Differential Effects of Heparin Saccharides on the Formation of Specific Fibroblast Growth Factor (FGF) and FGF Receptor Complexes

Olga Ostrovsky,Bluma Berman,John Gallagher,Barbara Mulloy,David G Fernig,Maryse Delehedde,Dina Ron

doi:10.1074/jbc.m108540200

Abstract

Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate the interaction of FGFs with their receptors (FGFRs) and are thought to play a critical role in receptor dimerization. Biochemical and crystallographic studies, conducted mainly with FGF-2 or FGF-1 and FGF receptors 1 and 2, suggests that an octasaccharide is the minimal length required for FGF- and FGFR-induced dimerization and subsequent activation. In addition, 6-O-sulfate groups are thought to be essential for binding of HS to FGFR and for receptor dimerization. We show here that oligosaccharides shorter than 8 sugar units support activation of FGFR2 IIIb by FGF-1 and interaction of FGFR4 with FGF-1. In contrast, only relatively long oligosaccharides supported receptor binding and activation in the FGF-1.FGFR1 or FGF-7.FGFR2 IIIb setting. In addition, both 6-O- and 2-O-desulfated heparin activated FGF-1 signaling via FGFR2 IIIb, whereas neither one stimulated FGF-1 signaling via FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required for activating FGFs is dictated by the specific FGF and FGFR combination. These different requirements may reflect the differences in the mode by which a given FGFR interacts with the various FGFs.

Highlights

Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is well studied for fibroblast growth factors (FGFs)
We first evaluated the concentration of native heparin that supports maximal biological response of FGFR2 IIIb to FGF-1 and FGF-7
We initially assessed the ability of 5 ␮g/ml heparin-derived oligosaccharides ranging in size from DP2 to DP18 to support the growth-stimulatory responses to FGF-1 and FGF-7

Summary

EXPERIMENTAL PROCEDURES

Materials—Human recombinant FGF-7 and FGF-1 were produced in bacteria and purified as described previously [11, 35]. Expression of Human FGFR4 in BaF3 Cells—Cells were transfected by electroporation (960 microfarads, 240 V) with pCEV plasmid bearing the human FGFR4 gene [11] and selected in BaF3 growth medium containing G418 (500 ␮g/ml) These cells were grown in RPMI plus 10% FCS, WEHI-3B conditioned medium, and G418. 6-O-Desulfated heparin was prepared by solvolysis of the pyridinium salt in n-methylpyrrolidinone-water [42] Approximately 100 mg of bovine lung heparin was passed several times through Dowex 50 Hϩ form, neutralized with pyridine, and evaporated to dryness under reduced pressure. The cooled reaction mixture was diluted with water and neutralized with NaOH to pH 9, concentrated by evaporation, and dialyzed against at least five changes of deionized water before lyophilization These conditions give a degree of N-desulfation besides 6-O-desulfation, so re-N-sulfation was carried out on the 6-O-desulfated product using trimethylamine-SO3 complex [43]. Loss of 2-O-sulfate in the solvolytically 6-Odesulfated sample was below the limit of detection

RESULTS

DISCUSSION

The present findings that signaling induced by the same