Chemical Induce And Regulate Function Of FGFR

Fibroblast growth factor receptors(FGFRs)belong to a large super family of receptor tyrosine kinases.Activated FGFRs that interacted with their FGF ligands participate in regulating cell proliferation,differentiation and survival and plays crucial roles in multiple physiological processes.Dysregulation of FGFR has been implicated in many types of human cancers.To better understand the mode of action and function of FGFR,this study applied the FKBP-RAPA-FRB system to the regulation of FGFR for the first time to construct a chemical-controlled FGFR,which is not interfered by endogenous ligands but can be activated by rapamycin(RAPA).First,HEK293 cells were transfected with plasmids expressing the chemical-controlled m FGFR1(PCDNA3.1-MYR-m FGFR1(ICD)-FKBP-FLAG and PCDNA3.1-MYR-m FGFR1(ICD)-FRB-HA).Western blot detected both the FKBP-tagged and the FRB-tagged m FGFR1 proteins in the transfected cells,which constitute an inactivated chemical-controlled FGFR1 system.To avoid auto-activation due to over-expression of the m FGFR1 protein,its CMV promoter was spliced in various ways to reduce its efficiency.This also represents another option to reduce background activity other than lowering transfection amount.Drug stimulation experiment demonstrated that 100 n M RAPA does not affect MAPK/ERK signaling pathway,but activates this system in cells transfected with the FGFR1 system and the downstream ERK signaling.The effects of different concentrations of RAPA for different durations on activating the system were also tested.It was found that treatment with 50 n M RAPA for 15 min was sufficient to activate the FGFR1 system and downstream ERK signaling.Co-immunoprecipitation further confirmed that RAPA mediated the dimerization and activation of the m FGFR1 protein,while FGFR inhibitor AZD4547 suppressed RAPA-mediated activation.To verify the possibility of activating FGFR1/2 heterodimer,we constructed plasmids for chemical-controlled h FGFR1 and h FGFR2(PCDNA3.1-MYR-h FGFR1(ICD)-FKBP-FLAG,PCDNA3.1-MYRh FGFR2(ICD)-FKBP-FLAG and PCDNA3.1-MYR-h FGFR1(ICD)-FRB-HA),and co-transfected them into HEK293 cells.Both FGFR1 homodimers and FGFR1/2 heterodimers could be detected in the transfected cells,and FGFR1/2 heterodimer can also be induced by RAPA and the downstream ERK signaling can be activated.Like the homodimers,activated heterodimers can also recruit Grb2,and be suppressed by AZD4547.To investigate the effects of the activated FGFR homodimers and heterodimers on tumor cells,pancreatic cancer Panc1 cells were stably transfected with chemical-controlled h FGFR1(Panc1-homo)or both h FGFR1 and h FGFR2(Panc1-hetero).Scratch and Transwell assays demonstrated that both homo-and hetero-dimerization of FGFR can enhance migration and invasion of the pancreatic cancer cells.