1. Volume 51, Issue 6, Pages 707-722 (September 2013)
Functional Proteomics Defines the Molecular Switch Underlying FGF Receptor Trafficking and Cellular Outputs 
Chiara Francavilla, Kristoffer T.G. Rigbolt, Kristina B. Emdal, Gianni Carraro, Erik Vernet, Dorte B. Bekker-Jensen, Werner Streicher, Mats Wikström, Michael Sundström, Saverio Bellusci, Ugo Cavallaro, Blagoy Blagoev, Jesper V. Olsen 
Molecular Cell 
Volume 51, Issue 6, Pages (September 2013)
DOI: /j.molcel
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2. Figure 1
Analysis of FGFR2b Signaling Dynamics Reveals a Functional Dichotomy between FGF-7 and FGF-10
(A–E) The immunoblotting for FGFR2, signaling molecules, and vinculin as loading control (A) is quantified in (B). Cell proliferation (C), BrdU incorporation (D), and cell migration (E) assays of stimulated cells are also shown. Data in (B–E) represent the mean ± SD of three experiments. ∗p < 0.05 and ∗∗p < 0.01 in comparison to control cells, represented by a black line.
(F) Experimental design of MS-based quantitative phosphoproteomics analysis of FGF-7- and FGF-10-induced signaling dynamics.
(G) Number of identified phosphorylated sites and proteins for each time point.
(H) Principal component analysis (PCA) of tyrosine phosphorylated proteins identified for each time point.
(I) A Venn diagram showing the 323 phosphorylated sites quantified at all time points. ∗, sites quantified upon FGF-7 and FGF-10 stimulation, respectively.
(J) GO term enrichment analysis of clustering shown in Figure S1H. The terms discussed in the text are shown in red.
See also Figure S1 and Tables S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 FGF-10 Specifically Induces FGFR2b Y734 Phosphorylation
(A) Functional network of tyrosine-phosphorylated proteins ordered on the basis of their function (signaling, localization, and proliferation) and color-coded according to the maximum difference in regulation between the ligands across the time course. The red square on FGFR2b highlights the Y734 dynamic profile.
(B and C) Representation (B) and quantification (C) of FGFR2b phosphorylated tyrosines.
(D) Representative MS/MS of the phosphorylated Y734 FGFR2b peptide.
See also Figure S1 and Tables S1 and S2.
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4. Figure 3 Y734 Phosphorylation Is Required for FGFR2b Functions
(A) Lysates from stimulated cells transfected with HA-FGFR2b, or its mutants were immunoblotted with the indicated antibodies.
(B) Internalization (cytoplasm) and recycling (plasma membrane) of FGF-7- or FGF-10-stimulated HA-FGFR2b (green). Arrows indicate internalized receptor. Asterisks indicate cells with the receptor recycled to the cell surface. The scale bar represents 5 μm.
(C) Colocalization of HA-FGFR2b (red) with Rab proteins (green) in cells stimulated for 40 min. The scale bar represents 5 μm.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 p85 Is Recruited to Y734 Phosphorylated FGFR2b
(A) The experimental design of the SILAC-based phosphopeptide pull-down screen with the FGFR2b peptide NSTNELY734MMMRDS and its tyrosine phosphorylated version.
(B) Phosphopeptide interactors quantified by MS. The insert shows immunoblotting confirming that p85 and p110 only interact with the FGFR2b phosphopeptide.
(C) ITC-binding isotherms and thermodynamic parameters for p85-SH2 domains titrated with the FGFR2b peptide NSTNELY734MMMRD (red triangles) or its tyrosine phosphorylated version (black circles). Data were fitted to a model describing a single binding isotherm (solid black line).
(D–G) Stimulated lysates from HeLa cells transfected with FGFR2b and its mutants (D, F, and G) or breast cancer cell lines (E) were immunoprecipitated and immunoblotted in order to show p85-FGFR2 and PLCγ-FGFR2 (D–F) or Gab1-Grb2-p85 (G) binding.
See also Figures S3 and S4 and Table S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 PI3K Controls FGFR2b Sorting
(A and B) Lysates from cells transfected with either HA-FGFR2b or the Y734F mutant, treated with LY294002, and stimulated with FGF-7 or FGF-10 were immunoprecipitated (or not) and immunoblotted as indicated.
(C and D) Quantification of the presence (total), internalization (internalized), and recycling (cell surface) of FGFR2b and its mutant in cells treated with LY and stimulated with FGF-7 (green) or FGF-10 (blue) for different time lengths. Values represent the means ± SEM of three different experiments (pictures not shown). A.U., arbitrary unit. For comparison, DMSO-treated cells curves (solid lines) from Figure S2F are reported.
(E) Colocalization of HA-FGFR2b (red) with Rab proteins (green) in cells stimulated for 8 (Rab5-GFP) or 40 (Rab7- and Rab11-GFP) min in the presence of LY For a comparison with DMSO-treated cells, see Figures 3C and S2G. The scale bar represents 5 μm.
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7. Figure 6 SH3BP4 Regulates FGFR2b Functions
(A) The experimental design of the p85-SH2 domain pull-down screen.
(B) FGF-10 over control or FGF-10 over FGF-7-specific p85 interactors. The insert shows FGFR2b-p85 binding.
(C) SH3BP4 interaction with endogenous p85. FLAG-SH3BP4∗, siRNA-resistant SH3BP4 transfected in SH3BP4-depleted cells.
(D) Peptide pull-downs were immunoblotted for SH3BP4. Dynamin (DYNII) was used as positive control (Tosoni et al., 2005). The numbers correspond to the amino acid sequence. In this panel, (A) indicates that all proline residues were replaced by alanine.
(E) Lysates incubated with the purified SH3 domain of SH3BP4 or its W92A mutant (SH3BP4_SH3∗) were immunoblotted for p85 and dynamin as a positive control. The Coomassie staining shows an equal expression of the SH3 domains.
(F) Colocalization of HA-FGFR2b (red) with Rab proteins (green) in 40 min stimulated cells depleted of SH3BP4 and transfected (or not) with FLAG-SH3BP4∗. Cells were pretreated or not with LY The scale bar represents 5 μm.
(G) Cells cotransfected with HA-FGFR2b and either SH3BP4- or control-siRNA (top) or cells depleted for SH3BP4 and cotransfected with HA-FGFR2b and FLAG-SH3BP4∗ (bottom) were stimulated with FGF-7 or FGF-10. Lysates were immunoblotted as indicated.
See also Figure S5 and Table S4.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 The FGFR2b-p85-SH3BP4 Complex Modulates FGFR2b Responses
(A–E) Cell proliferation (A and D) and migration (B, C, and E) assays show that FGF-10-induced migration depends on FGFR2b-Y734 phosphorylation, PI3K activity, and SH3BP4. Data represent the mean ± SD of three experiments. ∗p < 0.05 and ∗∗p < 0.01 in comparison to control cells represented by a black line. See also Figures S6A–S6D.
(F–M) Lung explants electroporated with pGFP (F–I) or FGFR2b_Y734F (J–M) were treated for 72 hr with FGF-7 or FGF-10. (G, I, K, and M) are magnifications of the areas surrounded by the black square in (F, H, J, and L), respectively. Black arrows indicate multiple buds. The scale bar represents 500 μm (F, H, J, and L) or 100 μm (G, I, K, and M). See also Figures S6E–S6O.
(N) FGF-10-induced receptor recycling, sustained signaling activation, and cell migration depend on p85 interaction with phosphorylated Y734 on FGFR2b, the adaptor protein SH3BP4 recruiting TfR and PI3K activity. The pink dotted arrow indicates the role of PI3K in receptor sorting from early to recycling endosomes. TfR, transferrin receptor. P, phosphorylation. See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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