EGF-induced activation of the EGF receptor does not trigger mobilization of caveolae.
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.
Kazazic
Maja
M
Institute of Pathology, Faculty of Medicine, University of Oslo, Rikshospitalet-Radiumhospitalet Medical Centre, N-0027 Oslo, Norway.
Roepstorff
Kirstine
K
Johannessen
Lene E
LE
Pedersen
Nina M
NM
van Deurs
Bo
B
Stang
Espen
E
Madshus
Inger H
IH
D021941:Caveolae / Q000187:drug effects / Q000502:physiology*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism; D033922:Clathrin Heavy Chains / Q000235:genetics / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000502:physiology; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D009761:Nystatin / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D014168:Transferrin / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology
C495540:CAV1 protein, human; D051242:Caveolin 1; D034741:RNA, Small Interfering; D014168:Transferrin; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D033922:Clathrin Heavy Chains; D009761:Nystatin; D004815:Epidermal Growth Factor; D002772:Cholera Toxin; D066246:ErbB Receptors