| Heparan sulfate proteoglycans (HSPG) interact with many heparin-binding growth factors including fibroblast growth factor-2 (FGF-2) and heparin-binding EGF-like growth factor (HB-EGF). The purpose of this study was to identify mechanisms by which HSPG modulate growth factor bioavailability in the extracellular matrix and on the cell surface. Transport of FGF-2 and HB-EGF through a proteoglycan-rich extracellular matrix was enhanced (10-fold, FGF-2; 2-fold, HB-EGF) in the presence of a diffusible heparin analog, sucrose octasulfate. Thus, interactions of growth factors with heparan sulfate can modulate the dynamics of extracellular transport based on the specific binding characteristics of the growth factor. To determine the extent to which growth factor binding to HSPG involves recognition of specific heparan sulfate structures versus general non-specific ionic associations, intercompetition binding studies were conducted with FGF-2, HB-EGF and EGF. FGF-2 and HB-EGF partially compete with one another however, each showed selective high affinity binding to a subclass of specific sites indicating that structurally diverse protein binding motifs exist within heparan sulfate.; In addition to the affinity and selectivity of growth factor binding sites, the ability of HSPG to localize to specific domains on the cell surface might also play a role in modulating growth factor action. Specifically, a novel mechanism involving the association of HSPG with lipid rafts was identified. Lipid rafts were disrupted with sterol-binding agents, filipin and methyl-β-cyclodextrin, and the effects of growth factor binding evaluated. The sterol-binding agents reduced binding of FGF-2 and HB-EGF to cell surface HSPG by altering the observed binding kinetics. In particular, the rate of FGF-2 dissociation from HSPG on vascular smooth muscle cells increased dramatically upon lipid raft disruption. Altered growth factor binding was associated with decreased localization of HSPG within cold detergent-insoluble rafts in cells treated with the sterol-binding agents. These results suggest that localization of HSPG within lipid rafts creates high local concentrations of binding sites such that growth factor binding is enhanced. Thus, HSPG and lipid rafts might represent key sites of regulation for control of vascular cell response to growth factors. |
