(A) Expression of FGFR1 and FGFR2 in wildtype RCS cells, RCS null for FGFR1-4, and RCS cells expressing only endogenous FGFR1 (RCSFgfr1). Actin serves as a loading control; n, number of independent experiments. (B) RCSFgfr1 cells were treated with FGF4, FGF8 and FGF9 for indicated times and ERK phosphorylation (pErk) was monitored by western blot. Vinculin serves as a loading control. pERK signal was quantified and graphed (right) as relative values compared to the 10’ FGF4 stimulation; data show average and SEM of six independent experiments. (C) RCSFgfr1 expressing the pKrox(MapERK)d1EGFP reporter were treated with FGF4, FGF8, and FGF9 and pKrox24 transactivation was monitored for 48 hours.

The oligomerization state of FGFR1, as measured by fluorescence intensity fluctuation (FIF) spectrometery. (A) Brightness distributions shown on the linear scale. Brightness scales with the oligomer size. LAT (gray) is a monomer control, TrkA+130 nM NGF (black) is a dimer control. EphA2 bound to ephrinA1-Fc (brown) is an oligomer control. All distributions are scaled to a maximum of 1. (B) Distributions of log(brightness). Points represent the experimental FIF data, and the solid lines are the best fit Gaussians. (C) Means of the best-fit Gaussians and the standard errors of the mean.

Phosphorylation of FGFR1 and downstream signaling substrates in HEK 293T cells. (A) Sample western blots for Y653/4 FGFR1 phosphorylation and FRS2 phosphorylation in response to FGF4, FGF8, and FGF9. (B) An example blot used for data scaling, where samples with maximum phosphorylation in response to FGF4, FGF8, and FGF9 are rerun on the same gel (C) Dose response curves from the Western blot experiments. The points represent the averaged data, while the solid lines are the best fit rectangular hyperbolic curves. Fit parameters are shown in Table 1.

Best fit parameters for dose response curves in Figures 3 and 5. EC50 is the potency of the ligand, and Etop is the efficacy (see equation 1)

FGFR1 phosphorylation as a function of time after ligand addition. (A) Phosphorylation time course of Y653/654 at high ligand concentration (130 nM) (B) Phosphorylation time course of Y653/654 at low ligand concentration (2.6 nM) (C) Phosphorylation time course of FRS2 at high ligand concentration (130 nM) (D) Phosphorylation time course of FRS2 at low ligand concentration (2.6 nM)

Calculated Bias Coefficients using equation 2. Gray shading indicates statistical significance between either FGF4 or FGF9 and the reference ligand FGF8 (see Supplemental Table S2 for p-values).

Functional FGFR1-mediated responses to different ligands. (A) FGFR1 concentration in the plasma membrane of HEK 293T cells at t=2 mins following ligand addition for FGF8, FGF9 and no ligand control. (B) HEK 293T cell viability after ligand exposure and six days of starvation for varying ligand concentrations. (C) Apoptosis of HEK 293T cells under starvation conditions, exposed to varying concentrations of FGF8 and FGF9. Results are summarized in Table S5. (D) RCSFgfr1 cells were treated with FGF4, FGF8 and FGF9 for 48 hours, and the levels of collagen type 2 were determined by western blot. Actin serves as a loading control. (E) Dose response curves describing collagen type 2 loss. (E) Dose response curves for growth arrest of RCSFgfr1 cells after 72 hours, in response to FGF4, FGF8 and FGF9.

Differences in FGFR1 dimer conformations in response to FGF ligands. (A) and (B). FRET data for ECTM-FGFR1-YFP and ECTM-FGFR1-mCherry in the presence of saturating FGF4 (orange), FGF8 (green) or FGF9 (blue) concentrations. (A) Measured FRET efficiencies versus total receptor (ECTM-FGFR1-YFP + ECTM-FGFR1-mCherry) concentrations and measured donor (ECTM-FGFR1-YFP) concentrations versus acceptor (ECTM-FGFR1-YFP) concentrations in single vesicles. (B) Histograms of single-vesicle intrinsic FRET values. Intrinsic FRET is a measure of the separation between the fluorescent proteins in the dimer. Different intrinsic FRET values were measured for FGF8 and FGF4/FGF9. (C) Graphical representation of experimental results showing that FGF8 induces a TM dimer conformation where the TM C-termini are positioned further apart from each other, as compared to the cases of FGF4 and FGF9.