Activation of the epidermal growth factor (EGF) receptor induces formation of EGF receptor- and Grb2-containing clathrin-coated pits.
In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the mu2 or alpha 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 alpha 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 alpha 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.
Johannessen
Lene E
LE
Institute of Pathology, The University of Oslo, Rikshospitalet, N-0027 Oslo, Norway.
Pedersen
Nina Marie
NM
Pedersen
Ketil Winther
KW
Madshus
Inger Helene
IH
Stang
Espen
E
D002966:Clathrin / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000819:agonists / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D016253:Microscopy, Immunoelectron; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017354:Point Mutation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011990:Receptors, Transferrin / Q000378:metabolism; D015398:Signal Transduction; D050656:Transcription Factor AP-2 / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains
D002966:Clathrin; D051380:GRB2 Adaptor Protein; D021122:Protein Subunits; D034741:RNA, Small Interfering; D011990:Receptors, Transferrin; D050656:Transcription Factor AP-2; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors