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.

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.

Rebinding of dissociated ligands from cell surface proteins can confound quantitative measurements of dissociation rates important for characterizing the affinity of binding interactions. This can be true also for in vitro techniques such as surface plasmon resonance (SPR). We present experimental results using SPR for the interaction of insulin-like growth factor-I (IGF-I) with one of its binding proteins, IGF binding protein-3 (IGFBP-3), and show that the dissociation, even with the addition of soluble heparin in the dissociation phase, does not exhibit the expected exponential decay characteristic of a 1:1 binding reaction. We thus consider the effect of (multiple) rebinding events and, within a self-consistent mean-field approximation, we derive the complete mathematical form for the fraction of bound ligands as a function of time. We show that, except for very low association rate and surface coverage, this function is non-exponential at all times, indicating that multiple rebinding events strongly influence dissociation even at early times. We compare the mean-field results with numerical simulations and find good agreement, although deviations are measurable in certain cases. Our analysis of the IGF-I-IGFBP-3 data indicates that rebinding is prominent for this system and that the theoretical predictions fit the experimental data well. Our results provide a means for analyzing SPR biosensor data where rebinding is problematic and a methodology to do so is presented.1 Present Address: Max Planck Institut für Physik komplexer Systeme, Nöthnitzer Stra e 38, 01187 Dresden, Germany. (i) IntroductionSignal transduction via transmembrane receptor proteins is initiated by extracellular binding with specific proteins known as growth factors. These interactions tend to be of high affinity and, in many systems, are regulated by binding proteins present in the extracellular environment. Insulin-like growth factor-I (IGF-I) constitutes one prominent example of such a growth factor. Cell signaling is transmitted by direct interaction with the IGF-I receptor but this binding can be impacted by solution and cell-associated IGF binding proteins (IGFBPs), of which there are at least six. Quantification of the interactions of IGF-I with IGFBPs is critical if one is to understand how changes in expression and secretion will impact IGF-I signaling. Surface plasmon resonance (SPR) is one technique amenable to such measurements. SPR is an optical sensor technique that has the advantage of being able to take real-time measurements using low concentrations of unlabeled biologicals [reviewed in Cooper 2003].Quantification of IGF-I interactions with both cell surface receptors and IGFBPs using SPR has been performed as a means of evaluating and predicting the competition between these molecules for IGF-I. Studies have used immobilized IGF-I [Wong et al. 1999;Dubaquie and Lowman 1999;Galanis et al. 2001;Fong et al. 2002; Vorwerk et al. 2002], IGF-I receptor [Jansson et al. 1997], or IGFBPs [Heding et al. 1996Jansson et al. ...