoelectron microscopy showed the same relative amount of bound EGF localizing to caveolae on incubation with high and low concentrations of EGF, not supporting rapid recruitment of EGFR to caveolae. Live-cell microscopy furthermore demonstrated that incubating HEp2 cells with high concentrations of EGF did not increase the mobility of caveolae. By RNA-interference-mediated knockdown of clathrin heavy chain in HEp2 and HeLa cells, we found that endocytosis of EGFR was efficiently inhibited both at high and low concentrations of EGF. Our results show that caveolae are not involved in endocytosis of EGF-bound EGFR to any significant degree and that high concentrations of EGF do not further mobilize caveolae.
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