FGF-21/FGF-21 receptor interaction and activation is determined by betaKlotho

Fibroblast growth factor-21 (FGF-21) is a metabolic regulator that can influence glucose and lipid control in diabetic rodents and primates. We demonstrate that betaKlotho is an integral part of an activated FGF-21-betaKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor. Cells lacking betaKlotho did not respond to FGF-21; the introduction of betaKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells. Interestingly, FGF-21-mediated effects are heparin independent suggesting that betaKlotho plays a role in FGF-21 activity similar to the one played by heparin in the signaling of conventional FGFs. Moreover, in addition to conferring specificity for FGF-21, betaKlotho appears to support FGF-19 activity and mediates the receptor selectivity profile of FGF-19. All together, these results indicate that betaKlotho and FGFRs form the cognate FGF-21 receptor complex, mediating FGF-21 cellular specificity and physiological effects.     

The kinetics of FGF-2 binding to heparan sulfate proteoglycans and MAP kinase signaling

Binding of growth factors to specific cell surface receptors is the first step in initiating cell signaling cascades that ultimately result in diverse activities such as proliferation, differentiation, and apoptosis. Dimerization and phosphorylation of tyrosine kinase transmembrane receptors is the typical paradigm for this activation but, for many growth factors, cell surface interactions are not limited to a single receptor type. In particular, heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2), bind to heparan sulfate proteoglycans (HSPG) on the cell surface and within the extracellular matrix (ECM), and these molecules have been viewed as accessory co-receptors serving to facilitate tyrosine kinase receptor binding. Recent studies, however, have indicated that HSPG can directly participate in signal transduction in response to FGF-2 binding. Thus, in the present study, we used mathematical modeling to examine whether the kinetics of formation of the various FGF-2 bound complexes on the cell surface correlate with the activation of the downstream mediators of FGF-2 response, Erk1/2. We find that FGF-2 binding to its receptor correlates well with Erk1/2 activation and that HSPG can modulate this response through its ability to stabilize these ligand receptor complexes. Moreover, we also observed that FGF-2 binding to HSPG correlates strongly with Erk1/2 activation under conditions where there is a loss of receptor activity, and we demonstrate that the relative amounts of signaling and non-signaling HSPG on the cell surface, as well as the presence of competing HSPG in the ECM, can impact the signal potential via this pathway. Thus, the selective regulation of specific HSPG might provide a mechanism for fine tuned modulation of heparin-binding growth factor signaling in cells where signal intensity and duration could direct cellular response toward growth, migration or differentiation.     

Ligand-induced coupling versus receptor pre-association: cellular automaton simulations of FGF-2 binding

The binding of basic fibroblast growth factor (FGF-2) to its cell surface receptor (CSR) and subsequent signal transduction is known to be enhanced by heparan sulfate proteoglycans (HSPGs). HSPGs bind FGF-2 with low affinity and likely impact CSR-mediated signaling via stabilization of FGF-2-CSR complexes via association with both the ligand and the receptor. What is unknown is whether HSPG associates with CSR in the absence of FGF-2. In this paper, we determine conditions by which pre-association would impact CSR-FGF-2-HSPG triad formation assuming diffusion-limited surface reactions. Using mean-field rate equations, we show that (i) when [HSPG] is much higher than [CSR], the presence of pre-formed complexes does not affect the steady state of FGF-2 binding, and (ii) when the concentrations are comparable, the presence of pre-formed complexes substantially increases the steady-state concentration of FGF-2 bound to CSR. These findings are supported by explicit cellular automaton simulations, which justify the mean-field treatment. We discuss the advantages of such a two-receptor system compared to a single-receptor model, when the parameters are comparable. Further, we speculate that the observed high concentration of HSPG in intact cells ([HSPG]-100[CSR]) provides a way to ensure that the binding levels of FGF-2 to its signaling receptor remains high, irrespective of the presence of pre-formed CSR-HSPG complexes on the cell surface, while allowing the cell to finely tune the response to FGF-2 via down-regulation of the signaling receptor.     

Structural basis for interaction of FGF-1, FGF-2, and FGF-7 with different heparan sulfate motifs.

Stromal cell-derived FGF-7 binds and activates only the resident FGFR2IIIb in epithelial cells while FGF-1 and FGF-2 exhibit a broader interaction with multiple isoforms of FGFR. Here we report the structure of FGF-7 that has been solved to 3.1 A resolution by molecular replacement with the structure of a dual function chimera of FGF-7 and FGF-1 (FGF-7/1) which was resolved to 2.3 A. Comparison of the FGF-7 structure to that of FGF-1 and FGF-2 revealed the strongly conserved Calpha backbone among the three FGF polypeptides and the surface hydrophobic patch that forms the primary receptor-binding domain. In contrast, a decrease and dispersion of the positive surface charge density characterized the heparin-binding domain of FGF-7 defined by homology to that of FGF-1 and FGF-2 in complexes with heparin. A simple heparin hexasaccharide that cocrystallized with FGF-1 and FGF-2 and protected both against protease in solution failed to exhibit the same properties with FGF-7. In contrast to FGF-1 and FGF-2, protection of FGF-7 was enhanced by heparin oligosaccharides of increased length with those exhibiting a 3-O-sulfate being the most effective. Protection of FGF-7 required interaction with specifically the fraction of crude heparin retained on antithrombin affinity columns. Conversely, heparin enriched by affinity for immobilized FGF-7 exhibited anti-factor Xa activity similar to that purified on an antithrombin affinity matrix. In contrast, an FGF-1 affinity matrix enriched the fraction of crude heparin with low anti-factor Xa activity. The results provide a structural basis to suggest that the unique FGF-7 heparin-binding (HB) domain underlies a specific restriction in respect to composition and length of the heparan sulfate motif that may impact specificity of localization, stability, and trafficking of FGF-7 in the microenvironment, and formation and activation of the FGFR2IIIb kinase signaling complex in epithelial cells.     

Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.     

Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2IIIb with stromal-derived fibroblast growth factor-7

Summary Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein, protamine sulfate, to reversibly block cellular heparan sulfates. Protamine sulfate, which does not bind significantly to FGF-7 or FGFR2IIIb, inhibited FGF-7 activities, but not those of epidermal growth factor. The inhibition was overcome by increasing the concentrations of FGF-7 or heparin. Heparin was essential for binding of FGF-7 to recombinant FGFR2IIIb expressed in insect cells or FGFR2IIIb purified away from cell products. These results suggest that, similar to other FGF polypeptides, heparan sulfate within the pericellular matrix is required for activity of FGF-7. Differences in response to heparin and alterations in the BULK heparan sulfate content of cells likely reflect FGF-specific differences in the cellular repertoire of multivalent heparan sulfate chains required for assembly and activation of the FGF signal transduction complex.     

Lysyl Oxidase Propeptide Inhibits FGF-2-induced Signaling and Proliferation of Osteoblasts

Pro-lysyl oxidase is secreted as a 50-kDa proenzyme and is then cleaved to a 30-kDa mature enzyme (lysyl oxidase (LOX)) and an 18-kDa propeptide (lysyl oxidase propeptide (LOX-PP)). The presence of LOX-PP in the cell layers of phenotypically normal osteoblast cultures led us to investigate the effects of LOX-PP on osteoblast differentiation. Data indicate that LOX-PP inhibits terminal mineralization in primary calvaria osteoblast cultures when added at early stages of differentiation, with no effects seen when present at later stages. LOX-PP was found to inhibit serum- and FGF-2-stimulated DNA synthesis and FGF-2-stimulated cell growth. Enzyme-linked immunosorbent assay and Western blot analyses show that LOX-PP inhibits FGF-2-induced ERK1/2 phosphorylation, signaling events that mediate the FGF-2-induced proliferative response. LOX-PP inhibits FGF-2-stimulated phosphorylation of FRS2α and FGF-2-stimulated DNA synthesis, even after inhibition of sulfation of heparan sulfate proteoglycans. These data point to a LOX-PP target at or near the level of fibroblast growth factor receptor binding or activation. Ligand binding assays on osteoblast cell layers with ¹²⁵I-FGF-2 demonstrate a concentration-dependent inhibition of FGF-2 binding to osteoblasts by LOX-PP. In vitro binding assays with recombinant fibroblast growth factor receptor protein revealed that LOX-PP inhibits FGF-2 binding in an uncompetitive manner. We propose a working model for the respective roles of LOX enzyme and LOX-PP in osteoblast phenotype development in which LOX-PP may act to inhibit the proliferative response possibly to allow cells to exit from the cell cycle and progress to the next stages of differentiation.     

Sulfated derivatives of Escherichia coli K5 polysaccharides as modulators of fibroblast growth factor signaling

Heparan sulfate (HS) proteoglycans are intimately involved in the regulation of fibroblast growth factor (FGF) signaling. HS and the related glycosaminoglycan heparin interact with FGFs and FGF receptors (FGFRs), and it is believed that both interactions are required for productive FGF signaling. Attempts to inhibit FGF activity have been made with modified heparin preparations, various heparin-like polysaccharide analogues and other polyanionic molecules, which may all act by interfering with the physiological HS-FGF-FGFR interactions on the cell surface. Here, we have studied the potential of sulfated derivatives of a bacterial polysaccharide (capsular polysaccharide from Escherichia coli K5 (K5PS)) in the modulation of FGF-heparin/HS interactions and FGF signaling. We demonstrate that O-sulfated and N,O-sulfated species of K5PS, with high degrees of sulfation, displaced FGF-1, FGF-2, and FGF-8b from heparin. However, only O-sulfated K5PS efficiently inhibited the FGF-induced proliferation of S115 mammary carcinoma cells and 3T3 fibroblasts, whereas N,O-sulfated K5PS had little or no inhibitory effect. Studies with CHO677 cells lacking endogenous HS, as well as with chlorate-treated S115 cells expressing undersulfated HS, indicated that whereas exogenously administered heparin and N,O-sulfated K5PS restored the cellular response toward FGF stimulation, O-sulfated K5PS was largely devoid of such stimulatory activity. Our data suggest that highly O-sulfated species of K5PS may be efficient inhibitors of FGF signaling.     

G-CSF stimulates Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation

Granulocyte colony-stimulating factor (G-CSF), the major cytokine regulator of neutrophilic granulopoiesis, stimulates both the proliferation and differentiation of myeloid precursors. A variety of signaling proteins have been identified as mediators of G-CSF signaling, but understanding of their specific interactions and organization into signaling pathways for particular cellular effects is incomplete. The present study examined the role of the scaffolding protein Grb2-associated binding protein-2 (Gab2) in G-CSF signaling. We found that a chemical inhibitor of Janus kinases inhibited G-CSF-stimulated Gab2 phosphorylation. Transfection with Jak2 antisense and dominant negative constructs also inhibited Gab2 phosphorylation in response to G-CSF. In addition, G-CSF enhanced the association of Jak2 with Gab2. In vitro, activated Jak2 directly phosphorylated specific Gab2 tyrosine residues. Mutagenesis studies revealed that Gab2 tyrosine 643 (Y643) was a major target of Jak2 in vitro, and a key residue for Jak2-dependent phosphorylation in intact cells. Mutation of Gab2 Y643 inhibited G-CSF-stimulated Erk1/2 activation and Shp2 binding to Gab2. Loss of Y643 also inhibited Gab2-mediated G-CSF-stimulated cell proliferation. Together, these results identify a novel signaling pathway involving Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation in response to G-CSF.     

Structural requirements in heparin for binding and activation of FGF-1 and FGF-4 are different from that for FGF-2

Size- and structure-defined oligosaccharides from heparin, 2-O-desulphated(2-O-DS-) heparin, 6-O-desulphated (6-O-DS-) heparin, carboxy-reduced(CR-) heparin, and carboxyamidomethylsulphonated (AMS-) heparinwere utilized in characterizing the structural properties ofheparin to specifically bind to basic fibroblast growth factor(FGF-2) and to modulate the mitogenic activity of FGF-2 (Ishihara,M.et al., Glycobiology, 4, 451–458, 1994). The previousresults showed that both 2-O-sulphate groups and the negativecharge of the carboxy group in iduronate residues are requiredfor specific interaction with FGF-2, but the 6-O-sulphate groupsin N-sulphated glucosamine (GlcNS) residues do not influencethe interaction with FGF-2. In the present study, the same oligosaccharideswere fractionated on a FGF-1- or FGF-4-affinity column, andwere assessed as promotors of FGF-1- or FGF-4-induced proliferationof adrenocortical endothelial (ACE) cells and chlorate-treatedACE cells. The present results suggest that the smallest heparin-derivedoligosaccharide binding to these growth factors with the highestaffinity and promoting their mitogenic activities is a fullyN-sulphated decasaccharide enriched in 2-O- and 6-O- sulphateddisaccharide units. In contrast to our results with FGF-2, ahigh content of 6-O-sulphate groups in GlcNS residues is requiredfor specific interaction with FGF-1 and FGF-4. FGF-1 FGF-4 heparin heparan sulphate oligosaccharides     

Structural observation of complexes of FGF-2 and regioselectively desulfated heparin in aqueous solutions

In order to clarify the mechanism of interaction between FGF-2 and heparin, the association structures between human FGF-2 and different kinds of regioselectively desulfated heparins were observed by small angle X-ray scattering. In the FGF-2-native heparin complex, the global FGF-2 molecules appeared to attach along heparin chain as strained unilaterally. The complexes with the 6-O-, or N-desulfated heparin seemed to have randomly associated structure as compared with above system. On the other hand, 2-O-desulfated heparin did not indicate the aggregation with FGF-2, indicating that the sulfate groups at O-2 of iduronate residues in heparin is most essential for association with FGF-2. These structural characteristics will be deeply related with signal transduction in the association with FGF-2 receptor.     

Inhibition of FGF-2-mediated chemotaxis of murine brain capillary endothelial cells by cyclic RGDfV peptide through blocking the redistribution of c-Src into focal adhesions

The alpha(v)beta(3) integrin is essential for fibroblast growth factor (FGF)-induced angiogenesis in vivo. However, the role of this integrin in FGF-2-mediated cellular responses by cultured endothelial cells is largely unknown. Cyclic RGDfV (cRGDfV) peptide is widely used to inhibit the binding of alpha(v)beta(3) integrin to vitronectin. To investigate the role of this integrin in FGF-2-mediated cellular responses, we used immortalized murine brain capillary endothelial cells, denoted IBE cells. Because IBE cells proliferate and migrate in response to FGF-2-treatment, when cultured on fibronectin-coated surface, we first examined the inhibitory activity of this peptide on the binding of alpha(v)beta(3) integrin to fibronectin as well as vitronectin. Solid phase binding assay revealed that cRGDfV peptide strongly inhibited the binding of purified alpha(v)beta(3) integrin to vitonectin- and fibronectin-coated plastic surfaces at a concentration of 50 microM. cRGDfV peptide at 50 microM inhibited spreading as well as adhesion of IBE cells on vitronectin-coated plastic surface but not on fibronectin. On fibronectin-coated substrata, cRGDfV at 50 microM attenuated FGF-2-mediated chemotaxis, but not FGF-2-induced proliferation, of IBE cells. We have previously demonstrated that mitogen-activated protein kinase (MAPK) activation within focal adhesions through c-Src activity was involved in FGF-2-induced chemotaxis of IBE cells. Treatment of cells with cRGDfV peptide was associated with reduced c-Src activity without tyrosine dephosphorylation. Immunofluorescent staining showed that cRGDfV inhibited redistribution of c-Src into focal adhesions. MAPK activation by FGF-2 within focal adhesions was also attenuated in the presence of cRGDfV peptide. Our results indicated that cRGDfV peptide inhibited redistribution of c-Src into focal adhesions, leading to impaired MAPK activation within focal adhesions and motility in FGF-2-treated endothelial cells.     

Intracoronary administration of FGF-2: a computational model of myocardial deposition and retention

This study uses a computational model to characterize the myocardial deposition and retention of basic fibroblast growth factor (FGF-2) at the cellular level after intracoronary (IC) administration of exogenous FGF-2. The model is applied to the in situ conditions present within the myocardium of a dog for which the plasma pharmacokinetics resulting from IC injection of FGF-2 were recorded. Our estimates show that the processes involved in FGF-2 signaling are not diffusion limited; rather, the response time is determined by the reaction time of FGF-2 binding to cell surface receptors. Additionally, the processes of receptor secretion and internalization are found to play crucial roles in the FGF-2 dynamics; future experiments are required to quantify these processes. The model predictions obtained in this study suggest that IC administration of FGF-2 via either a single bolus or repetitive injections causes a transient increase (time scale of hours) in myocardial FGF-2 concentration if the endogenous level of free interstitial FGF-2 is low enough to allow permeation of FGF-2 molecules from the microvascular to the interstitial spaces. The model shows that the majority (64%) of the extracellular FGF-2 ligands are located within the interstitium, and similar fractions are found in the basement membrane and extracellular matrix. Among the FGF-2 molecules found within the interstitium, 2% are free and 98% are bound to interstitial heparan sulfate proteoglycans. These results support the theory of extracellular control of the bioavailability of FGF-2 via dynamic storage of FGF-2 within the basement membrane and extracellular matrix.     

Pravastatin-induced proangiogenic effects depend upon extracellular FGF-2

The HMG-CoA reductase inhibitors (statins) have been shown to exert several protective effects on the vasculature that are unrelated to changes in the cholesterol profile, and to induce angiogenesis. The proangiogenic effect exerted by statins has been attributed to the activation of the PI3K/Akt pathway in endothelial cells; however, it is unclear how statins activate this pathway. Pravastatin-mediated activation of Akt and MAPK occurs rapidly (within 10 min.) and at low doses (10 nM). Here, we hypothesized that FGF-2 contributes to the proangiogenic effect of statins. We found that pravastatin, a hydrophilic statin, induced phosphorylation of the FGF receptor (FGFR) in human umbilical vein endothelial cells. SU5402, an inhibitor of FGFR, abolished pravastatin-induced PI3K/Akt and MAPK activity. Likewise, anti-FGF-2 function-blocking antibodies inhibited Akt and MAPK activity. Moreover, depletion of extracellular FGF-2 by heparin prevented pravastatin-induced phosphorylation of Akt and MAPK. Treatment with FGF-2 antibody inhibited pravastatin-enhanced endothelial cell proliferation, migration and tube formation. These observations indicate that pravastatin exerts proangiogenic effects in endothelial cells depending upon the extracellular FGF-2.     

Studies on FGF-2: Nuclear localization and function of high molecular weight forms and receptor binding in the absence of heparin

Multiple forms of FGF-2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF-2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF-2 as the AUG codon is 3′ to the CUG codons. Although the 18 kDa form FGF-2 is localized primarily in the cytosol, a significant fraction of the HMW FGF-2 has a nuclear location. The nuclear localization of HMW FGF-2 is determined by amino acid residues in the amino-terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino-terminal extension of HMW FGF-2. The nuclear localization of HMW FGF-2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF-2, or all forms of FGF-2, we have found that HMW FGF-2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF-2. These cells are transformed by what appears to be the intracellular action of HMW FGF-2. The interaction of FGF-2 with heparin has also been examined. Contrary to other reports claiming that FGF-2 required heparin or heparan-sulfate for interaction with its high-affinity receptor, we have found that FGF-2 binds to its receptor in the absence of glycosaminoglycans, and that this binding activates the receptor. © 1994 Wiley-Liss, Inc.     

LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor β (TGF-β), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-β, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9–14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR.