Irit Lax

A Lipid-Anchored Grb2-Binding Protein That Links FGF-Receptor Activation to the Ras/MAPK Signaling Pathway

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.

Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation

Heparin is required for fibroblast growth factor (FGF) stimulation of biological responses. Using isothermal titration calorimetry, we show that acidic FGF (aFGF) forms a 1:1 complex with the soluble extracellular domain of FGF receptor (FGFR). Heparin exerts its effect by binding to many molecules of aFGF. The resulting aFGF-heparin complex can bind to several receptor molecules, leading to FGFR dimerization. In two cell lines lacking endogenous heparan sulfate, exogenous heparin is required for FGFR dimerization, tyrosine kinase activation, c-fos mRNA transcription, and cell proliferation. Moreover, a synthetic heparin analog that binds monovalently to aFGF blocks FGFR dimerization, activation, and signaling via FGFR. We propose that heparin causes oligomerization of aFGF such that its binding to FGFR results in dimerization and activation. This represents a novel mechanism for transmembrane signaling and may account for the action of many heparin-bound growth factors.

Regulation of growth factor activation by proteoglycans: What is the role of the low affinity receptors?

Department of Pharmacology New York University Medical Center New York, New York 10016 Many cell membrane receptors, such as the nicotinic ace- tylcholine receptor and the T cell antigen receptor, are composed of several different subunits, and their correct assembly is necessary for the generation of functional re- ceptors. Most lymphokine receptors are composed of at least two components, and ligand-induced oligomeriza- tion is essential for receptor activation and signal transmis- sion. In recent years, it has become clear that various growth factors and lymphokines can bind to two different classes of cell surface receptors. For example, fibroblast growth factor (FGF) and transforming growth factor p (TGFB) both bind with high affinity to signaling receptors endowed with tyrosine or serinelthreonine kinase activi- ties. However, the same growth factors also bind with lower affinity to cell surface proteoglycans that cannot transmit signals alone, but somehow modulate the ability of the growth factor or the signaling receptor to generate a biological response (Klagsbrun and Baird, 1991; L6pez- Casillas et al., 1993; Yayon et al., 1991; Roghani et al., 1994). Proteoglycans are proteins that are found predomi- nantly on the cell surface and in the extracellular matrix and that contain carbohydrates called glycosaminogly- cans. Glycosaminoglycans are polymers of disaccharide repeats, which are mostly highly sulfated and negatively charged. The main glycosaminoglycans in proteoglycans are chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate (Ruoslahti, 1989). Binding of growth factors to proteoglycans is thought to have an important regulatory role (Ruoslahti and Yamaguchi, 1991). This has been particularly well explored for FGF, in which it has been shown that heparins (or heparan sulfate proteoglycans) are necessary for FGF-induced biologicl neurotrophin binding to ~75 (low affinity) and to the various nerve growth factor (NGF) receptor tyrosine kinases (trkA, trkB, and trkC, which are high affinity), as well as the binding of the insulin- like growth factor IGF2 to the IGF2 receptor (nonsignaling receptor) and to the IGFl receptor (a tyrosine kinase related to the insulin receptor). The existence of nonsignaling as well as signaling receptors for the same ligand is a feature common for many growth factors. How- ever, the physiological role of the nonsignaling receptors is poorly understood. The most discussed model for the role of the low affinity nonsignaling receptors is that they present ligand to high affinity signaling receptors. Binding of the ligand to the high affinity signaling receptors will then activate this receptor and trigger biological responses. However, when a given ligand at low concentration is allowed to bind to cells that display on their surface both low and high affinity receptors, the law of mass action dictates that at equilib- rium the ligand will bind, predominantly, to high affinity receptors rather than to the low affinity receptors. This fact makes it difficult to accept the so-called presentation model. At higher ligand concentration, both the high and low affinity receptors will be occupied. The binding con- stants of the high affinity receptors are usually lo- to 1 OO- fold higher than those of the low affinity receptors. There- fore, even when the density of the low affinity receptors on the cell surface is higher by an order of magnitude, a similar number of high and low affinity receptors will be occupied upon saturation of the high affinity receptors. These arguments demonstrate the need for a different model to understand the role of the low affinity receptors. An alternative and more plausible model is that the primary function of these receptors is to reduce the dimensionality of ligand diffusion from three to two dimensions (Adam and Delbruck, 1968; Richter and Eigen, 1974). When ligands such as lymphokines or growth factors are bound to cell surface receptors, their diffusion is restricted to two dimen- sions ratherthan diffusing in thethree-dimensionalvolume of the extracellular space. Once restricted to just two di- mensions, the ligand molecules are more likely to encoun- ter and bind to the less abundant high affinity signaling receptors. In otherwords, binding of ligands, such as FGF, to abundant low affinity receptors that are restricted two dimensions will increase the local concentration of the bound ligands at the plasma membrane, and the probabil- ity of their interaction with a high affinity receptor will be greatly enhanced. For example, 20,000 receptors per cell

Two EGF molecules contribute additively to stabilization of the EGFR dimer

Receptor dimerization is generally considered to be the primary signaling event upon binding of a growth factor to its receptor at the cell surface. Little, however, is known about the precise molecular details of ligand‐induced receptor dimerization, except for studies of the human growth hormone (hGH) receptor. We have analyzed the binding of epidermal growth factor (EGF) to the extracellular domain of its receptor (sEGFR) using titration calorimetry, and the resulting dimerization of sEGFR using small‐angle X‐ray scattering. EGF induces the quantitative formation of sEGFR dimers that contain two EGF molecules. The data obtained from the two approaches suggest a model in which one EGF monomer binds to one sEGFR monomer, and that receptor dimerization involves subsequent association of two monomeric (1:1) EGF‐sEGFR complexes. Dimerization may result from bivalent binding of both EGF molecules in the dimer and/or receptor‐receptor interactions. The requirement for two (possibly bivalent) EGF monomers distinguishes EGF‐induced sEGFR dimerization from the hGH and interferon‐γ receptors, where multivalent binding of a single ligand species (either monomeric or dimeric) drives receptor oligomerization. The proposed model of EGF‐induced sEGFR dimerization suggests possible mechanisms for both ligand‐induced homo‐ and heterodimerization of the EGFR (or erbB) family of receptors.

Binding of Shp2 Tyrosine Phosphatase to FRS2 Is Essential for Fibroblast Growth Factor-Induced PC12 Cell Differentiation

ABSTRACT FRS2 is a lipid-anchored docking protein that plays an important role in linking fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein (MAP) kinase signaling pathway. In this report, we demonstrate that FRS2 forms a complex with the N-terminal SH2 domain of the protein tyrosine phosphatase Shp2 in response to FGF stimulation. FGF stimulation induces tyrosine phosphorylation of Shp2, leading to the formation of a complex containing Grb2 and Sos1 molecules. In addition, a mutant FRS2 deficient in both Grb2 and Shp2 binding induces a weak and transient MAP kinase response and fails to induce PC12 cell differentiation in response to FGF stimulation. Furthermore, FGF is unable to induce differentiation of PC12 cells expressing an FRS2 point mutant deficient in Shp2 binding. Finally, we demonstrate that the catalytic activity of Shp2 is essential for sustained activation of MAP kinase and for potentiation of FGF-induced PC12 cell differentiation. These experiments demonstrate that FRS2 recruits Grb2 molecules both directly and indirectly via complex formation with Shp2 and that Shp2 plays an important role in FGF-induced PC12 cell differentiation.

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors.

ABSTRACT The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2α and FRS2β, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the α and β isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2α is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.

Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins

The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein kinase signaling cascade. In this report, we demonstrate that FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2α leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2α:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S/T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras/mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway.

Critical role for the docking-protein FRS2α in FGF receptor-mediated signal transduction pathways

The docking protein FRS2α has been implicated as a mediator of signaling via fibroblast growth factor receptors (FGFRs). We have demonstrated that targeted disruption of FRS2α gene causes severe impairment in mouse development resulting in embryonal lethality at E7.0–E7.5. Experiments with FRS2α-deficient fibroblasts demonstrate that FRS2α plays a critical role in FGF-induced mitogen-activated protein (MAP) kinase stimulation, phosphatidylinositol-3 (PI-3) kinase activation, chemotactic response, and cell proliferation. Following FGF stimulation, tyrosine phosphorylated FRS2α functions as a site for coordinated assembly of a multiprotein complex that includes Gab1 and the effector proteins that are recruited by this docking protein. Furthermore, we demonstrate that different tyrosine phosphorylation sites on FRS2α are responsible for mediating different FGF-induced biological responses. These experiments establish the central role of FRS2α in signaling via FGFRs and demonstrate that FRS2α mediates multiple FGFR-dependent signaling pathways critical for embryonic development.

Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor

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.

Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbb protein

The transforming protein v-erbB of avian erythroblastosis virus (AEV) displays extensive sequence homology with the presumptive protein-tyrosine kinase domain of the human EGF receptor and with the src protein-tyrosine kinase family of oncogenes. However, no kinase activity has previously been demonstrated for the v-erbB protein. Here antibodies generated against a synthetic peptide from the C terminus of human EGF receptor are shown to immunoprecipitate the EGF receptor from human and avian cells, as well as the v-erbB proteins from AEV-transformed cells that become phosphorylated on tyrosine residues upon the addition of gamma-32P-ATP. The immunoprecipitates are also able to phosphorylate exogenous tyrosine-containing substrates. Hence, it is likely that both avian EGF receptor and v-erbB proteins are protein tyrosine-specific protein kinases. Since the kinase activity of v-erbB protein cannot be regulated by EGF, it is proposed that the tyrosine protein kinase function of v-erbB may be constitutively activated.