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.

The insulin and insulin-like growth factor 1 receptors activate overlapping signalling pathways that are critical for growth, metabolism, survival and longevity. Their mechanism of ligand binding and activation displays complex allosteric properties, which no mathematical model has been able to account for. Modelling these receptors' binding and activation in terms of interactions between the molecular components is problematical due to many unknown biochemical and structural details. Moreover, substantial combinatorial complexity originating from multivalent ligand binding further complicates the problem. On the basis of the available structural and biochemical information, we develop a physically plausible model of the receptor binding and activation, which is based on the concept of a harmonic oscillator. Modelling a network of interactions among all possible receptor intermediaries arising in the context of the model (35, for the insulin receptor) accurately reproduces for the first time all the kinetic properties of the receptor, and provides unique and robust estimates of the kinetic parameters. The harmonic oscillator model may be adaptable for many other dimeric/dimerizing receptor tyrosine kinases, cytokine receptors and G-proteincoupled receptors where ligand crosslinking occurs.

The neural cell adhesion molecule, NCAM, mediates Ca(2+)-independent cell-cell and cell-substratum adhesion via homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. NCAM plays a key role in neural development, regeneration, and synaptic plasticity, including learning and memory consolidation. The crystal structure of a fragment comprising the three N-terminal Ig modules of rat NCAM has been determined to 2.0 A resolution. Based on crystallographic data and biological experiments we present a novel model for NCAM homophilic binding. The Ig1 and Ig2 modules mediate dimerization of NCAM molecules situated on the same cell surface (cis interactions), whereas the Ig3 module mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans interactions) through simultaneous binding to the Ig1 and Ig2 modules. This arrangement results in two perpendicular zippers forming a double zipper-like NCAM adhesion complex.

Human type 1 insulin-like growth factor receptor is a homodimeric receptor tyrosine kinase that signals into pathways directing normal cellular growth, differentiation and proliferation, with aberrant signalling implicated in cancer. Insulin-like growth factor binding is understood to relax conformational restraints within the homodimer, initiating transphosphorylation of the tyrosine kinase domains. However, no three-dimensional structures exist for the receptor ectodomain to inform atomic-level understanding of these events. Here, we present crystal structures of the ectodomain in apo form and in complex with insulin-like growth factor I, the latter obtained by crystal soaking. These structures not only provide a wealth of detail of the growth factor interaction with the receptor’s primary ligand-binding site but also indicate that ligand binding separates receptor domains by a mechanism of induced fit. Our findings are of importance to the design of agents targeting IGF-1R and its partner protein, the human insulin receptor.

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 neural cell adhesion molecule, NCAM, is a membraneassociated glycoprotein expressed on the surface of neurons and glial cells. NCAM plays a key role in morphogenesis of the nervous system, synaptic plasticity in relation to learning and memory consolidation, and regeneration of damaged neural tissue (Rønn et al. 1998).NCAM is a member of the immunoglobulin (Ig) superfamily and mediates cell)cell adhesion. It is involved in homophilic interactions (NCAM binding to NCAM) between opposing, NCAM-expressing cells and heterophilic binding between NCAM and a series of proteins including the fibroblast growth factor receptor (FGFR), the neuronal cell adhesion molecule L1 and different types of collagens and proteoglycans Crossin and Krushel 2000;Povlsen et al. 2003;Hinsby et al. 2004). Additionally, NCAM has recently been shown to bind glial cell Abbreviations used: Ab 25-35, Amyloid-b peptide; BDNF, brainderived neurotrophic factor; CGN, cerebellar granule neurons; Erk1/2, extracellular signal-regulated kinases 1 and 2; F3, fibronectin type III module; FAK, focal adhesion kinase; FGFR, fibroblast growth factor receptor; FGLd, FG loop peptide dendrimer; FGLm, FG loop peptide monomer; GDNF, glial cell line-derived neurotrophic factor; Ig, immunoglobulin; IGF-1, insulin-like growth factor 1; MAPK, Ras-mitogen activated protein kinase; NCAM, neural cell adhesion molecule; 6-OHDA, 6-hydroxydopamine; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PLCc, phospholipase Cc.

SummaryIn 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 cisdimers, 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 lM, 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.

To study the function of the first immunoglobulin (Ig)-like domain of the neural cell adhesion molecule (NCAM), it was produced as a recombinant fusion protein in a bacterial expression system and as a recombinant protein in a eukaryotic expression system of the yeast Pichia pastoris. For comparison, other NCAM domains were also produced as fusion proteins. By means of surface plasmon resonance analysis, it was shown that the first Ig-like NCAM domain binds the second Ig-like NCAM domain with a dissociation constant 5.5 +/- 1.6 x 10(-5) M. Furthermore, it was found that the first Ig-like domain binds heparin. It was also demonstrated that the second Ig-like NCAM domain binds heparin and that both domains bind collagen type I via heparin but not collagen type I directly.

We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å . DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel ␤-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel ␤-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.The ligands and receptors of the insulin peptide family constitute an ancient metazoan signaling system that plays a crucial pleiotropic role in cell growth, metabolism, reproduction, and longevity (1-7).The mammalian insulin receptor belongs to the family of receptor-tyrosine kinases and is composed of two ␣ subunits and two ␤ subunits linked together by disulfide bonds (for review, see Refs. 4 and 8 -10). The existence of a homologue of the mammalian insulin receptor in Drosophila melanogaster (DIR) 2 was suggested in 1985 by Petruzzelli et al. (11), who identified a glycoprotein of 350 -400 kDa that binds bovine insulin specifically with moderate affinity (15 nM). The cDNA sequence of the DIR is remarkably similar to that of the mammalian insulin and IGF-I receptors (with 33% sequence identity) except for substantial N-and C-terminal extensions (12, 13).In evolution, there is a single receptor from Cnidarians up to and including Amphioxus (Branchiostoma californiense), the phylum closest to vertebrates (for review see Refs. 1, 4 -6). In vertebrates, gene duplications resulted in three related receptors; that is, the insulin receptor, the type I IGF receptor, and the orphan insulin receptor-related receptor (1, 5).In humans, members of the insulin peptide family include insulin, the insulin-like growth factors I and II, and seven relaxin-related peptides (for review, see Ref. 14). The same basic fold is shared for all molecules in the superfamily whose structure is known; the B domain contains a single ␣-helix that lies across the two ␣-helices of the A domain (15) and two canonical disulfide bridges that connect the A-and Bchains, whereas an intrachain disulfide bridge is present in the A-chain.The D. melanogaster genome contains seven insulin-like genes that are expressed in a highly tissue-and stage-specific patterns, dilp1-7 (16). dilp2 is the most related to human insulin with 35% sequence identity, whereas dilp5 has 27.8% identity (16).So far, the structures of only two invertebrate insulin-like peptides have been determined by N...