Probing Fibroblast Growth Factor Dimerization and Role of Heparin-like Glycosaminoglycans in Modulating Dimerization and Signaling

Abstract

For a number of growth factors and cytokines, ligand dimerization is believed to be central to the formation of an active signaling complex. In the case of fibroblast growth factor-2 (FGF2) signaling, heparin/heparan sulfate-like glycosaminoglycans (HLGAGs) are involved through interaction with both FGF2 and its receptors (FGFRs) in assembling a tertiary complex and modulating FGF2 activity. Biochemical data have suggested different modes of HLGAG-induced FGF2 dimerization involving specific protein-protein contacts. In addition, several recent x-ray crystallography studies of FGF⅐FGFR and FGF⅐FGFR⅐HLGAG complexes have revealed other modes of molecular assemblage, with no FGF-FGF contacts. All these different biochemical and structural findings have clarified less and in fact raised more questions as to which mode of FGF2 dimerization, if any, is essential for signaling. In this study, we address the issue of FGF2 dimerization in signaling using a combination of biochemical, biophysical, and site-directed mutagenesis approaches. Our findings presented here provide direct evidence of FGF2 dimerization in mediating FGF2 signaling.Fibroblast growth factors (FGFs) 1 are involved in a wide range of physiological processes, including morphogenesis, as well as disease processes such as tumor angiogenesis (1-3). The FGF family consists of at least 20 members, including the well characterized acidic FGF (FGF1) and basic FGF (FGF2), both of which are potent mitogens of many cell types (4, 5). FGF signaling is mediated primarily through high affinity interactions with cell-surface FGF receptors (FGFRs), transmembrane polypeptides composed of immunoglobulin-like and tyrosine kinase domains (6, 7). FGF binding to different isoforms of FGFR is believed to trigger receptor dimerization, followed by transphosphorylation of specific tyrosine residues (8). In turn, phosphorylated tyrosine residues activate other signaling proteins, leading to cell proliferation, migration, and survival.For proper presentation to FGFR, FGF2 and other members of the FGF family interact with heparin/heparan sulfate-like glycosaminoglycans (HLGAGs). Consisting of a disaccharide repeat of glucosamine and uronic acid, HLGAGs are heterogeneous in length (10 -100 disaccharide units) and chemical composition (including differential sulfation, acetylation, and epimerization of each disaccharide unit) (9 -12). Found in the extracellular matrix and on the cell surface as part of proteoglycans, HLGAGs modulate FGF2 activity by low affinity interactions with specific FGF2-and FGFR-binding sites (13-15), facilitating FGF2 binding to FGFR. HLGAGs promote FGF2-induced activation of FGFR through a number of mechanisms, including regulating the diffusion rate of FGF2 (16, 17) and possibly dictating the specificity of FGF2-FGFR binding through interactions with both .Another hypothesis is that FGF2 binding to HLGAGs induces ligand oligomerization, which in turn induces dimerization and transphosphorylation of FGFR. Biochemical studies have demonstrated that HLGAGs...

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

“…Thus, according to our data, the strong ability of HM 8 to bind and dimerize FGF2 molecules in a cooperative fashion correlates with the higher efficacy of HM 8 relative to HM 6 to promote FGF2 signaling. Recent studies of heparin dendrimers (75) induced FGF-oligomers, synthetically polymerized FGFs (76), and covalently cross-linked FGF-dimers (77) suggest that oligomerization of FGF is required for an agonistic effect of HM on FGFR signaling. Therefore, the oligomerization of FGF in the presence of longer HM such as HM 6 and HM 8 needs to be considered in the formation of FGF⅐FGFR cell surface signaling assembly.…”

Modulation of the Heparanase-inhibiting Activity of Heparin through Selective Desulfation, Graded N-Acetylation, and Glycol Splitting

“…HS assembles ligands and receptors into ternary signaling complexes, best exemplified by the FGF-fibroblast growth factor receptor-heparin complex (62,63). Following cleavage by heparanase, the multitude of polypeptides sequestered and regulated by HS (17) become bioavailable (1,18), and this requires a tight regulation of their activity, applying, among other approaches, modified species of heparin and HS.…”

Fibroblast Growth Factor Receptors 1 and 2 Interact Differently with Heparin/Heparan Sulfate

“…However, a change in the conformation of FGF2 on binding HS is not seen in structures of co-crystals of FGF2 and heparin-derived oligosaccharides (22). Second, HS has been proposed to dimerize FGFs, thereby facilitating receptor dimerization (23,24). Finally, HS has been proposed to increase the affinity of FGF2 for FGFRs (25)(26)(27), possibly by reducing the dissociation rate constant through the formation of a ternary complex (25).…”