Evidence That Heparin Saccharides Promote FGF2 Mitogenesis through Two Distinct Mechanisms

Abstract

Heparin-like saccharides play an essential role in binding to both fibroblast growth factors (FGF) and their receptors at the cell surface. In this study we prepared a series of heparin oligosaccharides according to their size and sulfation level. We then investigated their affinity for FGF2 and their ability to support FGF2 mitogenesis of heparan sulfate-deficient cells expressing FGFR1c. Tetra-and hexasaccharides bound FGF2, but failed to dimerize the growth factor. Nevertheless, these saccharides promoted FGF2-mediated cell growth. Furthermore, whereas enzymatic removal of the non-reducing end 2-O-sulfate group had little effect on the 1:1 interaction with FGF2, it eliminated the mitogenic activity of these saccharides. This evidence supports the symmetric two-end model of ternary complex formation. In contrast, even at very low concentrations, octasaccharide and larger heparin fragments conferred a potent mitogenic activity that was independent of terminal 2-O-sulfation. This correlated with the ability to dimerize FGF2 in an apparently cooperative manner. This data suggests that potent mitogenic signaling results from heparin-mediated trans-dimerization of FGF2, consistent with the asymmetric model of ternary complex formation. We propose that, depending on saccharide structure, there are different architectures and modes of ternary complex assembly that differ in stability and/or efficiency of transmembrane signaling.FGF1 and FGF2 are the prototypic members of the fibroblast growth factor (FGF) 3 family. They can induce proliferation, differentiation, motility, adhesion, survival, and apoptosis of cells at both embryonic and adult stages (1, 2). A wide variety of cells express FGF1 and FGF2, for example, vascular smooth muscle cells, neurons, epithelial cells, and fibroblasts (3). FGF2 can also be released by macrophages in response to inflammation (4) or by platelets in response to vessel injury (5). FGF1 and FGF2 are potent angiogenic factors (6). Angiogenesis, the formation of new blood vessels, is important in human disease; it is induced by malignant tumors, and is a cause of arthritis and visual impairment in diabetic retinopathies. Tumors secrete pro-angiogenic molecules, such as the FGFs, in an autocrine or paracrine manner, and increased FGF levels are associated with poor prognosis for many cancers (2, 7).Heparan sulfate (HS) proteoglycans are membrane-associated co-receptors that coordinate the interaction of FGFs with their high-affinity tyrosine kinase receptors, the FGFRs (8). These high-affinity receptors are the products of distinct mammalian genes: FGFR1 (flg), FGFR2 (bek), FGFR3, and FGFR4. FGFR1-3 can be alternatively spliced to produce a, b, and c isoforms. The a isoform is secreted and may function as a soluble receptor antagonist (9). The b and c receptor variants are particularly important as they display different ligand binding specificities and affinities (10). For example, FGFR2c was reported to bind FGF1 and FGF2 with equal affinity, whereas FGFR2b bound FGF2 with a thousand-fol...

Extended N-Sulfated Domains Reside at the Nonreducing End of Heparan Sulfate Chains

“…The authors describe a 2:2:1 complex that is not limited to the NRE of HS chains. Evidence exists for both the Schlessinger and Pellegrini complexes (40,41), and it has recently been proposed that both complexes may be physiologically relevant, depending on the HS structures present (42).…”

Syndecan-4 proteoliposomes enhance fibroblast growth factor-2 (FGF-2)–induced proliferation, migration, and neovascularization of ischemic muscle

“…A major mechanistic theory of heparin's activity in FGF signaling is its ability to stabilize the ternary complex of dimerized FGF-FGFR pairs (4). In addition, recent studies suggest that heparin may increase FGF signaling through mechanisms in addition to receptor complex stabilization (24). Cell-surface heparan sulfates are also effective in facilitating FGF-2 signaling and can stabilize the ternary FGF-signaling complex (4).…”

Novel Mechanisms of Fibroblast Growth Factor Receptor 1 Regulation by Extracellular Matrix Protein Anosmin-1

“…In another scenario, HS first induces oligomerization of FGF molecules and subsequently enables FGFR dimerization, as proposed in the 2:2:1 FGF1⅐FGFR2c⅐heparin ternary complex model (9). These two binary FGF⅐FGFR and FGF⅐HS complexes may co-exist as the driving force for ternary signaling complex assembly, which can be regulated by the different length and sulfation patterns of heparin⅐HS saccharides (11). Here we have shown that heparinbound anosmin-1 facilitates FGF2⅐FGFR1 as the preferred fundamental binary complex, resulting in anosmin-1⅐FGF2⅐ FGFR1⅐heparin complex formation.…”

“…Two crystal structures have been proposed to demonstrate how the FGF⅐FGFR⅐heparin complex is assembled (9,10). Recent evidence suggests that both may be biologically relevant (11,12).…”

“…Two crystal structures have been proposed to demonstrate how the FGF⅐FGFR⅐heparin complex is assembled (9, 10). Recent evidence suggests that both may be biologically relevant (11,12).The diversity of FGF signaling pathways and consequent biological functions require that activation of FGFR should be tightly regulated. Such regulation can occur either at the level of the extracellular receptor-ligand complex assembly or via intracellular modulation of downstream effectors (13).…”