Receptor-mediated endocytosis of keratinocyte growth factor

Keratinocyte growth factor (KGF) is a fibroblast growth factor which acts s pecifically on epithelial cells, regulating their proliferation and differe ntiation. KGF elicits its activity through binding to and activation of KGF receptor, a splicing transcript variant of fibroblast growth factor recept or 2 (FGFR2), Here we analyzed the pathway of internalization of KGF and it s receptor using several approaches, including the utilization in immunoflu orescence 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 antiphosphot yrosine antibodies, as well as parallel double immunofluorescence and confo cal analysis of NIH3T3 KGFR transfectants treated with KGF at 4 degrees C, followed by incubations at 37 degrees C for different time points, showed t hat KGF induced endocytosis of tyrosine activated KGFRs, The use of KGF-HFc in immunofluorescence and in immunogold electron microscopy on KGFR transf ectants, A253 epithelial tumor cells and human cultured keratinocytes allow ed us to follow the early steps of KGF internalization and revealed that th is process occurred through clathrin-coated pits. A quantitative ELISA assa y confirmed that KGF-HFc binding on the cell surface rapidly decreased beca use of internalization. Our results demonstrate that KGF is internalized by receptor-mediated endocytosis and illustrate the involvement of clathrin-c oated pits in this process.