Structural basis for the activation of FGFR by NCAM

Abstract

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.

Figure 1.

Surface plasmon resonance analysis of the binding between the FGFR Ig2 module and the NCAM F3 modules. (A) Binding of the soluble FGFR Ig2 module to the immobilized NCAM F3 modules (with a fitting of the saturation plot shown in B). (C) Binding of the soluble NCAM F3 modules to the immobilized FGFR Ig2 module (with a fitting of the saturation plot shown in D). The experiment was repeated nine times.

Figure 2.

Mapping of the FGFR Ig2 module's residues involved in the NCAM binding. Changes in chemical shifts of the 10 μM ¹⁵N-labeled Ig2 module after addition of (A) 13 μM, (B) 25 μM, and (C) 40 μM unlabeled NCAM F3 modules and mapping of the significantly perturbed residues onto the FGFR Ig2 module structure (right panels). The crystal structure of human FGFR Ig2 module (PDB code: 1EVT) was used for mapping. MolMol software version 2K.2 was used for creating graphical representations of molecular structures. Origin software version 6.1 was used to create diagrams.

Figure 3.

Mapping of the various FGFR Ig2 module's binding sites onto the module's structure. For the NCAM binding site, the first and second clusters are shown in magenta and aquamarine, respectively. The crystal structure of the human FGFR Ig2 module (PDB code: 1EVT) was used for mapping. MolMol software version 2K.2 was used for creating graphical representations of molecular structures.

Figure 4.

Surface plasmon resonance analysis of the inhibitory effect of sucrose octasulphate (SOS) on binding of the FGFR Ig2 module to the NCAM F3 modules. The experimental setup was the same as described in Figure 1. The binding of 20 μM FGFR Ig2 module to the immobilized NCAM F3 modules was studied in the presence of the indicated concentrations of SOS. The experiment was repeated nine times.

Figure 5.

Competition isotherm (by SOS) of the FGFR Ig2 module binding to the NCAM. The isotherm shows the maximum binding level of Ig2 at 20 μM concentration versus SOS concentration. The data were fitted with a single site model in A and a two-site model in B. The data are shown as averages of six replicates, with error bars corresponding to standard deviations.

Figure 6.

Schematic representation of the proposed model for the interaction between the FGFR and NCAM. (A) The NCAM is not involved in the trans-homophilic binding, and there is no substantial binding between the FGFR and NCAM. (B) The NCAM is involved in trans-homophilic binding and the FGFR binds to two neighboring NCAM molecules belonging to the different cis-dimers. (C) The FGFR is bound to two NCAM molecules belonging to the same cis-dimer.