Abstract
NCAM1 and NCAM2 have ectodomains consisting of 5 Ig domains followed by 2 membrane-proximal FnIII domains. In this study we investigate and compare the structures and functions of these FnIII domains. The NCAM1 and -2 FnIII2 domains both contain a Walker A motif. In NCAM1 binding of ATP to this motif interferes with NCAM1 binding to FGFR. We obtained a structural model of the NCAM2 FnIII2 domain by NMR spectroscopy, and by titration with an ATP analogue we show that the NCAM2 Walker A motif does not bind ATP. Small angle X-ray scattering (SAXS) data revealed that the NCAM2 FnIII1-2 double domain exhibits a very low degree of flexibility. Moreover, recombinant NCAM2 FnIII domains bind FGFR in vitro, and the FnIII1-2 double domain induces neurite outgrowth in a concentration-dependent manner through activation of FGFR. Several synthetic NCAM1-derived peptides induce neurite outgrowth via FGFR. Only 2 of 5 peptides derived from similar regions in NCAM2 induce neurite outgrowth, but the most potent of these peptides stimulates neurite outgrowth through FGFR-dependent activation of the Ras-MAPK pathway. These results reveal that the NCAM2 FnIII domains form a rigid structure that binds and activates FGFR in a manner related to, but different from NCAM1.
Highlights
Cell adhesion molecules (CAMs) constitute a large class of plasma membrane-anchored proteins that mediate attachment of cells to neighboring cells and to the surrounding extracellular matrix
The folding of the protein expressed in E. coli was verified by circular dichroism (CD) experiments, and the proteins were used for Surface plasmon resonance (SPR) analysis, small-angle X-ray scattering (SAXS) experiments, as well as for cell culture studies
Using nuclear magnetic resonance (NMR) we here show that the Walker A motif in the Neural Cell Adhesion Molecule 2 (NCAM2) FnIII2 domain does not bind the ATP analogue AMP-PCP (Fig. S6)
Summary
Cell adhesion molecules (CAMs) constitute a large class of plasma membrane-anchored proteins that mediate attachment of cells to neighboring cells and to the surrounding extracellular matrix. A number of synthetic peptides derived from sequences in the NCAM1 FnIII domains are reported to bind and activate FGFR. A synthetic peptide, FGL, corresponding to the NCAM1 FnIII2 FG loop has been extensively studied. This peptide promotes e.g. neurite outgrowth and neuronal cell survival in vitro. It has multiple effects in vivo including reduction of inflammation, promotion of learning and memory formation, and the reduction of signs of Aβ-induced neuropathology and cognitive impairment in an Alzheimer’s disease model[21]
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