The Involvement of Heparan Sulfate (HS) in FGF1/HS/FGFR1 Signaling Complex

Abstract

Fibroblast growth factor (FGF) signaling begins with the formation of a ternary complex of FGF, FGF receptor (FGFR), and heparan sulfate (HS). Multiple models have been proposed for the ternary complex. However, major discrepancies exist among those models, and none of these models have evaluated the functional importance of the interacting regions on the HS chains. To resolve the discrepancies, we measured the size and molar ratio of HS in the complex and showed that both FGF1 and FGFR1 simultaneously interact with HS; therefore, a model of 2:2:2 FGF1⅐HS⅐FGFR1 was shown to fit the data. Using genetic and biochemical methods, we generated HSs that were defective in FGF1 and/or FGFR1 binding but could form the signaling ternary complex. Both genetically and chemically modified HSs were subsequently assessed in a BaF3 cell mitogenic activity assay. The ability of HS to support the ternary complex formation was found to be required for FGF1-stimulated cell proliferation. Our data also proved that specific critical groups and sites on HS support complex formation. Furthermore, the molar ratio of HS, FGF1, and FGFR1 in the ternary complex was found to be independent of the size of HS, which indicates that the selected model can take place on the cell surface proteoglycans. Finally, a mechanism for the FGF⅐FGFR signaling complex formation on cell membrane was proposed, where FGF and FGFR have their own binding sites on HS and a distinct ternary complex formation site is directly responsible for mitogenic activity.Heparan sulfate (HS) 1 is a linear and highly sulfated polysaccharide, consisting of 50 -150 basic disaccharide repeats of uronic acid and D-glucosamine units (1). Sulfation can occur at 2-O of the uronic acid and 3-O, 6-O, and N of the D-glucosamine and is catalyzed by a variety of sulfotransferases. Each modification is incomplete, which leads to sequence variation on HS, and it is very likely that critical sulfate groups determine the specificity of HS-protein interactions (2). Along the HS chain, the majority of sulfated residues are clustered in short functional domains separated by relatively less sulfated oligosaccharide sequences (3). Heparin resembles these functional domains and is widely used for the functional study of HS. One major function of HS is to interact with fibroblast growth factors (FGFs) and their receptors (FGFRs) and form FGF⅐HS⅐FGFR signaling complexes (4 -7). Defects in HS can cause complete losses of FGF, Hedgehog, and Wingless signaling pathways and lead to severe abnormality in embryonic development (8, 9). The involvement of HS in the FGF molecular signaling complex suggests that FGF activity and specificity may be modulated by HS and in turn by enzymes that synthesize and degrade HS.FGFs and FGFRs play critical roles in the control of many fundamental cellular processes, such as cell proliferation, differentiation, and migration (10 -13). There are 23 known FGFs and five types of FGFRs in humans (14). FGF1 and FGF2 were the first to be isolated and were called acidic and basic...

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