The V429E substitution in FGFR1 impedes recruitment and phosphorylation of FRS2α and FGF2-induced MAPK signaling(A) Analysis of the impact of the V429E mutation based on the NMR structure of the FRS2 phosphotyrosine binding (PTB) domain in complex with the juxtamembrane (JM) region peptide of FGFR1. The FRS2 PTB domain and FGFR1 JM peptide are shown as purple and green ribbons respectively, and side chains of the V429 of FGFR1 and L62, M105, V112 of FRS2 are rendered in sticks. The molecular surfaces of L62, M105, and V112 of FRS2 PTB are also shown, to highlight their hydrophobic contacts with V429 of FGFR1.(B) The V429E FGFR1c mutant fails to phosphorylate FRS2 in cell based assay. BaF3 were transfected with lentiviral vectors expressing WT or V429E FGFR1c and FRS2 phosphorylation was assessed upon FGF1 treatment by western blotting using anti phospho-FRS2-α specific antibodies . WT: wild type; EV: empty vector.(C) Analysis of the impact of the V429E mutation on the ability of FGFR1 to recruit FRS2α . The assay was based on FGFR2 V430E, which is equivalent to FGFR1 V429E. Increasing concentrations of FGFR2CDWT and FGFR2CDV430E (carrying the equivalent mutation to FGFR1-V429E) ranging from 12.5 to 400 nM were passed over a CM5 chip onto which FRS2α had been immobilized. As representative of the full dataset, binding responses obtained for injections of 200 nM of FGFR2CDWT or FGFR2CDV430E are shown. The rising and falling parts of the wild type curve (blue) represent the association and dissociation phases, respectively, of FGFR2CDWT-FRS2α binding over time. At 200 nM, FGFR2CDWT exhibits maximal binding of 55 response units (blue), whereas FGFR2CDV430E shows negligible binding (red). According to a steady-state equilibrium analysis of the full data sets (not shown), FGFR2CDWT binds FRS2α with a KD of 320 nM, whereas the FGFR2CDV430E negligible binding to FRS2α.(D) The V429E mutation reduces the ability of FGFR1 to phosphorylate FRS2 on Y196 in vitro. FGFR2WT and FGFR2V430E mutant kinases were allowed to phosphorylate FRS2 fragment PTB9-200 on Y196, a tyrosine phosphorylation site known to be required for Grb2 recruitment, at room temperature for 0, 1, 3, 5 10, 15, 20, 25, 30 minutes. Following tryptic digestion, samples were analyzed by Orbitrap mass spectrometry to quantify the phospho-Y196-containing tryptic peptide. FGFR2WT is shown in blue and FGFR2V430E is shown in red.(E-F) The V429E mutation is loss-of-function in the OCFRE reporter assay (FRS2α dependent MAPK signaling) and not different from WT in the NFAT reporter assay (FRS2α independent PLCγ /IP3/Ca2+ signaling). Data shown represent the means ± S.E.M. of 3 experiments. FGFR1 WT is shown in blue, FGFR1V429E in red, empty vector in black. Relative to the maximal stimulation of WT (%), ** p<0.001, NS: not significant.(G) Total abundance and maturation of recombinant FGFR1 proteins. COS-7 cells were transfected with FGFR1 constructs and the cell lysates were subjected to deglycosylation treatment followed by western blot analysis. PNGase-treated bands represent total protein abundance levels; 140kDa Endo H-treated bands represent the mature form while 100kDa Endo H-treated bands represent immature form of FGFR1. The experiment was performed three times, tested by Mann-Whitney test for statistic significance, no significant difference in overall expression and maturation index between WT and V429E. PNGase: Peptide N-Glycosidase F-treated; Endo H: endoglycosidase H-treated; WT: wild type; EV: empty vector.(H) Cell surface abundance of the transiently transfected FGFR1 mutant in COS-7 cells. Cell surface abundance levels were measured by a radiolabeled antibody binding assay and plotted as a percentage of wild type levels. The abundance level of cell surface expressed FGFR1V429E was significantly higher than FGFR1wt. Values shown are the means ± SEM of 3 experiments each performed in quadruplicate. The difference between the mutant and the wild type receptor expression was compared by Mann-Whitney test, * p<0.05.
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