Receptor-mediated endocytosis of keratinocyte growth factor.
Keratinocyte growth factor (KGF) is a fibroblast growth factor which acts specifically on epithelial cells, regulating their proliferation and differentiation. KGF elicits its activity through binding to and activation of KGF receptor, a splicing transcript variant of fibroblast growth factor receptor 2 (FGFR2). Here we analyzed the pathway of internalization of KGF and its receptor using several approaches, including the utilization in immunofluorescence and in immunoelectron microscopy of a functional KGF-HFc chimeric protein as a specific tool to follow the endocytosis of the growth factor and of its receptor. Western blot analysis with anti-FGFR2 and anti-phosphotyrosine antibodies, as well as parallel double immunofluorescence and confocal analysis of NIH3T3 KGFR transfectants treated with KGF at 4 degrees C, followed by incubations at 37 degrees C for different time points, showed that KGF induced endocytosis of tyrosine activated KGFRs. The use of KGF-HFc in immunofluorescence and in immunogold electron microscopy on KGFR transfectants, A253 epithelial tumor cells and human cultured keratinocytes allowed us to follow the early steps of KGF internalization and revealed that this process occurred through clathrin-coated pits. A quantitative ELISA assay confirmed that KGF-HFc binding on the cell surface rapidly decreased because of internalization. Our results demonstrate that KGF is internalized by receptor-mediated endocytosis and illustrate the involvement of clathrin-coated pits in this process.
Marchese
C
C
Istituto Nazionale Ricerca sul Cancro di Genova, Sezione di Biotecnologie, Roma, Italy.
Mancini
P
P
Belleudi
F
F
Felici
A
A
Gradini
R
R
Sansolini
T
T
Frati
L
L
Torrisi
M R
MR
D016475:3T3 Cells; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D051526:Fibroblast Growth Factor 10; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D016253:Microscopy, Immunoelectron; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002966:Clathrin; C496453:FGF7 protein, human; C496454:Fgf7 protein, mouse; D051526:Fibroblast Growth Factor 10; D006133:Growth Substances; D017468:Receptors, Fibroblast Growth Factor; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; C065104:keratinocyte growth factor receptor