Cooperative Dimerization of Fibroblast Growth Factor 1 (FGF1) upon a Single Heparin Saccharide May Drive the Formation of 2:2:1 FGF1·FGFR2c·Heparin Ternary Complexes

Abstract

The related glycosaminoglycans heparin and heparan sulfate are essential for the activity of the fibroblast growth factor (FGF) family as they form an integral part of the signaling complex at the cell surface. Using size-exclusion chromatography we have studied the capacities of a variety of heparin oligosaccharides to bind FGF1 and FGFR2c both separately and together in ternary complexes. In the absence of heparin, FGF1 had no detectable affinity for FGFR2c. However, 2:2:1 complexes formed spontaneously in solution between FGF1, FGFR2c, and heparin octasaccharide (dp8). The dp8 sample was the shortest chain length that bound FGFR2c, that dimerized FGF1, and that promoted a strong mitogenic response to FGF1 through FGFR2c. Heparin hexasaccharide and various selectively desulfated heparin dp12s failed to bind FGFR2c and could only interact with FGF1 monomerically. These saccharides formed 1:1:1 complexes with FGF1 and FGFR2c, which had no tendency to self-associate, suggesting that binding of two FGF1 molecules to the same saccharide chain is a prerequisite for subsequent FGFR2c dimerization. We found that FGF1 dimerization upon heparin was favored over monomeric interactions even when a large excess of saccharide was present. A cooperative mechanism of FGF1 dimerization could explain how 2:2:1 signaling complexes form at the cell surface, an environment rich in heparan sulfate.The fibroblast growth factor (FGF) 2 family of proteins control the proliferation, migration, differentiation, and survival of a wide range of cell types (1). The FGFs are of fundamental importance during embryogenesis where they orchestrate the complex processes of organ formation and limb development (2). Dysregulated FGF expression is often associated with malignant transformation, because it facilitates the growth, survival, and metastatic spread of cancer cells (3). In humans 22 members of the FGF family have been described along with 4 FGF receptor tyrosine kinases (FGFR1-4). FGF signal transduction is achieved through ligand-dependent dimerization of FGFR subunits and their subsequent activation through transphosphorylation (4).The FGFRs share a number of common structural features (5). All have an intracellular split tyrosine kinase domain, a transmembrane sequence, and an extracellular region composed of three immunoglobulin (Ig)-like domains (D1-D3). FGF binding is mediated by the D2 and D3 domains, with the D3 domain having the primary influence on ligand specificity (6, 7). The D1 domain of the FGFRs does not take part in ligand interactions, and engineered receptors that lack this domain bind FGFs at least as strongly as full-length forms (8). For FGFRs1-3, alternative splicing of the D3 domain gives rise to two variants (termed IIIb and IIIc) with different ligand specificities (9). Therefore seven distinct FGF receptors can be expressed on the cell surface, each of which binds specifically to a subset of the FGFs. Significant redundancy is present within this system, because each receptor can be activated by several differe...

Inhibition or Activation of Apert Syndrome FGFR2 (S252W) Signaling by Specific Glycosaminoglycans

“…2E) is consistent with previous observations (8) but is inconsistent with the very low affinity between FGF7 and the mutant FGFR2c (S252W) (24). In fact, the high degree of GAG dependence is more consistent with the "the asymmetric model," where despite the low affinity between FGF1 and FGFR2c, high mitogenic activity was observed (47 (24). Based on affinity alone, FGF9 should be more accommodating of different GAG structures as activators.…”

“…This co-crystal was directly formed from a solution containing FGF1, FGFR2c, and heparin decasaccharide. The difference between the two co-crystals was that in the first crystal, FGF2 and FGFR1c interact and form a binary crystal prior to GAG addition, whereas in the second co-crystal, GAG facilitates FGF1 and FGFR2c interaction because FGF1 and FGFR2c do not interact with each other (47). In the co-crystal structures, proline (253), in the highly conserved linker region of FGFR, FGF (1, 2, 4, 7, 9, or 10) and GAG (heparin, HS, CS-A, CS-B, or CS-E) adopted a trans-conformation in the symmetric two ends co-crystal and a cis-conformation in the asymmetric co-crystal.…”

Glycosaminoglycans inhibit the adherence and the spreading of osteoclasts and their precursors: Role in osteoclastogenesis and bone resorption

“…Proteoglycans and GAGs contribute to the maintenance of bone mass through their involvement in collagen organization (Corsi et al, 2002). They could exert several activities on bone cells as co-factors in cell-to-cell adhesion, or to modulate the binding and activation of several growth factors or receptors such as OPG by syndecan-1 (Bernfield et al, 1999;Hildebrand et al, 1994;Robinson et al, 2005;Mosheimer et al, 2005;Standal et al, 2002). Unfortunately, the data available on the effects of GAGs on osteoclastogenesis are very limited and controversial.…”

FGF2 binding, signaling and angiogenesis are modulated by heparanase in metastatic melanoma cells

“…Structural analysis indicates that HS interacts with both FGF and FGFR in forming the high-affinity ternary complex [38,39]. However, HS requirements for binding and signaling are not equivalent [40].…”