The angiogenesis inhibitor beta-cyclodextrin tetradecasulfate inhibits ecto-protein kinase activity.

The growth of new blood vessels plays an important role in the pathogenesis of several diseases including cancer, diabetes, and arthritis. Beta-cyclodextrin tetradecasulfate, when administered with an appropriate steroid inhibits angiogenesis, and can stimulate angiogenesis when given alone. The regulation of angiogenesis is not well understood, and the mechanism of action of beta-cyclodextrin tetradecasulfate is similarly not well defined. Ecto-protein kinase activity that utilizes extracellular ATP has recently been reported on several types of cells. Human neutrophils appear to possess two distinct ecto-protein kinase activities; one that phosphorylates exogenous substrates including vitronectin and basic fibroblast growth factor, and one that phosphorylates endogenous cell-surface proteins. This report shows that beta-cyclodextrin tetradecasulfate inhibits the phosphorylation of the exogenous substrates casein, vitronectin (the major ecto-protein kinase substrate in serum), and basic fibroblast growth factor by human neutrophil ecto-protein kinase activity. In contrast, beta-cyclodextrin tetradecasulfate had no effect on the phosphorylation of endogenous cell-surface proteins by the neutrophil ecto-protein kinase activity. Ecto-protein kinase activity that was inhibited by beta-cyclodextrin tetradecasulfate was also detected on porcine aortic and human umbilical vein endothelial cells. The effects of beta-cyclodextrin tetradecasulfate on ecto-protein kinase activities may play a role in its effects on angiogenesis.     

Glycosaminoglycan affinity of the complete fibroblast growth factor family

BACKGROUND: Many fibroblast growth factor family proteins (FGFs) bind to the heparan sulfate/heparin (HP) subtypes of sulfated glycosaminoglycans (GAGs), and a few have recently been reported to also interact with chondroitin sulfate (CS), another sulfated GAG subtype. METHODS: To gain additional insight into this interaction, we prepared all currently known FGFs (i.e., FGF1-FGF23) and assessed their affinity for HP, CS-B, CS-D and CS-E. In addition, midkine, hepatocyte growth factor and pleiotrophin were studied as other known HP-binding proteins. RESULTS: We found that members of the FGF19 subfamily (i.e., FGF15, 19, 21 and 23) had little or no affinity for HP; all of the other secretable growth factors tested had strong affinities for HP, as was indicated by the finding that their elution from HP-Sepharose columns required 1.0-1.5 M NaCl. We also found that FGF3, 6, 8 and 22 had strong affinities for CS-E, while FGF5 had a moderate affinity for CS-D. The interactions between FGFs and GAGs thus appear to be more diverse than previously understood. GENERAL SIGNIFICANCE: This is noteworthy, as the differential interactions of these growth factors with GAGs may be key determinants of their specific biological activities.     

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.     

In vitro changes in plasma membrane heparan sulfate proteoglycans and in perlecan expression participate in the regulation of fibroblast growth factor 2 mitogenic activity

Fibroblast growth factor 1 (FGF1) and 2 (FGF2) bind to two classes of receptors: the high affinity receptors, a family of four known transmembrane tyrosine kinases (FGF R1-R4), and the low affinity receptors, cell surface and basement membrane heparan sulfate proteoglycan (HSPG). During early (first and second) passages of retinal pigmented epithelial (RPE) cells, both FGF1 and FGF2 exhibited low mitogenic activity, while in later (fifth to ninth) passages the activity of FGF1 remained constant but FGF2 activity increased two- to threefold. We have investigated aspects of FGF receptor interactions and the role of heparin/heparan sulfate which modulates FGF activity on RPE cells during in vitro senescence. Northern blot analysis demonstrated that FGF receptor type 1 (FGF R1) is the major high affinity receptor expressed in RPE cells and that its level of expression did not change during serially passage. Both the FGF R1 and the FGF low affinity receptors' binding characteristics (i.e., Kd and number of sites per cell) for FGF1 were unaffected by passage number, whereas the capacity of FGF2 binding to FGF R1 and to the low affinity receptors increased by two- and fivefold, respectively, in late passages, although the affinities were unchanged. This change in the capacity of FGF2 to bind to FGF R1 and to HSPG was not due to a switch of all the IIIc splice form of FGF R1 to the IIIb splice form since the exon IIIc was the most predominant splice form of FGF R1 during RPE cell cultures. Furthermore the ratio of the IIIb to the IIIc splice form was not modified during cell subcultures. In parallel in the older RPE cell passages, expression of perlecan, the major FGF low affinity binding site localized on the extracellular matrix of RPE cells, was much elevated compared to early RPE cell passages. Moreover, the cell surface of late passage RPE cells had 79% more HSPG than early passage cells. Therefore, it is suggested that the increase in the number of FGF low affinity receptors present on the cell surface or basement membrane could account for a part of the greater proliferative response of aged RPE cells to FGF2. © 1996 Wiley-Liss, Inc.     

Control of fibroblast growth factor (FGF) 7- and FGF1-induced mitogenesis and downstream signaling by distinct heparin octasaccharide motifs

Variation in length, disaccharide composition, and sulfation of heparan sulfate (HS) affects fibroblast growth factor (FGF) signaling. However, it is unclear whether the specific distribution of groups within oligosaccharides or random variations in charge density underlies the effects. Recently we showed that a mixture of undersulfated octasaccharides exhibiting 7 and 8 sulfates (7,8-S-OctaF7) generated from heparin had the highest affinity for FGF7 monitored by salt resistance (>0.60 M salt) of octasaccharide-FGF7 complexes. 7,8-S-OctaF7 also had the highest specific activity for formation of a complex with dimeric FGFR2IIIb competent to bind FGF7. Here we show that when endogenous HS was inhibited by chlorate treatment, 7,8-S-OctaF7 specifically supported FGF7-stimulated DNA synthesis and downstream signaling in FGFR2IIIb-expressing mouse keratinocytes. It failed to support FGF1 signaling in both HS-deficient mouse keratinocytes and 3T3 fibroblasts. In contrast, abundant, more highly sulfated and heterogenous mixtures of octasaccharides with lower affinity (0.30-0.60 M salt) for FGF7 supported FGF1-induced signaling in both cell types. In contrast to the two-component 7,8-S-OctaF7 mixture from FGF7, the high affinity octasaccharide fraction from FGF1 was a heterogeneous mixture with components ranging from 8 to 12 sulfates with 11-S-octasaccharides the most abundant. The high affinity fraction exhibited similar properties to the lower affinity fractions from both FGF1 and FGF7. Octasaccharide mixtures eluting from FGF1 between 0.30 and 0.60 M and above 0.60 M salt were nearly equal in support of FGF1 signaling in fibroblasts and keratinocytes. Both were deficient in support of FGF7-induced signaling in keratinocytes. The results show that both variations in overall charge density and specific distribution of charged groups within HS motifs exhibit FGF-specific control over formation of FGF-HS-FGFR complexes and downstream signaling.     

Identification of a crucial amino acid responsible for the loss of specifying FGFR1–KLB affinity of the iodinated FGF21

Previous studies suggest that specific binding to the complex consisting of fibroblast growth factor receptor-1 (FGFR1) and the coreceptor beta-Klotho (KLB) is the premise for human FGF19 and FGF21 activating the downstream signaling cascades, and regulating the metabolic homeostasis. However, it was found that human FGF21 loses its ability to bind to FGFR1–KLB after iodination with Na¹²⁵I and chloramine T, whereas human FGF19 retained its affinity for FGFR1–KLB even after iodination. The molecular mechanisms underlying these differences remained elusive. In this study, we first demonstrated that an intramolecular disulfide bond was formed between cysteine-102 and cysteine-121 in FGF21, implying that the oxidation of the cysteine to cysteic acid, which may interfere with the active conformation of FGF21, did not occur during the iodination procedures, and thus ruled out the possibility of the two conserved cysteine residues mediating the loss of FGF21 binding affinity to FGFR1–KLB upon iodination. Site-directed mutagenesis and molecular modeling were further applied to determine the residue(s) responsible for the loss of FGFR1–KLB affinity. The results showed that mutation of a single tyrosine-207, but not the other five tyrosine residues in FGF21, to a phenylalanine retained the FGFR1–KLB affinity of FGF21 even after iodination, whereas replacing the corresponding phenylalanine residue with tyrosine in FGF19 did not alter its binding affinity to FGFR1–KLB, but decreased the receptor binding ability of the iodinated protein, suggesting that tyrosine-207 is the crucial amino acid responsible for the loss of specifying FGFR1–KLB affinity of the iodinated FGF21.     

Structural specificity in a FGF7-affinity purified heparin octasaccharide required for formation of a complex with FGF7 and FGFR2IIIb

Variations in sulfation of heparan sulfate (HS) affect interaction with FGF, FGFR, and FGF-HS-FGFR signaling complexes. Whether structurally distinct HS motifs are at play is unclear. Here we used stabilized recombinant FGF7 as a bioaffinity matrix to purify size-defined heparin oligosaccharides. We show that only 0.2%-4% of 6 to 14 unit oligosaccharides, respectively, have high affinity for FGF7 based on resistance to salt above 0.6M NaCl. The high affinity fractions exhibit highest specific activity for interaction with FGFR2IIIb and formation of complexes of FGF7-HS-FGFR2IIIb. The majority fractions with moderate (0.30-0.6M NaCl), low (0.14-0.30M NaCl) or no affinity at 0.14M NaCl for FGF7 supported no complex formation. The high affinity octasaccharide mixture exhibited predominantly 7- and 8-sulfated components (7,8-S-OctaF7) and formed FGF7-HS-FGFR2IIIb complexes with highest specific activity. Deduced disaccharide analysis indicated that 7,8-S-OctaF7 comprised of DeltaHexA2SGlcN6S in a 2:1 ratio to a trisulfated and a variable unsulfated or monosulfated disaccharide. The inactive octasaccharides with moderate affinity for FGF7 were much more heterogenous and highly sulfated with major components containing 11 or 12 sulfates comprised of predominantly trisulfated disaccharides. This suggests that a rare undersulfated motif in which sulfate groups are specifically distributed has highest affinity for FGF7. The same motif also exhibits structural requirements for high affinity binding to dimers of FGFR2IIIb prior to binding FGF7 to form FGF7-HS-FGFR2IIIb complexes. In contrast, the majority of more highly sulfated HS motifs likely play FGFR-independent roles in stability and control of access of FGF7 to FGFR2IIIb in the tissue matrix.     

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

The related glycosaminoglycans heparin and heparan sulfate are essential for the activity of the fibroblast growth factor (FGF) family as they form an integral part of the signaling complex at the cell surface. Using size-exclusion chromatography we have studied the capacities of a variety of heparin oligosaccharides to bind FGF1 and FGFR2c both separately and together in ternary complexes. In the absence of heparin, FGF1 had no detectable affinity for FGFR2c. However, 2:2:1 complexes formed spontaneously in solution between FGF1, FGFR2c, and heparin octasaccharide (dp8). The dp8 sample was the shortest chain length that bound FGFR2c, that dimerized FGF1, and that promoted a strong mitogenic response to FGF1 through FGFR2c. Heparin hexasaccharide and various selectively desulfated heparin dp12s failed to bind FGFR2c and could only interact with FGF1 monomerically. These saccharides formed 1:1:1 complexes with FGF1 and FGFR2c, which had no tendency to self-associate, suggesting that binding of two FGF1 molecules to the same saccharide chain is a prerequisite for subsequent FGFR2c dimerization. We found that FGF1 dimerization upon heparin was favored over monomeric interactions even when a large excess of saccharide was present. A cooperative mechanism of FGF1 dimerization could explain how 2:2:1 signaling complexes form at the cell surface, an environment rich in heparan sulfate.     

Presence of basic fibroblast growth factor receptors in bovine brain membranes

We described a protocol for purification of bovine brain membranes suitable to study the binding of iodinated basic fibroblast growth factor (FGF) to bovine brain membrane preparation. The binding of 125I basic FGF to brain membranes reached equilibrium within 30 min at 20 degrees C, was reversible, and displaced by an excess of unlabeled basic FGF. Scatchard analysis of the data revealed that two classes of binding sites could be detected with an apparent Kd of 30 pM and a capacity of 0.24 pmol/mg of membrane proteins for the high affinity binding site and Kd of 3 nM with a capacity of 51 pmol/mg of membrane proteins for the low affinity binding site. Cross-linking experiments of labeled basic FGF to brain membrane receptor yield the formation of a single major complex with an apparent molecular mass of 170 kDa which is similar to the value obtained for the high affinity binding site for basic FGF on target cells in tissue culture. Hence these data present the first biochemical evidence suggesting that membrane purified from bovine brain contain two classes of specific binding sites for basic FGF and confirm results described with cells grown in vitro.     

FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

Distribution of basic fibroblast growth factor binding sites in various tissue membrane preparations from adult guinea pig

In order to localize a rich source of basic FGF receptor, we examined the distribution of basic FGF binding sites in brain, stomach, lung, spleen, kidney, liver and intestine membrane preparations from adult guinea pig. Comparative binding studies using iodinated basic FGF showed that a specific binding was detected in all the membrane preparations tested. Scatchard plots from iodinated basic FGF competition experiment with native basic FGF in various membrane preparations, suggested the presence of one class of binding sites in some tissues such as liver, kidney, spleen, lung, stomach, and intestine with an apparent dissociation constant (appKD) value ranging from 4 to 7.5 nM and the existence of a second class of higher affinity sites in brain membranes with appKD value of 15 pM. Characterization of these basic FGF high affinity interaction sites was performed using a cross-linking reagent. These results show for the first time that specific interaction sites for basic FGF are widely distributed, suggesting that this growth factor might play a role in the physiological functions of a number of adult organs.     

Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor.

The role of low affinity, heparin-like binding sites for basic fibroblast growth factor (bFGF) was investigated in CHO cells mutant in their metabolism of glycosaminoglycans. Heparan sulfate-deficient mutants transfected to express a cloned mouse FGF receptor cDNA are not able to bind bFGF. It is demonstrated that free heparin and heparan sulfate can reconstitute a low affinity receptor that is, in turn, required for the high affinity binding of bFGF. These studies suggest that the low affinity receptor is an accessory molecule required for binding of bFGF to the high affinity site. Such an obligatory interaction of low and high affinity FGF receptors suggests a physiological role for heparin-like, low affinity receptors and constitutes a novel mechanism for the regulation of growth factor-receptor interactions.     

Differential specificity of endocrine FGF19 and FGF21 to FGFR1 and FGFR4 in complex with KLB

Background

Recent studies suggest that betaKlotho (KLB) and endocrine FGF19 and FGF21 redirect FGFR signaling to regulation of metabolic homeostasis and suppression of obesity and diabetes. However, the identity of the predominant metabolic tissue in which a major FGFR-KLB resides that critically mediates the differential actions and metabolism effects of FGF19 and FGF21 remain unclear.

Methodology/Principal Findings

We determined the receptor and tissue specificity of FGF21 in comparison to FGF19 by using direct, sensitive and quantitative binding kinetics, and downstream signal transduction and expression of early response gene upon administration of FGF19 and FGF21 in mice. We found that FGF21 binds FGFR1 with much higher affinity than FGFR4 in presence of KLB; while FGF19 binds both FGFR1 and FGFR4 in presence of KLB with comparable affinity. The interaction of FGF21 with FGFR4-KLB is very weak even at high concentration and could be negligible at physiological concentration. Both FGF19 and FGF21 but not FGF1 exhibit binding affinity to KLB. The binding of FGF1 is dependent on where FGFRs are present. Both FGF19 and FGF21 are unable to displace the FGF1 binding, and conversely FGF1 cannot displace FGF19 and FGF21 binding. These results indicate that KLB is an indispensable mediator for the binding of FGF19 and FGF21 to FGFRs that is not required for FGF1. Although FGF19 can predominantly activate the responses of the liver and to a less extent the adipose tissue, FGF21 can do so significantly only in the adipose tissue and adipocytes. Among several metabolic and endocrine tissues, the response of adipose tissue to FGF21 is predominant, and can be blunted by the ablation of KLB or FGFR1.

Conclusions

Our results indicate that unlike FGF19, FGF21 is unable to bind FGFR4-KLB complex with affinity comparable to FGFR1-KLB, and therefore, at physiological concentration less likely to directly and significantly target the liver where FGFR4-KLB predominantly resides. However, both FGF21 and FGF19 have the potential to activate responses of primarily the adipose tissue where FGFR1-KLB resides.     

Basic fibroblast growth factor (bFGF) dissociates rapidly from heparan sulfates but slowly from receptors. Implications for mechanisms of bFGF release from pericellular matrix.

The effect of heparin on the rate of binding of basic fibroblast growth factor (bFGF) to high affinity (receptor) and low affinity (heparan sulfate) binding sites on endothelial cells and CHO cells transfected with FGF receptor-1 or FGF receptor-2 was investigated. Radiolabeled bFGF bound rapidly to both high and low affinity sites on all three types of cells. Addition of 10 micrograms/ml heparin eliminated binding to low affinity sites and decreased the rate of binding to high affinity sites to about 30% of the rate observed in the absence of heparin. However, the same amount of 125I-bFGF bound to high affinity sites at equilibrium in the presence and absence of heparin. The effect of heparin on the initial rate of binding to high affinity sites was related to the log of the heparin concentration. Depletion of the cells of heparan sulfates by treatment with heparinase also decreased the initial rate of binding to high affinity receptors. These results suggest that cell-surface heparan sulfates facilitate the interaction of bFGF with its receptor by concentrating bFGF at the cell surface. Dissociation rates for receptor-bound and heparan sulfate-bound bFGF were also measured. Dissociation from low affinity sites was rapid, with a half-time of 6 min for endothelial cell heparan sulfates and 0.5 min for Chinese hamster ovary heparan sulfates. In contrast, dissociation from receptors was slow, with a half-time of 46 min for endothelial cell receptors, 2.5 h for FGF receptor-1, and 1.4 h for FGF receptor-2. These results suggest that degradative enzymes may not be needed to release bFGF from the heparan sulfates in instances where receptors and heparan sulfate-bound bFGF are in close proximity because dissociation from heparan sulfates occurs rapidly enough to allow bFGF to bind to unoccupied receptors by laws of mass action.     

Interleukin-4 is a potent mitogen for capillary endothelium

Interleukin-4 (IL-4) is a mitogen for both microvascular (human adrenal capillary, HACE) and large vessel (human umbilical vein, HUVEC) endothelial cells. Comparison of growth promotion by IL-4 to that by the potent endothelial mitogen fibroblast growth factor (FGF) showed the activity of IL-4 on HACE cells to be strong (50% of that with FGF) but on HUVEC's weak (12% of that with FGF). Growth stimulation was characterised by both 3H-thymidine incorporation and by cell number, and was maximal at 1 nM IL-4. The presence of IL-4 receptors on HACE cells and HUVEC's was confirmed by specific binding of radioiodinated IL-4. Scatchard analysis confirmed a single high affinity binding receptor on both HACE cells (Kd = 80 pM, 358 receptors/cell) and HUVEC's (Kd = 88 pM, 2,580 receptors/cell). Potent activity on capillary as opposed to large vessel endothelium places IL-4 in a unique position amongst endothelial mitogens.     

Purification of a heparin binding FGF receptor (HB-FGFR) from adult bovine brain membranes

A new form of high affinity fibroblast growth factor receptor has been purified from adult bovine brain membranes. Purification was performed by chromatography on DEAE-Trisacryl and wheat germ agglutinin-agarose followed by FGF-2 affinity chromatography. Affinity labeling of purified fractions with 125I-FGF-2 showed after cross-linking a 170-kDa complex, suggesting the existence of a 150-kDa FGF receptor. No cross-reactivity with anti-FGF receptor 1 (FGFR-1 or flg) or with anti-receptor 2 (FGFR-2 or bek) antibodies could be detected with this partially purified receptor. Heparitinase treatment of the partially purified FGF receptor abolished the formation of the ligand receptor complex. The complex was restored in the presence of heparin in a dose dependent fashion, supporting the idea that heparin-like molecules are needed for proper binding. Further purification of the receptor was achieved by heparin-Sepharose affinity chromatography and yielded a purification of over 320,000-fold. The purified receptor fraction was radiolabeled and loaded on RPLC C4 column. Eluted fractions were analysed by SDS-PAGE. A major 150-kDa band was detected. These data show for the first time a new form of FGF receptor isolated from bovine brain membranes. This purified receptor displays affinity for heparin and was therefore named heparin binding FGF receptor (HB-FGFR). It remains unclear whether the receptor is a proteo-heparin sulfate or whether heparans are strongly associated and therefore are copurified. Large scale preparations are in progress for core protein structure studies.     

Molecular level interaction of the human acidic fibroblast growth factor with the antiangiogenic agent, inositol hexaphosphate

Acidic fibroblast growth factor (FGF1) regulates a wide array of important biological phenomena such as angiogenesis, cell differentiation, tumor growth, and neurogenesis. Generally, FGFs are known for their strong affinity for the glycosaminoglycan heparin, as a prerequisite for recognition of a specific tyrosine kinase on the cell surface and are responsible for the cell signal transduction cascade. Inositol hexaphosphate (IP6) is a natural antioxidant and is known for its antiangiogenic role, in addition to its ability to control tumor growth. In the present study, we investigated the interaction of IP6 with the acidic fibroblast growth factor (FGF1) using various biophysical techniques including isothermal calorimetry, circular dichroism, and multidimensional NMR spectroscopy. Herein, we have reported the three-dimensional solution structure of the FGF1-IP6 complex. These data show that IP6 binds FGF1 and enhances its thermal stability. In addition, we also demonstrate that IP6 acts as an antagonist to acidic fibroblast growth factor by inhibiting its receptor binding and subsequently decreasing the mitogenic activity. The inhibition likely results in the ability of IP6 to antagonize the angiogenic and mitogenic activity of FGF1.     

The crystal structure of fibroblast growth factor (FGF) 19 reveals novel features of the FGF family and offers a structural basis for its unusual receptor affinity

The 22 members of the FGF family have been implicated in cell proliferation, differentiation, survival, and migration. They are required for both development and maintenance of vertebrates, demonstrating an exquisite pattern of affinities for both protein and proteoglycan receptors. FGF19, one of the most divergent human FGFs, is unique in binding solely to one receptor, FGFR4. We have used molecular replacement to solve the crystal structure of FGF19 at 1.3 A resolution using five superimposed FGF structures as the search model. The structure shows that two novel disulfide bonds found in FGF19, one of which appears to be conserved among several of the other FGFs, stabilize extended loops. The key heparin-binding loops of FGF19 have radically different conformations and charge patterns, compared to other FGFs, correlating with the unusually low affinity of FGF19 for heparin. A model for the complex of FGF19 with FGFR4 demonstrates that unique sequences in both FGF19 and FGFR4 are key to the formation of the complex. The structure therefore offers a clear explanation for the unusual affinity of FGF19 for FGFR4 alone.     

Reconstitution of fibroblast growth factor receptor interactions in the yeast two hybrid system

Fibroblast growth factors (FGF) activate their receptors through the formation of trimolecular complexes, composed of a ligand, a receptor, and a heparan sulfate oligosaccharide, all of which are members of particularly large families capable of multiple interactions in a combinatorial fashion. Understanding this large network of interactions not only presents a great challenge, but is practically beyond the capacity of most classical techniques routinely used to study ligand receptor interactions. We have used the yeast two hybrid system to study protein-protein interaction in the FGF family. Both ligand and receptor ectodomains are properly folded and functional in the yeast. Basic FGF (bFGF) expressed in the yeast dimerizes spontaneously. This self-assembly occurs at low affinity, which can be greatly enhanced by the introduction of heparin, supporting a defined role for heparin in bFGF dimerization. Screening a rat embryo cDNA library with bFGF in the yeast two hybrid system identified a short variant of FGF receptor 1, found most frequently in embryonal and tumor cells and which possesses affinity toward bFGF that is significantly greater than that of the more abundant, full-length receptor. We find the yeast two hybrid system, a most suitable alternative method for the analysis of growth factor-receptor interactions as well as for screening for novel interacting proteins and modulators of FGF and its receptors.