Fibroblast growth factors 1 and 2 are distinct in oligomerization in the presence of heparin-like glycosaminoglycans

Fibroblast growth factor (FGF) 1 and FGF-2 are prototypic members of the FGF family, which to date comprises at least 18 members. Surprisingly, even though FGF-1 and FGF-2 share more than 80% sequence similarity and an identical structural fold, these two growth factors are biologically very different. FGF-1 and FGF-2 differ in their ability to bind isoforms of the FGF receptor family as well as the heparin-like glycosaminoglycan (HLGAG) component of proteoglycans on the cell surface to initiate signaling in different cell types. Herein, we provide evidence for one mechanism by which these two proteins could differ biologically. Previously, it has been noted that FGF-1 and FGF-2 can oligomerize in the presence of HLGAGs. Therefore, we investigated whether FGF-1 and FGF-2 oligomerize by the same mechanism or by a different one. Through a combination of matrix-assisted laser desorption ionization mass spectrometry and chemical crosslinking, we show here that, under identical conditions, FGF-1 and FGF-2 differ in the degree and kind of oligomerization. Furthermore, an extensive analysis of FGF-1 and FGF-2 uncomplexed and HLGAG complexed crystal structures enables us to readily explain why FGF-2 forms sequential oligomers whereas FGF-1 forms only dimers. FGF-2, which possesses an interface capable of protein association, forms a translationally related oligomer, whereas FGF-1, which does not have this interface, forms only a symmetrically related dimer. Taken together, these data show that FGF-1 and FGF-2, despite their sequence homology, differ in their mechanism of oligomerization.     

Fibroblast growth factors, their receptors and signaling

Fibroblast growth factors (FGFs) are small polypeptide growth factors, all of whom share in common certain structural characteristics, and most of whom bind heparin avidly. Many FGFs contain signal peptides for secretion and are secreted into the extracellular environment, where they can bind to the heparan-like glycosaminoglycans (HLGAGs) of the extracellular matrix (ECM). From this reservoir, FGFs may act directly on target cells, or they can be released through digestion of the ECM or the activity of a carrier protein, a secreted FGF binding protein. FGFs bind specific receptor tyrosine kinases in the context of HLGAGs and this binding induces receptor dimerization and activation, ultimately resulting in the activation of various signal transduction cascades. Some FGFs are potent angiogenic factors and most play important roles in embryonic development and wound healing. FGF signaling also appears to play a role in tumor growth and angiogenesis, and autocrine FGF signaling may be particularly important in the progression of steroid hormone-dependent cancers to a hormone-independent state.     

Heparinase treatment of bovine smooth muscle cells inhibits fibroblast growth factor-2 binding to fibroblast growth factor receptor but not FGF-2 mediated cellular proliferation

On the surface of smooth muscle cells there are two types of receptors for the mitogenic and angiogenic growth factor fibroblast growth factor-2 (FGF-2); a high affinity tyrosine kinase FGF receptor (FGFR1) and low affinity heparin./heparan-like glycosaminoglycan (HLGAG) component of surface expressed proteoglycans. It is believed that all three components; FGFR1, FGF-2, and the HLGAG chains, must form a ternary complex for maximal cellular stimulation. To carefully examine the role surface HLGAGs play in FGF-2-mediated proliferation of SMCs we have utilized HLGAG degrading enzymes heparinase I, II and III. We report that heparinase treatment of bovine smooth muscle cells inhibits the binding of ¹²⁵I-FGF-2 to FGFR1, but does not inhibit FGF-2 induced cellular proliferation. Through the use of both sodium chlorate and FGF-2 mutants with deficient HLGAG-binding capabilities, we show the FGF-2-HLGAG interaction is important for FGF-2's ability to induce SMC proliferation. Finally, we report conditioned media from heparinase treated SMCs is capable of supporting FGF-2 induced proliferation in an HLGAG-free lymphoid F32 cells, suggesting that the heparinase generated fragments are responsible for the proliferative response. The data presented here suggest FGF-2 is capable of stimulating smooth muscle cell proliferation through an FGFR independent, HLGAG dependent mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct evidence for a predominantly exolytic processive mechanism for depolymerization of heparin-like glycosaminoglycans by heparinase I

Heparinase I from Flavobacterium heparinum has important uses for elucidating the complex sequence heterogeneity of heparin-like glycosaminoglycans (HLGAGs). Understanding the biological function of HLGAGs has been impaired by the limited methods for analysis of pure or mixed oligosaccharide fragments. Here, we use methodologies involving MS and capillary electrophoresis to investigate the sequence of events during heparinase I depolymerization of HLGAGs. In an initial step, heparinase I preferentially cleaves exolytically at the nonreducing terminal linkage of the HLGAG chain, although it also cleaves internal linkages at a detectable rate. In a second step, heparinase I has a strong preference for cleaving the same substrate molecule processively, i.e., to cleave the next site toward the reducing end of the HLGAG chain. Computer simulation showed that the experimental results presented here from analysis of oligosaccharide degradation were consistent with literature data for degradation of polymeric HLGAG by heparinase I. This study presents direct evidence for a predominantly exolytic and processive mechanism of depolymerization of HLGAG by heparinase I.     

Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin III binding site

Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is DeltaU(2S)H(NS,6S)I(2S)H(NS, 6S)I(2S)H(NS,6S)IH(NAc,6S)GH(NS,3S,6S) (+/-DDD4-7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.     

Acidic and basic fibroblast growth factors are survival factors with distinctive activity in quiescent BALB/c 3T3 murine fibroblasts.

Platelet-derived growth factor (PDGF), epidermal growth factor, and insulin-like growth factor have previously been identified as survival factors with distinctive activities for the density-inhibited quiescent BALB/c 3T3 murine fibroblasts. Fibroblast growth factor (FGF), like PDGF, renders quiescent BALB/c 3T3 cells competent to respond to epidermal growth factor and insulin-like growth factor, which mediate cell-cycle traverse through G1 into S phase [Stiles, C. D., Pledger, W. J., VanWyk, J. J., Antoniades, H. N. & Scher, C. D. (1979) Proc. Natl. Acad. Sci. USA 76, 1279-1283]. We now show that FGF possess marked cell survival-enhancing activity distinctive from that of PDGF. Both acidic FGF (aFGF) and basic FGF (bFGF) markedly enhance short-term (3-hr) survival of quiescent cells. bFGF is the more active of the two factors and shows marked long-term (20-hr) survival-promoting activity alone, whereas aFGF requires heparin for long-term activity. Protection by bFGF or aFGF plus heparin is not associated with cell-cycle traverse into S phase. Both the short-term (3-hr) and long-term (20-hr) protective actions of aFGF and bFGF critically depend on protein synthesis, whereas those of PDGF do not. The accumulated evidence shows that several growth factors can contribute to maintenance of the integrity of quiescent murine fibroblasts and that their action can involve protein kinase A- and C-mediated processes as well as protein synthesis. Different growth factors display distinctive modes of action.     

The heparins and cancer

Heparin, and particularly low molecular weight heparin, is widely used for the treatment of patients with deep vein thrombosis (DVT) and the prevention of DVT that commonly accompanies malignancy. Malignant growth has also been linked experimentally to the function of heparin-like glycosaminoglycans (HLGAGs). In addition to the voluminous general literature on this subject, the heparin-cancer link has been the theme of at least three entire journal issues in recent months. These include the June 15, 2001 issue of Thrombosis Research, the November 2001 Supplement to Haemostasis, and the February 2002 issue of Seminars in Thrombosis and Hemostasis. Previous reviews have documented the historical development of this link that includes evidence from basic biochemistry and cell biology, studies in experimental animal model systems, and clinical trials. This concise review updates recent basic and clinical data on the heparin-cancer link that clarify mechanisms by which HLGAGs regulate the malignant behavior of cells. Electronic access to information that is increasing geometrically has become indispensable. Evidence for control of tumor cell growth by heparin-binding growth factors, tumor cell invasion by heparin-inhibitable enzyme systems, tumor cell metastasis by heparin-binding cell surface selectins, tumor angiogenesis, and tumor matrix formation related to deposition of fibrinogen/fibrin provides a secure theoretical foundation for systematic testing of this class of compounds in patients with cancer. HLGAGs may be a fundamental intermediate in the abnormal growth regulation characteristic of neoplasia that is susceptible to targeted intervention.     

Molecular characteristics of fibroblast growth factor-fibroblast growth factor receptor-heparin-like glycosaminoglycan complex

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF-FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF-FGFR interaction mediated by the 'conserved' primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the 'variable' secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1beta receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF-FGFR interactions. In the FGF-FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.     

Growth factors in the regulation of plasminogen-plasmin system in tumor cells.

Growth regulatory polypeptides, which act in an autocrine or paracrine fashion, are increasingly implicated in the control of pericellular proteolysis. Representatives of major growth factor families, like EGF, PDGF, IGF, FGF, IL, and TGF beta, and in addition, TNFs have effects both on cell proliferation and proteolytic events. Some of them participate in the control of proteolytic events by affecting pericellular PA activity. These factors regulate the synthesis, secretion, and activity of both PAs and their inhibitors in a cell and factor-specific manner. Interestingly, most of these affect simultaneously the secretion of both PAs and their inhibitors, sometimes concomitantly. In addition to PAs, growth factors modulate secretion of collagenases, transin, and stromelysin, and their respective inhibitors, TIMP. The balance of pericellular proteolytic activity is regulated according to the nature and interaction of various growth factors. Pericellular proteolysis can be modulated by growth factors at different levels. Several growth factors are able to regulate the amount and composition of the extracellular matrices. This, in turn, may affect the interactions of certain growth factors with the pericellular matrix structures. Altered structure of the matrix due to excessive proteolytic activity may thus limit the amount and activity of matrix-associated growth factors. Several growth factors exist in latent forms, and activation of these growth factors often requires proteolytic processing. A regulatory loop is thus formed where active growth factors affect the secretion of proteolytic enzymes and thus the concentrations of active ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth factor-specific regulation of insulin receptor substrate-1 expression in MCF-7 breast carcinoma cells: effects on the insulin-like growth factor signaling pathway

IGFs are potent mitogens that play a crucial role in cell proliferation and/or differentiation and tumorigenesis. Insulin receptor substrate-1 (IRS-1) is a key protein in the IGF signaling pathway in the estrogen-dependent MCF-7 breast carcinoma cell line. In this study, three growth factors [fibroblast growth factor (FGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF)] were tested for their ability to modulate IRS-1 protein expression and the IGF-I signaling pathway. FGF and, to a lesser extent, EGF were found to increase IRS-1 protein, whereas PDGF had no effect. This indicates that growth factors can specifically modulate IRS-1 protein content. The increases provoked by EGF and FGF were dependent on the MAPK signaling pathway but independent of phosphatidylinositol 3-kinase (PI 3-kinase) signaling and required de novo protein synthesis. We noted that the kinetics of MAPK activation was continuous in response to FGF but transient in response to EGF. In addition, transfection of cells with a constitutively active form of MAPK kinase, which results in continuous MAPK activity, increased IRS-1 expression. Taken together, these results suggest that stimulation of IRS-1 expression was therefore stronger when MAPK activity was sustained. Pretreatment of cells with EGF, FGF, or PDGF for 24 h reduced IGF-I-induced tyrosine phosphorylation per molecule of IRS-1. However, IGF-I-induced PI 3-kinase activity was decreased by 24 h of pretreatment with EGF or PDGF but not with FGF. Our results therefore demonstrate that different growth factors are capable of specifically modulating the IGF-I signaling via IRS-1. They further suggest that the FGF-induced increase in IRS-1 counterbalances the inhibition of IRS-1 tyrosine phosphorylation to allow normal stimulation of IGF-I-induced PI 3-kinase activity.     

Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria

Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity.     

Analysis of the biochemical mechanisms for the endocrine actions of fibroblast growth factor-23

Fibroblast growth factor (FGF)-23 has emerged as an endocrine regulator of phosphate and of vitamin D metabolism. It is produced in bone and, unlike other FGFs, circulates in the bloodstream to ultimately regulate phosphate handling and vitamin D production in the kidney. Presently, it is unknown which of the seven principal FGF receptors (FGFRs) transmits FGF23 biological activity. Furthermore, the molecular basis for the endocrine mode of FGF23 action is unclear. Herein, we performed surface plasmon resonance and mitogenesis experiments to comprehensively characterize receptor binding specificity. Our data demonstrate that FGF23 binds and activates the c splice isoforms of FGFR1-3, as well as FGFR4, but not the b splice isoforms of FGFR1-3. Interestingly, highly sulfated and longer glycosaminoglycan (GAG) species were capable of promoting FGF23 mitogenic activity. We also show that FGF23 induces tyrosine phosphorylation and inhibits sodium-phosphate cotransporter Npt2a mRNA expression using opossum kidney cells, a model kidney proximal tubule cell line. Removal of cell surface GAGs abolishes the effects of FGF23, and exogenous highly sulfated GAG is capable of restoring FGF23 activity, suggesting that proximal tubule cells naturally express GAGs that are permissive for FGF23 action. We propose that FGF23 signals through multiple FGFRs and that the unique endocrine actions of FGF23 involve escape from FGF23-producing cells and circulation to the kidney, where highly sulfated GAGs most likely act as cofactors for FGF23 activity. Our biochemical findings provide important insights into the molecular mechanisms by which dysregulated FGF23 signaling leads to disorders of hyper- and hypophosphatemia.     

Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3

The kinase domain of human epidermal growth factor receptor (HER) 3/ErbB3, a member of the EGF receptor (EGFR) family, lacks several residues that are critical for catalysis. Because catalytic activity in EGFR family members is switched on by an allosteric interaction between kinase domains in an asymmetric kinase domain dimer, HER3 might be specialized to serve as an activator of other EGFR family members. We have determined the crystal structure of the HER3 kinase domain and show that it appears to be locked into an inactive conformation that resembles that of EGFR and HER4. Although the crystal structure shows that the HER3 kinase domain binds ATP, we confirm that it is catalytically inactive but can serve as an activator of the EGFR kinase domain. The HER3 kinase domain forms a dimer in the crystal, mediated by hydrophobic contacts between the N-terminal lobes of the kinase domains. This N-lobe dimer closely resembles a dimer formed by inactive HER4 kinase domains in crystal structures determined previously, and molecular dynamics simulations suggest that the HER3 and HER4 N-lobe dimers are stable. The kinase domains of HER3 and HER4 form similar chains in their respective crystal lattices, in which N-lobe dimers are linked together by reciprocal exchange of C-terminal tails. The conservation of this tiling pattern in HER3 and HER4, which is the closest evolutionary homolog of HER3, might represent a general mechanism by which this branch of the HER receptors restricts ligand-independent formation of active heterodimers with other members of the EGFR family.     

The neuronal cell-surface molecule mitogenic for Schwann cells is a heparin-binding protein.

The cell surface of embryonic peripheral neurons provides a mitogenic stimulus for Schwann cells. We report (i) the solubilization of this mitogenic activity from rat dorsal root ganglion neurons grown in tissue culture and (ii) the solubilization and partial purification of mitogenic activity from neonatal rat brains. Extracted mitogenic activity is peripheral rather than intrinsic to the membrane, stable after extraction, and active as a mitogen in the absence of serum (the most stringent criterion defining the neuronal mitogen). We have previously provided evidence suggesting that a neuronal cell-surface heparan sulfate proteoglycan is required for expression of the neurons' mitogenic activity. We now show that mitogenic activity can be extracted from the membrane dissociated from proteoglycan as assayed by its ability to bind to immobilized heparin. After dissociation, low concentrations of heparin (1 micrograms/ml) inhibit the ability of the mitogen to stimulate Schwann cell division. Basic fibroblast growth factor (FGF) is weakly mitogenic for Schwann cells, but it is not present in mitogenic brain extracts (based on immunoblotting). Immunodepletion experiments with specific antibodies to FGF indicate that the mitogenic activity extracted from neurons is not a form of this heparin-binding mitogen. Acidic FGF is not mitogenic for Schwann cells and is not present in mitogenic brain extracts. We suggest that these and previous data indicate the neurite mitogen is a proteoglycan-growth factor complex that limits mitogenic activity to the axonal surface, protects mitogen against inactivation by other proteoglycans, and provides for effective presentation of mitogen to the Schwann cell.     

Structural views of the ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and both glutamate and Gd3+

Crystal structures of the extracellular ligand-binding region of the metabotropic glutamate receptor, complexed with an antagonist, (S)-(alpha)-methyl-4-carboxyphenylglycine, and with both glutamate and Gd3+ ion, have been determined by x-ray crystallographic analyses. The structure of the complex with the antagonist is similar to that of the unliganded resting dimer. The antagonist wedges the protomer to maintain an inactive open form. The glutamate/Gd3+ complex is an exact 2-fold symmetric dimer, where each bi-lobed protomer adopts the closed conformation. The surface of the C-terminal domain contains an acidic patch, whose negative charges are alleviated by the metal cation to stabilize the active dimeric structure. The structural comparison between the active and resting dimers suggests that glutamate binding tends to induce domain closing and a small shift of a helix in the dimer interface. Furthermore, an interprotomer contact including the acidic patch inhibited dimer formation by the two open protomers in the active state. These findings provide a structural basis to describe the link between ligand binding and the dimer interface.     

Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor.

Expression cloning of cDNAs encoding a basic fibroblast growth factor (FGF) binding protein confirms previous hypotheses that this molecule is a cell-surface heparan sulfate proteoglycan. A cDNA library constructed from a hamster kidney cell line rich in FGF receptor activity was transfected into a human lymphoblastoid cell line. Clones expressing functional basic FGF binding proteins at their surfaces were enriched by panning on plastic dishes coated with human basic FGF. The amino acid sequence deduced from the isolated cDNAs revealed several interesting features, including hydrophobic signal and transmembrane domains that flank an extracellular region containing six potential attachment sites for glycosaminoglycan side chains. The structure also contains a short hydrophilic cytoplasmic tail sequence homologous to previously reported actin binding domains. Binding of basic FGF to cells expressing the binding protein could be inhibited by heparin and heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. In addition to binding basic FGF, this protein or related surface proteins may function as an initial cellular attachment site for other growth factors and for viruses, such as herpes simplex virus.     

Escherichia coli ribosomal protein L7/L12 dimers remain fully active after interchain crosslinking of the C-terminal domains in two orientations.

Cysteine site-directed mutagenesis was used to create variants of Escherichia coli ribosomal protein L7/L12 that have single cysteine substitutions, at residues 63 or 89, located in different exposed loops in the structure of the globular C-terminal domain indicated by the crystallographic structure. That structure shows a possible dimer interaction in which the two sites of cysteine substitution appear to be too distant for disulfide bond formation. After mild oxidation in solution both of the overexpressed purified cysteine-substituted proteins formed interchain disulfide crosslinked dimers in high yield. Both crosslinked dimers were fully active in restoring activity in poly(U)-directed polyphenylalanine synthesis to ribosomal core particles depleted of wild-type L7/L12. These results show that the two C-terminal domains have independent mobility. The activity of dimeric L7/L12 does not require the independent movement of the two globular C-terminal domains in an L7/L12 dimer; moreover, it appears independent of their mutual orientation when joined by crosslinking at the two loops. A third variant with a cysteine substitution at residue 33 near the junction between the alpha-helical N-terminal domain and the flexible hinge was prepared and tested. This protein was active in the protein synthesis assay in the reduced state. Oxidation produced the interchain crosslinked dimer in high yield, but this crosslinked dimer was inactive in polyphenylalanine synthesis. The inactivation was due to the inability of the Cys33-Cys33 oxidized dimer to bind to the core particle.