Heparin and fibroblast growth factor-1 interactions: The role of heparin in mediating FGF-1 mitogenic signal transduction

Fibroblast growth factors (FGFs) are a family of polypeptides with multiple functions on a variety of cell types. There are currently 16 described members that have 30-60% amino acid sequence identity and have relatively high affinity for immobilized heparin. This study investigated the functional role of heparin and cell-surface heparan sulfate proteoglycans (HSPGs) in the mitogenic activities of FGFs. The presence of heparin activates or inhibits the mitogenic activities of different FGF members. However, the heparin dependence of FGF-1 mitogenic activity has been found to vary with cell type. This finding is inconsistent with the current and generally accepted model of heparin and cell-surface HSPGs in FGF function. In order to gain a better understanding of heparin:FGF-1 interaction, the affinity of FGF-1 for heparin was examined. Site-directed mutations were made in FGF-1 within the three putative heparin-binding domains as predicted by consensus sequences. The results indicate that a significant reduction in apparent heparin affinity is only observed when basic residues in one of the three regions are mutated. These findings indicate that heparin-binding regions cannot be defined by single consensus motifs. A heparin-binding peptide was identified that was able to quantitatively inhibit the heparin-dependent mitogenic activity of FGF-1. Mutation of a non-basic residue within the heparin-binding peptide abolished heparin affinity and demonstrated the importance of non-basic residues for heparin affinity.;Within the same cell type, the mitogenic activity of FGF-1 and FGF-7 varies in response to the presence of heparin, and mutants of FGF-1 have been identified that are able to escape the heparin dependence exhibited by wildtype FGF-1 on certain cell types. These results indicate that the role of heparin is not restricted to receptor dimerization in FGF function. In addition, conditioned media experiments have identified the existence of an unknown inhibitory factor for FGF-1 activity that is not affected by the presence of heparin. These findings indicate that other factors also contribute to FGF function.;The co-crystallization of FGF-2 and heparin-derived tetra- and hexasaccharides identified contact residues on FGF-2 and supported our identification of amino acids on FGF-1 that contribute to heparin affinity. Site-directed mutagenesis of the putative heparin contact residues on FGF-1 and FGF-7 predictively altered heparin affinity. These findings indicate that a generality exists between the co-crystal structure of FGF-2 and heparin-derived saccharides that can be applied to other FGF family members. Since the crystal structure studies are compatible with solution studies, these results indicate that the structure can be used for future mutagenesis studies of activities other than heparin binding.