Ligand-induced clathrin-mediated endocytosis of the keratinocyte growth factor receptor occurs independently of either phosphorylation or recruitment of eps15.
Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells. Following ligand binding, KGFR is rapidly activated and internalized by clathrin-mediated endocytosis. Among the possible receptor substrates which could be involved in the regulation of KGFR endocytosis and down-modulation, we analyzed here the eps15 protein in view of the proposed general role of eps15 in regulating clathrin-mediated endocytosis as well as that of eps15 tyrosine phosphorylation in the control of regulated endocytosis. Immunoprecipitation and Western blot analysis showed that activated KGFR was not able to phosphorylate eps15, suggesting that eps15 is not a receptor substrate. Double immunofluorescence and confocal microscopy revealed that activated KGFR, differently from epidermal growth factor receptor (EGFR), did not induce recruitment of eps15 to the cell plasma membrane. Microinjection of a monoclonal antibody directed against the C-terminal DPF domain which contains the AP2 binding region of eps15 led to inhibition of both pathways of receptor-mediated endocytosis, the EGFR ligand-induced endocytosis and the transferrin constitutive endocytosis, but did not appear to block the KGFR ligand-induced internalization. Taken together our results indicate that the clathrin-mediated uptake of KGFR is not mediated by eps15.
Dipartimento di Medicina Sperimentale e Patologia, Università di Roma 'La Sapienza', Viale Regina Elena 324, 00161 Rome, Italy. francesca.belleudi@uniroma1.it
Francesca
F
Belleudi
Vincenzo
V
Visco
Mara
M
Ceridono
Laura
L
Leone
Raffaella
R
Muraro
Luigi
L
Frati
Maria Rosaria
MR
Torrisi
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D051379:Mice; D008845:Microinjections; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D002966:Clathrin; C486206:EPS15 protein, human; C082729:Eps15 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D010750:Phosphoproteins; D017468:Receptors, Fibroblast Growth Factor; D014443:Tyrosine; D066246:ErbB Receptors; D051497:Receptor, Fibroblast Growth Factor, Type 2; C065104:keratinocyte growth factor receptor