Activation of the Epidermal Growth Factor (EGF) Receptor Induces Formation of EGF Receptor- and Grb2-Containing Clathrin-Coated Pits

Abstract

In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the μ2 or α subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the α subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the α subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required.

Alexandre Benmerah, Andrew Chantry, Robin Scaife, Alan Hall, and Frances M. Brodsky are acknowledged for gifts of valuable reagents. We are grateful to Harald Stenmark for critical reading of the manuscript and to Marianne Skeie Rødland for excellent technical assistance.

This work was supported by The Research Council of Norway (including the functional genomics program [FUGE]), The Norwegian Cancer Society, Medinnova, NOVO Nordic Foundation, Anders Jahre's Foundation for the Promotion of Science, Torsted's Legacy, Blix Legacy, and Bruun's Legacy.