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The crystal structure of a dimeric 2:2:2 FGF:FGFR:heparin ternary complex at 3 A resolution has been determined. Within each 1:1 FGF:FGFR complex, heparin makes numerous contacts with both FGF and FGFR, thereby augmenting FGF-FGFR binding. Heparin also interacts with FGFR in the adjoining 1:1 FGF:FGFR complex to promote FGFR dimerization. The 6-O-sulfate group of heparin plays a pivotal role in mediating both interactions. The unexpected stoichiometry of heparin binding in the structure led us to propose a revised model for FGFR dimerization. Biochemical data in support of this model are also presented. This model provides a structural basis for FGFR activation by small molecule heparin analogs and may facilitate the design of heparin mimetics capable of modulating FGF signaling.

Heparin is required for the binding of basic fibroblast growth factor (bFGF) to high-affinity receptors on cells deficient in cell surface heparan sulfate proteoglycan. So that this heparin requirement could be evaluated in the absence of other cell surface molecules, we designed a simple assay based on a genetically engineered soluble form of murine FGF receptor 1 (mFRl) tagged with placental alkaline phosphatase. Using this assay, we showed that FGF-receptor binding has an absolute requirement for heparin. By using a cytokine-dependent lymphoid cell line engineered to express mFRl, we also showed that FGF-induced mitogenic activity is heparin dependent. Furthermore, we tested a series of small heparin oligosaccharides of defined lengths for their abilities to support bFGF-receptor binding and biologic activity. We found that a heparin oligosaccharide with as few as eight sugar residues is sufficient to support these activities. We also demonstrated that heparin facilitates FGF dimerization, a property that may be important for receptor activation.Heparin or heparan sulfate is required for basic fibroblast growth factor (bFGF) high-affinity receptor binding (38) and for bFGF-induced fibroblast growth and myoblast differentiation (29). These observations suggest that heparan sulfate proteoglycans (HSPGs) may be important regulators of bFGF biologic activity, acting directly at the level of the cell surface receptor (32). Not surprisingly, heparin and HSPGs are important regulators of cell growth (37). Depending on the tissue or cell type, heparin can either stimulate or inhibit cell proliferation (reviewed in references 14 and 31). Some of these effects may be mediated by FGF; however, other growth factors (including granulocyte-macrophage colonystimulating factor, [GM-CSF], interleukin 3 [IL-3], pleiotrophin, platelet factor 4, keratinocyte autocrine factor or amphiregulin, and heparin-binding-epidermal growth factor [EGF]) are also known to interact with heparin and may mediate some of these effects (6, 16; reviewed in reference 32). With the exception of FGF and amphiregulin, it is not known that heparin modulates growth factor activity at the level of a cell surface receptor. Nevertheless, heparin decreases the binding of amphiregulin to its receptor (6) and is required for bFGF-receptor binding (38). In addition, bFGF and the hematopoietic growth factors GM-CSF and IL-3, are biologically active when bound to HSPG (26,30,36).bFGF is known to reside in the extracellular matrix (ECM) of a wide variety of cells and tissues. This reservoir for bFGF serves to limit the diffusibility of the growth factor and thus to regulate its bioavailability. Biologically active bFGF can be displaced from the ECM by heparin or be released from the ECM by heparin-degrading enzymes (reviewed in references 14 and 20). Recently, syndecan, an HSPG, has been identified as a low-affinity binding site for bFGF (18 The observation that HSPGs serve as low-affinity binding sites for bFGF has been confirmed by comparing low-affinity FGF bi...

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