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Activation of the Ras/MAPK signaling cascade is essential for growth factor-induced cell proliferation and differentiation. In this report, we describe the purification, cloning, and characterization of a novel protein, designated FRS2, that is tyrosine phosphorylated and binds to Grb2/Sos in response to FGF or NGF stimulation. We find that FRS2 is myristylated and that this modification is essential for membrane localization, tyrosine phosphorylation, Grb2/Sos recruitment, and MAPK activation. FRS2 functions as a lipid-anchored docking protein that targets signaling molecules to the plasma membrane in response to FGF stimulation to link receptor activation with the MAPK and other signaling pathways essential for cell growth and differentiation. Finally, we demonstrate that FRS2 is closely related and probably indentical to SNT, the long-sought target of FGF and NGF receptors.

Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.

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The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4 interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.

The family of fibroblast growth factors (FGFs) consists of at least 10 different growth factors that control cellular processes such as growth, differentiation, and cell migration (reviewed in reference 2). FGFs induce their biological responses by binding to and activating a family of cell surface receptors with intrinsic protein tyrosine kinase activity (reviewed in reference 12). By contrast to other growth factors such as platelet-derived growth factor (PDGF) or epidermal growth factor, acidic FGF (aFGF; also called FGF1) binds to the FGF receptor (FGFR1) monovalently, and FGFR dimerization and activation are mediated by multivalent interactions between heparin sulfate proteoglycans and FGF (reviewed in reference 26).Upon activation, receptor tyrosine kinases undergo rapid autophosphorylation on numerous tyrosine residues. Autophosphorylation sites located within the catalytic domain are crucial for stimulation of kinase activity, while autophosphorylation sites located in other regions are usually involved in the recruitment of cellular target proteins (21). FGFR1 (encoded by flg) contains at least seven autophosphorylation sites. Two are located in the catalytic domain (Y653 and Y654) and are essential for kinase activation (17). One phosphorylation site in the C-terminal tail (Y766) functions as a high-affinity binding site for the SH2 domain of phospholipase C-␥ (19). Phosphorylation of Y766 is essential for phosphatidylinositol hydrolysis but not for FGF-induced DNA synthesis in myoblasts or differentiation of PC12 cells, indicating that these biological responses are mediated by different FGF-dependent signaling pathways (18,22). Interestingly, elimination of all known tyrosine autophosphorylation sites on FGFR1 by site-directed mutagenesis (except the two sites in the catalytic domain) does not impair FGF-induced mitogen-activated protein (MAP) kinase activation, mitogenesis, or PC12 cell differentiation (17).The Ras/MAP kinase signaling pathway plays an important role in signaling via FGF receptors (1,20). It is well established that the adapter protein Grb2 (6, 16) links receptor tyrosine kinases with the Ras signaling pathway by binding to the guanine nucleotide-releasing factor Sos through its SH3 domains and to tyrosine-phosphorylated receptors or docking molecules via its SH2 domain (25). We have recently identified a lipid-anchored docking protein, termed FRS2, that links FGFR molecules with the Ras/MAP kinase signaling pathway (14). We demonstrated that FRS2 is tyrosine phosphorylated and forms a complex with Grb2 and Sos in response to FGF stimulation (14). In this report, we demonstrate that in addition to the direct interactions with Grb2, tyrosine-phosphorylated FRS2 forms a complex with the SH2 domain-containing protein tyrosine phosphatase Shp2. This interaction results in tyrosine phosphorylation of Shp2 and complex formation between Shp2 and Grb2. Moreover, an FRS2 mutant impaired in Grb2 and Shp2 binding induces weak and transient MAP kinase response and fails to induce neuronal diffe...

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