We investigated the mechanism by which heparin enhances the binding of vascular endothelial growth factor (VEGF) to the extracellular matrix protein fibronectin. In contrast to other systems, where heparin acts as a protein scaffold, we found that heparin functions catalytically to modulate VEGF binding site availability on fibronectin. By measuring the binding of VEGF and heparin to surface-immobilized fibronectin, we show that substoichiometric amounts of heparin exposed cryptic VEGF binding sites within fibronectin that remain available after heparin removal. Measurement of association and dissociation kinetics for heparin binding to fibronectin indicated that the interaction is rapid and transient. We localized the heparin-responsive element to the C-terminal 40-kDa Hep2 domain of fibronectin. A mathematical model of this catalytic process was constructed that supports a mechanism whereby the heparininduced conformational change in fibronectin is accompanied by release of heparin. Experiments with endothelial extracellular matrix suggest that this process may also occur within biological matrices. These results indicate a novel mechanism whereby heparin catalyzes the conversion of fibronectin to an open conformation by transiently interacting with fibronectin and progressively hopping from molecule to molecule.
Abstract-Fibroblast growth factor-2 (FGF2) activates the extracellular signal-regulated kinases 1 and 2 (ERK1/2) through its specific receptors. Interaction of FGF2 with cell-surface heparan sulfate proteoglycans has also been suggested to induce intracellular signals. Thus, we investigated whether FGF2 can stimulate ERK1/2 activation through heparan sulfate proteoglycans using mechanisms that do not depend on receptor activation in vascular smooth muscle cells. FGF2 also binds to heparan sulfate proteoglycans (HSPGs), complex molecules composed of a core protein and covalently attached heparan sulfate chains. 4 HSPGs are found in the extracellular matrix and on cell surfaces of most tissues, where they serve many functions. 4,5 In the vascular system, HSPG and heparin have been demonstrated to inhibit vascular smooth muscle growth in vitro and in vivo. 6 -8 Although one mechanism suggests that heparin and heparan sulfate can sequester growth factors, such as FGF2, to prevent cell stimulation, 9 others studies have shown that HSPGs are necessary for the maximal mitogenic activity of FGF2. 2,4,10 Indeed, when agents were delivered to degrade or decrease HSPG, both enhanced and inhibited smooth muscle cell hyperplasia was observed. 11-14 Thus, it is clear that HSPGs play important roles in the blood vessel wall, yet the underlying mechanisms used by HSPG remain poorly understood.Cell-surface HSPGs have been demonstrated to modulate FGF2 activity by functioning as coreceptors to enhance FGF2 binding to FGFRs. 2,10 However, recent studies suggest that the role of HSPG in the FGF2 system might be more complex. Indeed, FGF2 might signal directly via cell-surface HSPG by inducing aggregation of syndecan-4 and activation of protein kinase C␣. 15,16 Thus, a more complete understanding of how HSPGs modulate the vascular response to FGF2 is required before approaches can be designed to treat vascular disease by targeting HSPGs.The syndecans are a widely distributed 4-member family of transmembrane proteins carrying both heparan and chondroitin sulfate chains. 4 Although there are significant differences within their ectodomains, the four syndecans share a highly conserved cytoplasmic tail, suggesting important functions. 4 Indeed, Horowitz and Simons 16,17 showed that the cytoplasmic tail of syndecan-4 is phosphorylated on Ser 183 and that FGF2 can cause a 2-to 3-fold reduction in phosphorylation, inducing multimerization and activation of protein kinase C␣. FGF2-mediated signaling via syndecan-4
Receptor-ligand binding is a critical first step in signal transduction and the duration of the interaction can impact signal generation. In mammalian cells, clustering of receptors may be facilitated by heterogeneous zones of lipids, known as lipid rafts. In vitro experiments show that disruption of rafts significantly alters the dissociation of fibroblast growth factor-2 (FGF-2) from heparan sulfate proteoglycans (HSPGs), co-receptors for FGF-2. In this article, we develop a continuum stochastic formalism to address how receptor clustering might influence ligand rebinding. We find that clusters reduce the effective dissociation rate dramatically when the clusters are dense and the overall surface density of receptors is low. The effect is much less pronounced in the case of high receptor density and shows nonmonotonic behavior with time. These predictions are verified via lattice Monte Carlo simulations. Comparison with FGF-2-HSPG experimental results is made and suggests that the theory could be used to analyze similar biological systems. We further present an analysis of an additional cooperative internal-diffusion model that might be used by other systems to increase ligand retention when simple rebinding is insufficient.
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