Structural basis for a direct interaction between FGFR1 and NCAM and evidence for a regulatory role of ATP

The neural cell adhesion molecule (NCAM) promotes axonal outgrowth, presumably through an interaction with the fibroblast growth factor receptor (FGFR). NCAM also has a little-understood ATPase activity. We here demonstrate for the first time a direct interaction between NCAM (fibronectin type III [F3] modules 1 and 2) and FGFR1 (Ig modules 2 and 3) by surface plasmon resonance (SPR) analysis. The structure of the NCAM F3 module 2 was determined by NMR and the module was shown by NMR to interact with the FGFR1 Ig module 3 and ATP. The NCAM sites binding to FGFR and ATP were found to overlap and ATP was shown by SPR to inhibit the NCAM-FGFR binding, indicating that ATP probably regulates the NCAM-FGFR interaction. Furthermore, we demonstrate that the NCAM module was able to induce activation (phosphorylation) of FGFR and to stimulate neurite outgrowth. In contrast, ATP inhibited neurite outgrowth induced by the module.     

A Peptide Motif from the Second Fibronectin Module of the Neural Cell Adhesion Molecule,NCAM, NLIKQDDGGSPIRHY,is a Binding Site for the FGF Receptor

The mechanism of fibroblast growth factor receptor (FGFR) activation by the neural cell adhesion molecule (NCAM) is not well understood. A motif in the second NCAM fibronectin type III (FN3) module, termed FGL, has by means of nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analyses been demonstrated to be involved in NCAM–FGFR interactions. An FGFR activation motif (FRM) in the first NCAM FN3 module also has been suggested to take part in NCAM interactions with FGFR. Here, we show for the first time that a peptide motif in the second NCAM FN3 module, different from the previously described FGL motif (NLIKQDDGGSPIRHY; termed BCL) binds and activates FGFR and induces FGFR-dependent neurite outgrowth in cultures of cerebellar granule neurons. Our results provide evidence that the BCL motif is one of the multiple FGFR binding sites in NCAM. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

NCAM-derived peptides function as agonists for the fibroblast growth factor receptor

The neural cell adhesion molecule (NCAM) directly interacts with the fibroblast growth factor receptor (FGFR). Both fibronectin type III (FN3) modules of NCAM are involved in this interaction. One of the NCAM–FGFR contact sites has been localized recently to the upper N-terminal part of the second NCAM FN3 module encompassing the F and G β-strands and the interconnecting loop region. Here, we investigated whether any of the six putative strand-loop-strand regions in the first NCAM FN3 module are involved in FGFR interactions. Peptide sequences encompassing these regions, termed encamins, were synthesized and tested for their ability to bind and activate FGFR. Encamins localized to the N-terminal part of the first FN3 module did not interact with FGFR, whereas encamins localized to the C-terminal part, termed EncaminA, C and E, bound to and activated FGFR. The encamins induced FGFR-dependent neurite outgrowth, and EncaminC and E promoted neuronal survival and enhanced pre-synaptic function. In conclusion, the interaction between NCAM and FGFR probably involves multiple contact sites at an interface formed by the two NCAM FN3 modules and FGFR, and encamins could constitute important pharmacological tools for the study of specific functional aspects of NCAM, including neuroprotection and modulation of plasticity.     

Structural basis for the activation of FGFR by NCAM

The fibroblast growth factor receptor (FGFR) can be activated through direct interaction with the neural cell adhesion molecule (NCAM). The extracellular part of the FGFR consists of three immunoglobulin-like (Ig) modules, and that of the NCAM consists of five Ig and two fibronectin type III (F3) modules. NCAM-FGFR interactions are mediated by the third FGFR Ig module and the second NCAM F3 module. Using surface plasmon resonance and nuclear magnetic resonance analyses, the present study demonstrates that the second Ig module of FGFR also is involved in binding to the NCAM. The second Ig module residues involved in binding were identified and shown to be localized on the "opposite sides" of the module, indicating that when NCAMs are clustered (e.g., due to homophilic binding), high-affinity FGFR binding sites may be formed by the neighboring NCAMs.     

The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface plasmon resonance analysis that NCAM can bind to FGFR2 with an affinity similar to that for the NCAM-FGFR1 interaction. However, the kinetic parameters for the NCAM-FGFR2 binding are different from those of the NCAM-FGFR1 binding. Both receptors were shown to cycle relatively fast between the NCAM bound and unbound states, although FGFR2 cycling was clearly faster (13 times) than the FGFR1 cycling. Moreover, ATP was more effective in inhibiting the binding of NCAM to FGFR1 than to FGFR2, indicating that the binding sites in NCAM for the two receptors are similar, but not identical.     

Modulation of the homophilic interaction between the first and second Ig modules of neural cell adhesion molecule by heparin

The second Ig module (IgII) of the neural cell adhesion molecule (NCAM) is known to bind to the first Ig module (IgI) of NCAM (so-called homophilic binding) and to interact with heparan sulfate and chondroitin sulfate glycoconjugates. We here show by NMR that the heparin and chondroitin sulfate-binding sites (HBS and CBS, respectively) in IgII coincide, and that this site overlaps with the homophilic binding site. Using NMR and surface plasmon resonance (SPR) analyses we demonstrate that interaction between IgII and heparin indeed interferes with the homophilic interaction between IgI and IgII. Accordingly, we show that treatment of cerebellar granule neurons (CGNs) with heparin inhibits NCAM-mediated outgrowth. In contrast, treatment with heparinase III or chondroitinase ABC abrogates NCAM-mediated neurite outgrowth in CGNs emphasizing the importance of the presence of heparan/chondroitin sulfates for proper NCAM function. Finally, a peptide encompassing HBS in IgII, termed the heparin-binding peptide (HBP), is shown to promote neurite outgrowth in CGNs. These observations indicate that neuronal differentiation induced by homophilic NCAM interaction is modulated by interactions with heparan/chondroitin sulfates.     

An NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth and neuronal survival in primary rat neurons

The Neural Cell Adhesion Molecule (NCAM) plays a crucial role in development of the central nervous system regulating cell migration, differentiation and synaptogenesis. NCAM mediates cell-cell adhesion through homophilic NCAM binding, subsequently resulting in activation of the fibroblast growth factor receptor (FGFR). NCAM-mediated adhesion leads to activation of various intracellular signal transduction pathways, including the Ras-mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K)-Akt pathways. A synthetic peptide derived from the second fibronectin type III module of NCAM, the FGL peptide, binds to and induces phosphorylation of FGFR without prior homophilic NCAM binding. We here present evidence that this peptide is able to mimic NCAM heterophilic binding to the FGFR by inducing neuronal differentiation as reflected by neurite outgrowth through a direct interaction with FGFR in primary cultures of three different neuronal cell types all expressing FGFR subtype 1: dopaminergic, hippocampal and cerebellar granule neurons. Moreover, we show that the FGL peptide promotes neuronal survival upon induction of cell death in the same three cell types. The effects of the FGL peptide are shown to depend on activation of FGFR and the MAPK and PI3K intracellular signalling pathways, all three kinases being necessary for the effects of FGL on neurite outgrowth and neuronal survival.     

The fibroblast growth factor receptor acid box is essential for interactions with N-cadherin and all of the major isoforms of neural cell adhesion molecule

Interactions between the neural cell adhesion molecules NCAM and N-cadherin with the fibroblast growth factor receptor (FGFR) are important for a number of developmental events and have also been implicated in tumor progression. The factors regulating these interactions are not known. We have used co-immunoprecipitation and co-clustering paradigms to show that both adhesion molecules can interact with the 3Ig IIIC isoform of the FGFR1 in a number of cell types. Interestingly, whereas the interaction can be seen over most of the cell surface, it is not seen at points of cell-cell contact where the adhesion molecules accumulate at stable junctions. We also demonstrate for the first time that all of the major isoforms of NCAM can interact with the FGFR. Using deletion mutagenesis we have found that the adhesion molecule/FGFR interaction can withstand the removal of most of any one of the FGFR immunoglobulin-like domains (D1-D3). In contrast, the FGFR interaction with N-cadherin and NCAM (but not FGF) is absolutely dependant on the presence of the acid box motif that can be found in the linker region between D1 and D2. As this motif can be spliced out of all four FGFRs, it suggests that this is one mechanism that can regulate the interaction of the receptor with different ligand classes.     

Agonists of fibroblast growth factor receptor induce neurite outgrowth and survival of cerebellar granule neurons

Fibroblast growth factor receptor (FGFR) signaling is pivotal in the regulation of neurogenesis, neuronal differentiation and survival, and synaptic plasticity both during development and in adulthood. In order to develop low molecular weight agonists of FGFR, seven peptides, termed hexafins, corresponding to the β6‐β7 loop region of the FGF 1, 2, 3, 8, 9, 10, and 17, were synthesized. This region shares a homologous amino acid sequence with the FG‐loop region of the second fibronectin Type III module of the neural cell adhesion molecule (NCAM) that binds to the FGFR. Hexafins were shown by surface plasmon resonance to bind to FGFR1‐IIIc‐Ig2‐3 and FGFR2‐IIIb‐Ig2‐3. The heparin analog sucrose octasulfate inhibited hexafin binding to FGFR1‐IIIc‐Ig2‐3 indicating overlapping binding sites. Hexafin‐binding to FGFR1‐IIIc resulted in receptor phosphorylation, but inhibited FGF1‐induced FGFR1 phosphorylation, indicating that hexafins act as partial agonists. Hexafin2, 3, 8, 10, and 17 (but not 1 or 9) induced neurite outgrowth from cerebellar granule neurons (CGNs), an effect that was abolished by two inhibitors of FGFR, SU5402 and inositol hexaphosphate (IP6) and a diacylglycerol lipase inhibitor, RHC‐80267. The neuritogenic effects of selected hexafins could also be inhibited by FGF1 which by itself did not induce neurite outgrowth. Moreover, hexafin1, 3, 9, 10, and 17 (but not 2 or 8) promoted survival of CGNs induced to undergo apoptosis. Thus, selected hexafins induced neuronal differentiation and survival, making them promising pharmacological tools for the study of functional FGFR regulation in development of the nervous system. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Structural biology of NCAM homophilic binding and activation of FGFR

In this review, we analyse the structural basis of the homophilic interactions of the neural cell adhesion molecule (NCAM) and the NCAM-mediated activation of the fibroblast growth factor receptor (FGFR). Recent structural evidence suggests that NCAM molecules form cis-dimers in the cell membrane through a high affinity interaction. These cis-dimers, in turn, mediate low affinity trans-interactions between cells via formation of either one- or two-dimensional 'zippers'. We provide evidence that FGFR is probably activated by NCAM very differently from the way by which it is activated by FGFs, reflecting the different conditions for NCAM-FGFR and FGF-FGFR interactions. The affinity of FGF for FGFR is approximately 10(6) times higher than that of NCAM for FGFR. Moreover, in the brain NCAM is constantly present on the cell surface in a concentration of about 50 microm, whereas FGFs only appear transiently in the extracellular environment and in concentrations in the nanomolar range. We discuss the structural basis for the regulation of NCAM-FGFR interactions by two molecular 'switches', polysialic acid (PSA) and adenosine triphosphate (ATP), which determine whether NCAM acts as a signalling or an adhesion molecule.     

Nectin-1 Binds and Signals through the Fibroblast Growth Factor Receptor

Nectins belong to a family of immunoglobulin (Ig)-like cell-adhesion molecules comprising four members, nectin-1 through nectin-4. Nectins are involved in formation of the mechanical adhesive puncta adherentia junctions of synapses. Nectins share the same overall structural topology with an extracellular region containing three Ig modules, a transmembrane region, and a cytoplasmic region. In nectin-1, the first and second Ig module in the extracellular region are necessary for the trans-interaction with nectin-3 and formation of cis-dimers, respectively. The function of the third Ig module of nectin-1 remains unknown. We here report the structure in solution of the third, membrane-proximal Ig module of mouse nectin-1 (nectin-1 Ig3) solved by means of nuclear magnetic resonance (NMR) spectroscopy. It belongs to the C1 set of the Ig superfamily. Nectin-1 Ig3 was produced as a recombinant protein and induced neurite outgrowth in primary cultures of hippocampal and cerebellar granule neurons, an effect abolished by treatment with the fibroblast growth factor receptor (FGFR) inhibitor SU5402, or by transfection with a dominant-negative FGFR1 construct. We showed by surface plasmon resonance (SPR) analysis that nectin-1 Ig3 directly interacted with various isoforms of FGFR. Nectin-1 Ig3 induced phosphorylation of FGFR1c in the same manner as the whole nectin-1 ectodomain, and promoted survival of cerebellar granule neurons induced to undergo apoptosis. Finally, we constructed a peptide, nectide, by employing in silico modeling of various FGFR ligand-binding sites. Nectide mimicked all the effects of nectin-1 Ig3. We suggest that FGFR is a downstream signaling partner of nectin-1.     

ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation

Homophilic binding in trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen-activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)-mediated neurite outgrowth in the PC12-E2 cell line. We found that both FGFR substrate-2 and Grb2 play important roles in NCAM as well as in FGF2-stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/Fyn signaling pathway.     

Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule

Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant > 100 μM). Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.     

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.     

Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM-FGFR-dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM-dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM-responsive axons.     

Costimulation of T cell receptor-triggered IL-2 production by Jurkat T cells via fibroblast growth factor receptor 1 upon its engagement by CD56

Recent studies have demonstrated that neural cell adhesion molecule (NCAM) is involved in multiple adhesive interactions with several different classes of ligands on the cell surface and in the extracellular matrix. One of these ligands is fibroblast growth factor receptor (FGFR) that is expressed on neural cells. While it is known that CD56 is a molecular isoform of NCAM expressed on human NK cells and a subset of T cells, it remains poorly characterized, with its ligand unidentified. Therefore, we were prompted to examine if CD56 molecules on NK cells interact with FGFR expressed on T cells. We demonstrate that ligation of FGFR1 beta on J.C2-14 Jurkat T cells by CD56 on fixed NK-92 cells costimulates TCR/CD3-triggered IL-2 production. CD56-binding mAbs inhibited the costimulatory effect of NK-92 cells in 50-75%. Flow cytometric analysis and cell adhesion assays showed that FGFR1 beta/Fc and FGFR2 beta/Fc chimeric proteins bind to NK-92 cells. The binding of FGFR1 beta/Fc protein to CD56 molecules was verified by immunoprecipitation of CD56 with anti-CD56 mAb followed by Western blotting with FGFR1 beta/Fc. These findings suggest that ligation of FGFR1 by CD56 may contribute to the interaction between NK cells and T cells that we have postulated in our previous studies.     

The binding of NCAM to FGFR1 induces a specific cellular response mediated by receptor trafficking

Neural cell adhesion molecule (NCAM) associates with fibroblast growth factor (FGF) receptor-1 (FGFR1). However, the biological significance of this interaction remains largely elusive. In this study, we show that NCAM induces a specific, FGFR1-mediated cellular response that is remarkably different from that elicited by FGF-2. In contrast to FGF-induced degradation of endocytic FGFR1, NCAM promotes the stabilization of the receptor, which is recycled to the cell surface in a Rab11- and Src-dependent manner. In turn, FGFR1 recycling is required for NCAM-induced sustained activation of various effectors. Furthermore, NCAM, but not FGF-2, promotes cell migration, and this response depends on FGFR1 recycling and sustained Src activation. Our results implicate NCAM as a nonconventional ligand for FGFR1 that exerts a peculiar control on the intracellular trafficking of the receptor, resulting in a specific cellular response. Besides introducing a further level of complexity in the regulation of FGFR1 function, our findings highlight the link of FGFR recycling with sustained signaling and cell migration and the critical role of these events in dictating the cellular response evoked by receptor activation.     

Co-localization of neural cell adhesion molecule and fibroblast growth factor receptor 2 in early embryo development

During development there is a multitude of signaling events governing the assembly of the developing organism. Receptors for signaling molecules such as fibroblast growth factor receptor 2 (FGFR2) enable the embryo to communicate with the surrounding environment and activate downstream pathways. The neural cell adhesion molecule (NCAM) was first characterized as a cell adhesion molecule highly expressed in the nervous system, but recent studies have shown that it is also a signaling receptor. Using a novel single oocyte adaptation of the proximity ligation assay, we here show a close association between NCAM and FGFR2 in mouse oocytes and 2-cell embryos. Real-time PCR analyses revealed the presence of messenger RNA encoding key proteins in downstream signaling pathways in oocytes and early mouse embryos. In summary these findings show a co-localization of NCAM and FGFR2 in early vertebrate development with intracellular signaling pathways present to enable a cellular response.     

Neural cell adhesion molecule induces intracellular signaling via multiple mechanisms of Ca2+ homeostasis

The neural cell adhesion molecule (NCAM) plays a pivotal role in the development of the nervous system, promoting neuronal differentiation via homophilic (NCAM-NCAM) as well as heterophilic (NCAM-fibroblast growth factor receptor [FGFR]) interactions. NCAM-induced intracellular signaling has been shown to affect and be dependent on the cytoplasmic Ca2+ concentration ([Ca2+]i). However, the molecular basis of this remains unclear. In this study, we determined [Ca2+]i regulating mechanisms involved in intracellular signaling induced by NCAM. To mimic the effect of homophilic NCAM interaction on [Ca2+]i in vitro, we used a peptide derived from a homophilic binding site of NCAM, termed P2, which triggers signaling cascades similar to those activated by NCAM-NCAM interaction. We found that P2 increased [Ca2+]i in primary hippocampal neurons. This effect depended on two signaling pathways. The first pathway was associated with activation of FGFR, phospholipase Cgamma, and production of diacylglycerol, and the second pathway involved Src-family kinases. Moreover, NCAM-mediated Ca2+ entry required activation of nonselective cation and T-type voltage-gated Ca2+ channels. These channels, together with the Src-family kinases, were also involved in neuritogenesis induced by physiological, homophilic NCAM interactions. Thus, unanticipated mechanisms of Ca2+ homeostasis are shown to be activated by NCAM and to contribute to neuronal differentiation.     

Elucidation of the mechanism of the regulatory function of the Ig1 module of the fibroblast growth factor receptor 1

The extracellular part of the fibroblast growth factor (FGF) receptor (FGFR) consists of up to three Ig modules (Ig1-Ig3), in which the Ig2 and Ig3 modules determine affinity and specificity for FGF and heparin. The FGFR isoforms lacking the Ig1 module have higher affinity for FGF and heparin than the triple Ig-module isoforms, suggesting that the Ig1 module is involved in the regulation of the FGFR-ligand interaction. We show here by surface plasmon resonance and NMR analyses that the Ig1 module binds to the Ig2 module, and identify by NMR the binding sites involved in the Ig1-Ig2 interaction. The identified binding site in the Ig2 module was found to be in the area of the FGF-Ig2 and Ig2-heparin contact sites, thus providing direct structural evidence that the Ig1 module functions as a competitive autoinhibitor of the FGFR-ligand interaction. Furthermore, the Ig1 binding site of the Ig2 module overlaps the Ig2-Ig2 contact site. This suggests that the function of the Ig1 module is not only regulation of the FGFR-ligand binding affinity but also prevention of spontaneous FGFR dimerization (through a direct Ig2-Ig2 interaction) in the absence of FGF.