Editors' ChoiceCell Biology

Dictating the Route

Science Signaling  01 Oct 2013:
Vol. 6, Issue 295, pp. ec236
DOI: 10.1126/scisignal.2004768

Fibroblast growth factors (FGFs) control various processes during development. Although combinatorial complexity among the many different ligands and receptors produces some of the specificity in the response, some FGFs bind the same receptor yet produce distinct cellular outcomes. Francavilla et al. used a mass spectrometry–based proteomics approach to identify how FGF7 and FGF10 could produce proliferation and cell migration, respectively, when signaling through the same receptor, FGFR2b. Stimulation of HeLa cells transfected with FGFR2b showed that FGF7 produced transient activation of several downstream signals, including phosphorylation of the kinase Akt and the adaptor Shc, but sustained phosphorylation of extracellular signal–regulated kinase (ERK). In contrast, FGF10 produced sustained Akt and Shc phosphorylation and transient ERK phosphorylation. Furthermore, FGF7 stimulated receptor internalization followed by degradation, whereas FGF10 stimulated receptor internalization followed by receptor recycling to the cell surface. Proteomic analysis of the cells at three time points after stimulation with either ligand showed that although both ligands stimulated the phosphorylation of the receptor autocatalytic tyrosines and the site where phospholipase C binds the receptor, only FGF10 stimulated the phosphorylation of Tyr734. Immunofluorescence microscopy showed that, similar to the receptor in FGF7-stimulated cells, cells expressing a Y734F mutant receptor and stimulated with either ligand showed rapid internalization, transient colocalization with degradative compartment markers, and ultimately loss of the receptor from the cell, suggesting that this residue dictates the trafficking route after internalization. The catalytic (p110) and regulatory (p85) subunits of phosphoinositide 3-kinase (PI3K) specifically interacted with a peptide containing phosphorylated Tyr734, as indicated by mass spectrometry analysis of proteins that affinity purified with either the phosphorylated or nonphosphorylated peptide. FGF10, but not FGF7, stimulated an interaction between FGFR2b and p85, and this interaction was detected not only in HeLa cells but also between endogenous proteins in several breast cancer cell lines. p85 has a SH3 domain, and mass spectrometry identified SH3-domain–binding protein 4 (SH3BP4) as associated with p85 only in cells stimulated with FGF10 and not those stimulated with FGF7. This interaction depended on Tyr734, and depletion of SH3BP4 disrupted the trafficking of the receptor to the recycling endosome in response to FGF10 stimulation. The importance of this trafficking for cellular response was verified by showing that knockdown of SH3BP4 or expression of the Y734F mutant resulted in FGF10 producing a proliferative instead of migratory response. In addition, introduction of the Y734F mutant into or knockdown of SH3BP4 in mouse embryonic lung explants also compromised the response to FGF10, making the cells respond like they did to FGF7. Thus, phosphorylation of a single tyrosine residue on the receptor governs the cellular trafficking route and response to distinct ligands.

C. Francavilla, K. T. G. Rigbolt, K. B. Emdal, G. Carraro, E. Vernet, D. B. Bekker-Jensen, W. Streicher, M. Wikström, M. Sundström, S. Bellusci, U. Cavallaro, B. Blagoev, J. V. Olsen, Functional proteomics defines the molecular switch underlying FGF receptor trafficking and cellular outputs. Mol. Cell 51, 707–722 (2013). [PubMed]