Elsevier

Biochimie

Volume 127, August 2016, Pages 214-226
Biochimie

Review
Paradigms in the structural biology of the mitogenic ternary complex FGF:FGFR:heparin

https://doi.org/10.1016/j.biochi.2016.05.017Get rights and content

Highlights

  • Two ternary complex models for FGF:FGFR:heparin have been proposed.

  • The symmetric 2:2:2 and the asymmetric 2:2:1 models.

  • Data concerning the ligand binding motifs to FGF 1 and 2 are divergent.

  • Polarization of sulfation on the saccharide chains is relevant to mitogenesis.

  • Both symmetric and asymmetric models can occur and are mitogenic active.

Abstract

The main achievements regarding the molecular interaction involving fibroblast growth factors (FGFs), canonical receptors (FGFRs) and the glycosaminoglycans (GAGs) heparan sulfate (HS)/heparin (Hp) are overviewed. Despite the recent works concerning the subject, conflicting paradigms in the structural biology of the resultant ternary complex FGF:FGFR:HS/Hp seem to persist up to these days. The principal dilemma, centered on the functional intermolecular complex of mitogenesis and angiogenesis, has been lasting for approximately a decade and a half since the publications of the two contradicting crystal structures, the asymmetric 2:2:1 versus the symmetric 2:2:2 complex model. When the principal results regarding this ternary complex are analyzed as a whole and through an impartial manner, conclusion heavily and reliably supports the existence and activity of both complex models. Selection of each complex is driven by multiple factors of different degrees of impact. Specificity in protein-binding motifs in ligands (although the minimal binding sequences are yet controversial), slight differences on the structure of the GAG-binding sites of FGF and of FGFR isoforms as well as on the possible ligand-induced conformational changes of FGFR are examples of these factors. Here, the structural biology of the mitogenic FGF:FGFR:HS/Hp ternary complex is revisited. Discussion is focused on the major attributes of this intermolecular complex including the existing conflicts about the righter biologically active model and information regarding ligand structure, conformation and minimal length required for binding to the growth factors and receptors. This review is very timely in light of the 100(th) anniversary of the discovery of Hp.

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

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