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Fibroblast growth factor receptors (FGFRs) are important for many different mechanisms, including cell migration, proliferation, differentiation, and survival. Here, we show a new link between FGFR1 and the cell adhesion molecule neurofascin, which is important for neurite outgrowth. After overexpression in HEK293 cells, embryonal neurofascin isoform NF166 was able to associate with FGFR1, whereas the adult isoform NF186, differing from NF166 in additional extracellular sequences, was deficient. Pharmacological inhibitors and overexpression of dominant negative components of the FGFR signaling pathway pointed to the activation of FGFR1 after association with neurofascin in neurite outgrowth assays in chick tectal neurons and rat PC12-E2 cells. Both extra-and intracellular domains of embryonal neurofascin isoform NF166 were able to form complexes with FGFR1 independently. However, the cytosolic domain was both necessary and sufficient for the activation of FGFR1. Cytosolic serine residues 56 and 100 were shown to be essential for the neurite outgrowth-promoting activity of neurofascin, whereas both amino acid residues were dispensable for FGFR1 association. In conclusion, the data suggest a neurofascin intracellular domain, which activates FGFR1 for neurite outgrowth, whereas the extracellular domain functions as an additional, regulatory FGFR1 interaction domain in the course of development.The four known fibroblast growth factor receptors (FGFRs), 2 which are targeted by a large family of 22 fibroblast growth factor ligands, represent a highly diverse signaling system important for migration, proliferation, differentiation, and survival of many different cell types (1, 2). fibroblast growth factor activation of FGFR leads to the activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and phospholipase C␥ (PLC␥), depending on the cellular system under study. Non-canonical FGFR interactions with NCAM, cadherins, and syndecan via extracellular domains were also described (1). However, the contribution of intracellular interactions of FGFR1 with further membrane co-receptors is poorly understood. Only cytosolic interaction between FGFRs and EphA4 have been described that are involved in mutual transphosphorylation (3).The cell adhesion molecule neurofascin is important for cellcell communication in the nervous system (4, 5). Neurofascin regulates many different functions in the brain, suggesting that it functions as a key regulator for both developing and differentiated neural cells. Different alternatively spliced neurofascin isoforms are expressed in different cells and at different times of development (6). Embryonal neurofascin NF166 is important for neurite outgrowth and guidance (7,8). Recently, a role for neurofascin NF166 for early processes of inhibitory synaptogenesis at the axon hillock and for the positioning of inhibitory synapses at the axon initial segment has been proven (9, 10).In the more developed nervous system, NF166 is replaced by NF186, which is inhibitory...

Neurofascin is a member of the immunoglobulin superfamily involved in axon extension and fasciculation. Here we apply adenoviral short hairpin RNA (shRNA) expression in primary neurons, PC12-NIH/3T3 co-cultures in combination with Luminex assays, to demonstrate homophilic interactions of neurofascin for neurite outgrowth. An adenoviral vector was constructed for the expression of shRNA in primary tectal cells that inhibits gene expression similar to short interfering RNA. We demonstrate that after shRNA-mediated knockdown neuronal neurofascin expression is important for neurite outgrowth on a neurofascin substrate. Neurite outgrowth assays reveal that neurite formation of PC12 cells is increased when neurofascin is overexpressed on both outgrowing PC12 cells and substrate NIH/3T3 cells, suggesting that neurofascin expression is also sufficient for neurite induction. Luminex technology for the analysis of protein-protein interactions showed homophilic binding of neurofascin to itself.