Solution Structure of the Ligand Binding Domain of the Fibroblast Growth Factor Receptor:  Role of Heparin in the Activation of the Receptor^,

Abstract

The three-dimensional solution structure of the ligand binding D2 domain of the fibroblast growth factor receptor (FGFR) is determined using multidimensional NMR techniques. The atomic root-mean-square distribution for the backbone atoms in the structured region is 0.64 A. Secondary structural elements in the D2 domain include 11 beta-strands arranged antiparallely into two layers of beta-sheets. The structure of the D2 domain is characterized by the presence of a short flexible helix that protrudes out of the layers of beta-sheets. Results of size exclusion chromatography and sedimentation velocity experiments show that the D2 domain exists in a monomeric state both in the presence and in the absence of bound sucrose octasulfate (SOS), a structural analogue of heparin. Comparison of the solution structure of the D2 domain with the crystal structure of the protein (D2 domain) in the FGF signaling complex reveals significant differences, suggesting that ligand (FGF) binding may induce significant conformational changes in the receptor. SOS binding sites in the D2 domain have been mapped on the basis of the 1H-15N chemical shift perturbation data. SOS binds to the positively charged residues located in beta-strand III and the flexible helix. Isothermal titration calorimetry data indicate that the ligand (hFGF-1) binds strongly (Kd approximately 10(-9) M) to the D2 domain even in the absence of SOS. Binding of SOS to either the D2 domain or hFGF-1 does not seem to be the driving force for the formation of the D2-hFGF-1 binary complex. The function of SOS binding appears to stabilize the preformed D2-FGF binary complex.

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

“…Crystal structures of several FGF⅐FGFR complexes have established that both D2 and D3 are needed for FGF binding (8). In contrast to the well resolved Ig domains D1 (64) and D2 (60,65), the D3 domain of all investigated FGFR (FGFR1/2/4) constructs (D1D2D3, D2D3, and D3) lead to line broadening beyond detection in solution. The structural integrity of the investigated D3 domain was confirmed by x-ray analysis (successful crystallization of FGFR2 D2D3⅐FGF1).…”

“…This observation suggests that, although D2 alone is not sufficient for high affinity FGF binding by itself, the presence of heparin increases the binding affinity of FGF to D2. Indeed a pervious ITC study has shown that the presence of sucrose octasulfate, a heparin analog, enhances binding of FGFR2 D2 to FGFR1 (60).…”

“…Expression and purification of FGF1 (58) and FGF2 (59) was performed as previously described. The successful expression, purification, and refolding of the D2 domain is similar for FGFR1, FGFR2, and FGFR4 with minor modifications (60,61).…”

Combined Treatment With Sustained-Release Basic Fibroblast Growth Factor and Heparin Enhances Neovascularization in Hypercholesterolemic Mouse Hindlimb Ischemia

“…Because ischemia caused by femoral arterial extraction induces the production and release of bFGF, vascular growth factor and platelet-derived growth factor, 34,35 it is likely that heparin potentiates the angiogenic effects of these endogenous growth factors. [16][17][18][19][20][21]36,37 Because the half-life of intravenously administrated heparin is short, that is, 90 min, in the present study, we used heparin calcium that has a longer half-life. 33 The dose of heparin calcium used in the present study was approximately 400 U/kg, which was determined based on the data of previous studies (250-300 U/kg for rats, 14 300 U/kg for humans 33 ).…”

“…10 Heparin treatment has been reported to accelerate neovascularization in a variety of experimental models including those of hindlimb ischemia. [12][13][14][15] Although the precise beneficial mechanisms of heparin treatment have not been completely clarified, the interaction of heparin, endogenous bFGF, [16][17][18][19] and hepatocyte growth factor 20,21 has been proposed.…”