Vladislav Soroka

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.

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 106 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 µm, 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.

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.

Molecular mechanisms of Ca2+ signaling in neurons induced by the S100A4 protein

ABSTRACT The S100A4 protein belongs to the S100 family of vertebrate-specific proteins possessing both intra- and extracellular functions. In the nervous system, high levels of S100A4 expression are observed at sites of neurogenesis and lesions, suggesting a role of the protein in neuronal plasticity. Extracellular oligomeric S100A4 is a potent promoter of neurite outgrowth and survival from cultured primary neurons; however, the molecular mechanism of this effect has not been established. Here we demonstrate that oligomeric S100A4 increases the intracellular calcium concentration in primary neurons. We present evidence that both S100A4-induced Ca2+ signaling and neurite extension require activation of a cascade including a heterotrimeric G protein(s), phosphoinositide-specific phospholipase C, and diacylglycerol-lipase, resulting in Ca2+ entry via nonselective cation channels and via T- and L-type voltage-gated Ca2+ channels. We demonstrate that S100A4-induced neurite outgrowth is not mediated by the receptor for advanced glycation end products, a known target for other extracellular S100 proteins. However, S100A4-induced signaling depends on interactions with heparan sulfate proteoglycans at the cell surface. Thus, glycosaminoglycans may act as coreceptors of S100 proteins in neurons. This may provide a mechanism by which S100 proteins could locally regulate neuronal plasticity in connection with brain lesions and neurological disorders.

Structure and interactions of NCAM modules 1 and 2, basic elements in neural cell adhesion

The structure in solution of the second Ig-module fragment of residues 117–208 of NCAM has been determined. Like the first Ig-module of residues 20–116, it belongs to the I set of the immunogloblin superfamily. Module 1 and module 2 interact weakly, and the binding sites of this interaction have been identified. The two–module fragment NCAM(20–208) is a stable dimer. Removal of the charged residues in these sites in NCAM(20–208) abolishes the dimerization. Modeling the dimer of NCAM(20–208) to fit the interactions of these charges produces one coherent binding site for the formation of two antiparallel strands of the first two NCAM modules. This mode of binding could be a major element in trans–cellular interactions in neural cell adhesion.

Role of Glial Cell Line-Derived Neurotrophic Factor (GDNF)–Neural Cell Adhesion Molecule (NCAM) Interactions in Induction of Neurite Outgrowth and Identification of a Binding Site for NCAM in the Heel Region of GDNF

The formation of appropriate neuronal circuits is an essential part of nervous system development and relies heavily on the outgrowth of axons and dendrites and their guidance to their respective targets. This process is governed by a large array of molecules, including glial cell line-derived neurotrophic factor (GDNF) and the neural cell adhesion molecule (NCAM), the interaction of which induce neurite outgrowth. In the present study the requirements for NCAM-mediated GDNF-induced neurite outgrowth were investigated in cultures of hippocampal neurons, which do not express Ret. We demonstrate that NCAM-mediated GDNF-induced signaling leading to neurite outgrowth is more complex than previously reported. It not only involves NCAM-140 and the Src family kinase Fyn but also uses NCAM-180 and the fibroblast growth factor receptor. We find that induction of neurite outgrowth by GDNF via NCAM or by trans-homophilic NCAM interactions are not mutually exclusive. However, whereas NCAM-induced neurite outgrowth primarily is mediated by NCAM-180, we demonstrate that GDNF-induced neurite outgrowth involves both NCAM-140 and NCAM-180. We also find that GDNF-induced neurite outgrowth via NCAM differs from NCAM-induced neurite outgrowth by being independent of NCAM polysialylation. Additionally, we investigated the structural basis for GDNF–NCAM interactions and find that NCAM Ig3 is necessary for GDNF binding. Furthermore, we identify within the heel region of GDNF a binding site for NCAM and demonstrate that a peptide encompassing this sequence mimics the effects of GDNF with regard to NCAM binding, activation of intracellular signaling, and induction of neurite outgrowth.

Epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting S100A4 protein.

The function of S100A4, a member of the calcium‐binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro‐metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms and protein partners (receptors) has remained elusive. To identify putative targets for extracellular S100A4, we screened a phage display peptide library using S100A4 as bait. We identified three independent peptide motifs with varying affinities for the S100A4 protein. Sequence analyses indicated that the most abundant peptide mimicked the F/YCC motif present in the epidermal growth factor domain of ErbB receptor ligands. S100A4 selectively interacted with a number of epidermal growth factor receptor (EGFR) ligands, demonstrating highest affinity for amphiregulin. Importantly, we found that S100A4 stimulated EGFR/ErbB2 receptor signaling and enhanced the amphiregulin‐mediated proliferation of mouse embryonic fibroblasts. S100A4‐neutralizing antibodies, as well as EGFR‐ and ErbB2 receptor‐specific tyrosine kinase inhibitors, blocked these effects. The present results suggest that extracellular S100A4 regulates tumor progression by interacting with EGFR ligands, thereby enhancing EGFR/ErbB2 receptor signaling and cell proliferation.

Neuroprotective properties of a novel, non-haematopoietic agonist of the erythropoietin receptor

Erythropoietin, a member of the type 1 cytokine superfamily, controls proliferation and differentiation of erythroid progenitor cells through binding to and dimerization of the erythropoietin receptor. Both erythropoietin and its receptor are also expressed in the central nervous system, where they are involved in tissue protection. However, the use of erythropoietin as a neuroprotective agent may be hampered by its erythropoietic activity. Therefore, developing non-haematopoietic erythropoietin mimetics is important. Based on the crystal structure of the complex of erythropoietin and its receptor, we designed a peptide, termed Epotris, corresponding to the C α-helix region (amino-acid residues 92-111) of human erythropoietin. The peptide specifically bound to the erythropoietin receptor and promoted neurite outgrowth and survival of primary neurons with the same efficiency as erythropoietin, but with 10(3)-fold lower potency. Knockdown of the erythropoietin receptor or interference with its downstream signalling inhibited the Epotris-induced neuritogenic and pro-survival effect. Similarly to erythropoietin, Epotris penetrated the blood-brain barrier. Moreover, treatment with the peptide attenuated seizures, decreased mortality and reduced neurodegeneration in an in vivo model of kainic acid-induced neurotoxicity. In contrast to erythropoietin, Epotris did not stimulate erythropoiesis upon chronic administration. Thus, Epotris is a novel neuroprotective non-haematopoietic erythropoietin mimetic that may offer new opportunities for the treatment of neurological disorders.

Neuroplastin-55 binds to and signals through the fibroblast growth factor receptor

Neuroplastin (Np) is a glycoprotein belonging to the immunoglobulin superfamily of cell adhesion molecules (CAMs) and existing in two isoforms, Np55 and Np65, named according to their molecular weights. The extracellular part of Np65 contains three immunoglobulin (Ig)‐like modules (Igl, Ig2, and Ig3), whereas Np55 lacks the Igl module. Of these two isoforms, only Np65 is involved in homophilic interactions resulting in cell adhesion, whereas the role of Np55 is poorlyunderstood. The present studyreports for the first time the crystal structure of the ectodomain of Np55 at 1.95‐Å resolution and demonstrates that Np55 binds to and activates the fibroblast growth factor receptor 1 (FGFR1). Furthermore, we identify a sequence motif in the Ig2 module of Np55 interacting with FGFR1 and show that a synthetic peptide encompassing this motif, termed narpin, binds to and activates FGFR1. We show that both Np55 and the narpin peptide induce neurite outgrowth through FGFR1 activation and that Np55 increases synaptic calcium concentration in an FGFR1‐dependent manner. Moreover, we demonstrate that narpin has an antidepressive‐like effect in rats subjected to the forced swim test, suggesting that Np55‐induced signaling may be involved in synaptic plasticity in vivo. Owczarek, S., Kiryushko, D., Larsen, M. H., Kastrup, J. S., Gajhede, M., Sandi, C., Berezin, V., Bock, E., Soroka, V. Neuroplastin‐55 binds to and signals through the fibroblast growth factor receptor. FASEB J. 24, 1139–1150 (2010). www.fasebj.org

The metastasis-promoting S100A4 protein confers neuroprotection in brain injury

Identification of novel pro-survival factors in the brain is paramount for developing neuroprotective therapies. The multifunctional S100 family proteins have important roles in many human diseases and are also upregulated by brain injury. However, S100 functions in the nervous system remain unclear. Here we show that the S100A4 protein, mostly studied in cancer, is overexpressed in the damaged human and rodent brain and released from stressed astrocytes. Genetic deletion of S100A4 exacerbates neuronal loss after brain trauma or excitotoxicity, increasing oxidative cell damage and downregulating the neuroprotective protein metallothionein I+II. We identify two neurotrophic motifs in S100A4 and show that these motifs are neuroprotective in animal models of brain trauma. Finally, we find that S100A4 rescues neurons via the Janus kinase/STAT pathway and, partially, the interleukin-10 receptor. Our data introduce S100A4 as a therapeutic target in neurodegeneration, and raise the entire S100 family as a potentially important factor in central nervous system injury.