Fibroblast growth factor receptors (FGFRs) regulate fundamental cellular processes, including proliferation, differentiation and angiogenesis and have emerged as growth factor receptors central to oncogenesis. This study developed a live-cell assay system for studying FGFR endocytosis and trafficking by employing both confocal and total internal reflection fluorescence (TIRF) microscopy in cells expressing a previously characterised GFP-tagged FGFR2 construct. Data from this work have demonstrated that endocytosis of activated FGFR occurs through clathrin-mediated endocytosis.
Interestingly, FGF treatment also significantly increased the number of CCPs as well as the number of clathrin-mediated endocytic events. However, treatment of cells with the Src family inhibitor Dasatinib or depletion of Src kinase target Eps8, prevents the FGF induced increase in plasma membrane clathrin and reduces the internalization of FGFR. This study also shows that both Src and Eps8 are required for receptor to exit from EEA I positive peripheral compartment into the Rab 1 1 positive PNRC. Eps8 depletion also inhibits the early phases of ERK activation in response to FGFR activation, placing this signalling event early in the trafficking pathway of the receptor. Thus, these results have identified the endocytic pathway for endocytosis of FGFR2 and described Eps8 and Src as key mediators of the early phases of activated FGFR trafficking and signalling
