Fibroblast growth factor-2 binds to small heparin-derived oligosaccharides and stimulates a sustained phosphorylation of p42/44 mitogen-activated protein kinase and proliferation of rat mammary fibroblasts

Abstract

We examine the relationship between the chain length of heparin-derived oligosaccharides, fibroblast growth factor (FGF)-2 binding kinetics and the ability of the oligosaccharides to allow FGF-2-induced proliferation of chlorate-treated rat mammary fibroblasts. First, using an optical biosensor, we show that FGF-2 did not bind disaccharides, but definitively bound to tetrasaccharides. As the chain length increased from tetrasaccharide to octasaccharide, there was a substantial increase in k(ass) (564000 M(-1) x s(-1) to 2000000 M(-1) x s(-1), respectively) and affinity (K(d) 77 nM to 11 nM, respectively) for FGF-2. From decasaccharides and longer, the k(ass) and affinity for FGF-2 was reduced substantially (tetradecasaccharide k(ass) 470000 M(-1) x s(-1), K(d) 30 nM). In chlorate-treated, and hence sulphated, glycosaminoglycan-deficient cells, FGF-2 alone or in the presence of disaccharides did not stimulate DNA synthesis and it only elicited an early transient dual phosphorylation of p42/44 mitogen-activated protein kinase (MAPK). In the same cells FGF-2 in the presence of tetrasaccharides and longer oligosaccharides was able to restore DNA synthesis and enable the sustained dual phosphorylation of p42/44(MAPK). However, the oligosaccharides from tetrasaccharides to octasaccharides were less potent in proliferation assays than deca- and longer oligosaccharides. Therefore, there was no correlation between the binding parameters and the potency of the oligosaccharides in DNA synthesis assays. These results demonstrate that tetrasaccharides are able to bind FGF-2 and enable FGF-2 to stimulate cell proliferation, which sets important boundary conditions for models of the FGF-2-heparan sulphate-FGF receptor complex.

N-Glycosylation of Fibroblast Growth Factor Receptor 1 Regulates Ligand and Heparan Sulfate Co-receptor Binding

“…No more than 300 arc s FGF-2 was immobilized on the surface (1 arc s ϭ 1/3600°, 600 arc s ϭ 1 ng protein/mm 2 ). Heparin-derived dodecasaccharides were biotinylated at their reducing ends and immobilized on streptavidin surfaces, as described (22,30).…”

“…A third extracellular regulatory input is likely to be the assembly of the FGF receptor ligand system into different complexes that have different signaling potentials. This is suggested by the identification of two different crystal structures of complexes of FGFR⅐FGF⅐heparin oligosaccharide (19,20) and by biophysical (21) and biochemical evidence (9,22). Finally, genetic evidence indicates that mutation of an asparagine residue in the extracellular domain of FGFR2 and FGFR3 increases its activity, to result in skeletal growth defects, which has been linked to the disruption of N-glycosylation (23,24).…”

HSulf-1 modulates HGF-mediated tumor cell invasion and signaling in head and neck squamous carcinoma

“…The best example of such a regulation is the role of HSGAGs in FGF-2 signaling (Lundin et al, 2000;Delehedde et al, 2002). FGF-2 binding to its receptors FGFR and heparan sulfate proteoglycans (HSPGs) promotes the formation of a ternary complex that is essential for cell proliferation and angiogenesis.…”

FGF2 binding, signaling and angiogenesis are modulated by heparanase in metastatic melanoma cells

“…Conversely, Hep III produces HS fragments that are rich in IdoUA(2S)a 1,4 GlcNSO 3 (±6S) disaccharides [50], and HS degradation is significantly more extensive than with HPSE, resulting in the production of shorter HS fragments. Although highly sulfated HS with as few as four disaccharides units can bind to FGF2, longer oligomers are required for effective formation of the FGF2-FGFR stoichiometry required for subsequent signaling [51].…”