ReviewParadigms in the structural biology of the mitogenic ternary complex FGF:FGFR:heparin
Graphical abstract
Introduction
The activity of chemotactic and growth-stimulating factors like fibroblast growth factors (FGFs), mostly the isoforms FGF1 and basic FGF (bFGF or FGF2), is essential to cell proliferation processes in numerous biological events like mitogenesis and angiogenesis [1], [2]. These growth factors have to interact and be activated by an active ternary complex made up with the canonical receptors (fibroblast growth factor receptors, FGFRs) and the glycosaminoglycans (GAGs) on endothelial surface proteoglycans in order to trigger cell division and further neovascularization process [3], [4], [5], [6], [7], [8]. Assuming that these growth factors are circulating unattached to other molecules, after proper cell biosynthesis, release, and tight attachment on surface proteoglycan GAGs, their signaling actions are triggered. In addition, GAG interaction is highly required for stabilizing the FGF-FGFR complex by counter-balancing the surface charges of such proteins. This interaction, besides making the resultant intermolecular complex with active biological function, will also limit the activity of the growth factors to only certain sites in which cell proliferation and/or neovascularization have to occur. Examples of these situations in which these biological events are necessary are the healing process after tissue wounds or injury and cancer cell growth and metastasis during tumor development. In the former situation, the actions of the growth factors are commonly seen in the cell differentiation event named mesenchymal-epithelial transition, a process which permits the formation of new endothelial cells from angioblasts, and these in turn form mesodermal cells [9]. Besides assisting the cell differentiation processes, FGFs are also key players in the molecular networks involved in formation of novel vessels [10] as seen in the later situations concerning cancer progress. Angiogenesis is a pivotal process to cancer growth (of the primary tumor) and metastasis [11], [12]. Formation of the new vessels intended to feed the tumor cell growth and this process is needed for the evolution of this severe and devastating disease [11], [12].
Among the existence of various GAG types, chondroitin sulfate and heparan sulfate (HS) are the principal ones physiologically involved in interactions and controlling mechanisms of GAG-binding proteins. Among these two GAG types, HS seems the preferable one biologically responsible for binding and regulating the activities of the different FGF isoforms [13], [14], [15], [16], [17], [18]. In fact, FGF binding is accomplished by a very select number of sequences (protein-binding motifs) within the HS backbone [19], [20]. Conversely, preparation for biophysical studies of HS-derived oligosaccharides with biological action is usually challenging and laborious. Heparin (Hp), as the sister GAG molecule of HS (Fig. 1A), and derivatives are generally more manageable, especially in terms of quantities. Thus Hp is widely used as the principal model GAG in studies concerning both structure and function of the various GAG-binding proteins, including the studies involving FGFs [21]. This preference results primarily from the fact that heparin can be found commercially available at large amounts and at reasonable cost. Hence, several studies published so far concerning the biological and structural properties of the interactions involving FGFs, FGFRs and GAGs have employed Hp and/or its derivatives as the preferable ligand.
Here, the main important achievements regarding the science of the mitogenic ternary complex FGF-FGFR-Hp are chronologically, systematically and critically described. For clarity, this presentation is divided in three major sections. The first one focuses on structure of the Hp derivatives investigated and used as functional ligands to the complex. In this section, discussion covers ligand structures derived either from natural or synthetic sources, the contradiction of publications describing the minimal binding motifs necessary for interaction, the influencing conformational properties of the composing monosaccharide iduronic acid (IdoA) in ligands as well as of the overall conformations of ligand chains upon attachment to FGFs and/or FGFRs, and finally the specific sulfation patterns and consequential 3D structures necessary for binding. The second section aims more at the biological consequences of the GAG ligands on FGF isoforms (FGF1 and bFGF) and FGFR isoforms (FGFR1 and FGFR2). In this section, information about the controlling and regulatory mechanisms of action of the various Hp ligands onto the growth factors and their receptors are revisited. Mechanism for FGF inhibition is also described in this second section. In the third section, discussion centers primarily on the contradicting paradigms persisting in the structural biology of the biologically active FGF-FGFR-Hp complex models. Discussion aims primarily at comparing the characteristics of the two FGF-FGFR-Hp complex models (the asymmetric model with 2:2:1 molecular ratio and the symmetric model with 2:2:2 molecular ratio) derived from the crystallographic studies undertaken roughly 15 years ago, and at lower extension on the different oligomerization levels of the growth factors and receptors required for triggering the signaling activities in mitogenesis and angiogenesis. This report is ended by a conclusion section dedicated to prove that both complex exist and are active in the body. A fresh publication covering the biological contribution of Hp in modulating the activity of FGFs, like this review article, is very timely in the literature considering the current celebration of one-century for the discovery of Hp.
Section snippets
Structural background
Hp and HS share basically the same precursor disaccharide composed of alternating glucosamine (GlcN) and glucuronic acid (GlcA) to build-up their backbones. This precursor is named heparosan and is structurally composed of the following unit [→4)-β-D-GlcA-(1 → 4)-α-D-GlcNAc-(1→] in which N-acetyl GlcN (GlcNAc) occurs significantly throughout the chain. The principal difference between HS and Hp in terms of structure is the different degrees of chain modification occurred on a polymer made up of
Structural controlling mechanisms
The crystal structure of Hp-derived tetrasaccharide and hexasaccharide with bFGF generated in the publication of Faham and coworkers have indicated the contributing amino acids of bFGF involved in binding with Hp ligands [3]. In the binding with the tetrasaccharide, asparagine-28, arginine-121, lysine-126 and glutamine-135 were observed as part of a single binding site within the surface of bFGF. Besides this binding site mapped in the work, the hexasaccharide was also able to interact with
Revisiting the asymmetric 2:2:1 complex model and related attributes
Dr. Pellegrini, co-authors and Profs. Mulloy and Blundell have shown through a publication in Nature in 2000 the resultant crystal structure of the complex made between FGF1, FGFR2 ectodomain and an Hp decasaccharide [4] (Fig. 4A). From the crystal structure the authors were able to undertake in-depth analyses on several aspects involved in the FGF:FGFR:Hp ternary complex including the mechanisms required for the proper molecular assembly (Fig. 5A–D). Based on this study, it has been postulated
Concluding remarks: evidences strongly support the existence and activity of both complex models
In FGF-dependent cell proliferation events seen commonly in mitogenesis and angiogenesis, a ternary complex involving FGF, its canonical receptor and the GAG HS, usually investigated by its sister molecule Hp, is required. The structural biology regarding the assembly of this ternary complex as well as some related attributes and controlling factors have been reviewed in this report. Across the revision, three major conflicting areas seem to exist in the structural biology of the ternary
Acknowledgments
VHP is grateful to the financial supports provided from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), to Prof. Barbara Mulloy for the brief discussion regarding heparin and the two distinct complexes with FGFs/FGFRs, and to Dr. Paulo A.G Soares for his assistance with Fig. 6 of the manuscript. VHP apologizes the authors whose publications regarding the molecular mechanisms involving FGF, FGFR and GAGs
References (55)
- et al.
Function of fibroblast growth factors and vascular endothelial growth factors and their receptors in angiogenesis
Crit. Rev. Oncol. Hematol.
(2000) - et al.
Crystal structure of a ternary FGF-FGFR-Heparin complex reveals a dual role for heparin in FGFR binding and dimerization
Mol. Cell
(2000) - et al.
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
J. Biol. Chem.
(2005) - et al.
Understanding the biology of angiogenesis: review of the most important molecular mechanisms
Blood Cells Mol. Dis.
(2007) - et al.
Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor
J. Biol. Chem.
(1993) - et al.
A protein canyon in the FGF-FGF receptor dimer selects from an à la carte menu of heparan sulfate motifs
Curr. Opin. Struct. Biol.
(2005) - et al.
Current structural biology of the heparin interactome
Curr. Opin. Struct. Biol.
(2015) - et al.
Heparin and heparan sulfate: biosynthesis, structure and function
Curr. Opin. Chem. Biol.
(2000) - et al.
Requirement for anticoagulant heparan sulfate in the fibroblast growth factor receptor complex
J. Biol. Chem.
(1999) - et al.
Minimal heparin/heparan sulfate sequences for binding to fibroblast growth factor-1
Biochem. Biophys. Res. Commun.
(2002)