©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heparan Sulfates Mediate the Binding of Basic Fibroblast Growth Factor to a Specific Receptor on Neural Precursor Cells (*)

(Received for publication, May 22, 1995; and in revised form, August 11, 1995)

Yardenah G. Brickman (1)(§) Miriam D. Ford (1) David H. Small (2) Perry F. Bartlett (3) Victor Nurcombe (1)(¶)

From the  (1)Department of Anatomy and Cell Biology, University of Melbourne, 3052 Australia, the (2)Department of Pathology, University of Melbourne, 3052 Australia, and (3)The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Melbourne, 3050 Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Heparan sulfate proteoglycans are thought to be obligatory for receptor binding and subsequent mitogenic activity of basic fibroblast growth factor (FGF-2). In a previous study (Nurcombe V., Ford, M. D., Wildschut, J., Bartlett, P. F.(1993) Science 260, 103-106) we have shown that primary cultures of mouse neuroepithelial cells and a cell line derived from them, 2.3D, secrete a heparan sulfate proteoglycan with a high affinity for FGF-2. In this study, a combination of affinity chromatography and gel chromatography was used to further isolate heparan sulfate side chains with high affinity for FGF-2. These active chains had an average molecular weight of 18,000-20,000. In order to determine whether heparan sulfate chains with specificity for FGF-2 also displayed selectivity for the different FGF receptors, peptides designed to the heparin-binding region of the receptors were used in competitive inhibition studies. The structure of the predicted heparin-binding domain of the FGF receptor 1 was modeled on the basis of its presumed secondary and tertiary structure homology with immunoglobulin loops. These results suggested that many of the basic residues within the second immunoglobulin loop of the FGF receptor 1 form a basic domain in the molecule and therefore form part of a heparin-binding site. Peptides homologous to this region of FGF receptor 1 were shown to inhibit mitogenesis in 2.3D cells, while those to FGF receptor types 2, 3, and 4 did not. A reverse transcriptase-polymerase chain reaction assay designed to detect expression of the four FGF receptors types demonstrated that FGF receptors 1 and 3 were present on the 2.3D cell line but that receptors 2 and 4 were not. These findings indicate that unique heparan sulfate domains interact with specific cell-surface receptors to direct cellular responses.


INTRODUCTION

The fibroblast growth factor (FGF) (^1)family consists of at least nine members including acidic FGF (FGF-1) and basic FGF (FGF-2), which are known to control the proliferation, migration, and differentiation of a broad variety of cell types, including vertebrate neuroepithelial cells(1) . The biological effects of the FGFs are derived from their interactions with specific, high affinity cell-surface receptors(2, 3) . The FGF receptor family consists of at least four types: FGFR1 (flg)(4, 5) , FGFR2 (bek)(6) , FGFR3(7) , and FGFR4(8, 9) . These integral transmembrane proteins have been identified within the mammalian central nervous system(10) . Three of these receptors, FGFR1, FGFR2, and FGFR3, can transduce the FGF-2 signal in vitro(6, 11, 12) and can be alternatively spliced to produce translation products with either two or three immunoglobulin domains with a variably spliced C-terminal region within the third immunoglobulin domain (isoforms) that binds FGF-2 with different affinities(13, 14, 15) . The temporal and spatial regulation of these isoforms suggest that they are likely to play a major role in the specificity of bioactivation of the FGFs in different tissues throughout the body(16, 17, 18) .

FGFs are also known to interact with a large number of low affinity sites on the cell surface and within the surrounding extracellular matrix. These sites have been identified as heparan sulfate proteoglycans (HSPG), and are proposed to form complexes with FGFs to protect them against proteolysis and thermal denaturation(19, 20, 21) . More recently it has been shown that HSPGs are an obligatory part of the FGF interaction with FGFRs(11, 22) . Yayon et al. (23) and Rapraeger et al. (24) demonstrated that cells which express FGF receptors but lack heparan sulfates (HS) do not bind or respond to FGF-2. Exogenous heparin or HS restores the binding and mitogenic activity of FGF-2, strongly suggesting that an interaction between HS and either FGF or the high affinity FGFR is required to elicit a biological response. All four FGF receptors contain a stretch of basic amino acids between the first and second immunoglobulin loop which, in FGFR1, is proposed to form a heparin-binding site(25) . The homologous regions in each of the receptor types contain similar but uniquely different sequences rich in basic amino acids. These differences suggest a means by which cells might differentially activate receptors with unique HS domains. The site which binds FGF has been mapped to the variable C terminus of the third immunoglobulin loop (26) . The dependence of FGF-receptor interactions on heparin has recently been questioned by Roghani and colleagues (27, 28) who demonstrated that heparin merely increased the binding affinity of FGF for FGFR in both myeloid and CHO cells, but was not required for it.

We have recently shown that embryonic day 9 murine neuroepithelial cells are capable of releasing an HSPG which selectively binds FGF-2 through HS side chains and elicits a biological response to FGF-2(29) . This HSPG appears to play a direct role in the interactions of FGF-1 and FGF-2 with responsive cells. Between embryonic days 9 and 11, the core protein of the HSPG is variably glycosylated with HS chains that switch their affinity from FGF-2 to FGF-1. The change in affinity of the HSPG from FGF-2 to FGF-1 also correlates very closely with the period when neuroepithelial cells begin to differentiate into a neuronal phenotype(30) . The aim of this present study was to determine whether HS chains bearing affinity for FGF-2 display selectivity for a receptor type and mitogenic response. Receptor isoforms present in the 2.3D cell line were detected with a reverse transcriptase-polymerase chain reaction assay (RT-PCR). We show that even though FGF receptor 3 is present on the cells, the cells use FGF receptor 1 to transduce the FGF signal in the presence of an appropriate HS.


EXPERIMENTAL PROCEDURES

Materials

Peptides were obtained from Chiron-Mimotopes (Clayton, Victoria, Australia). Bovine pituitary FGF-1 and FGF-2, sodium heparin from porcine intestinal mucosa (molecular mass = 12 kDa) and Pronase were from Sigma. Heparitinase (Flavobacterium heparinum) was from Calbiochem. Dulbecco's modified Eagle's medium and fetal calf serum were from Commonwealth Serum Laboratories (Parkville, Victoria, Australia). [^3H]Thymidine and [^3H]glucosamine was from Amersham Pty. Ltd. (Sydney, Australia). Culture plates were from Falcon LabWare (Becton Dickinson).

Molecular Modeling of the FGFR1 Heparin-binding Domain

The structure of the second immunoglobulin loop in the FGFR1 (residues 152-230) which overlaps a predicted heparin-binding domain (25) was modeled on the known x-ray crystal structure of the C(L) subunit of Fab New(31, 32) . The homologous amino acid residues in Fab New (protein data bank accession 3FAB) were mutated to the corresponding residues found in FGFR1 and the structure energy-minimized in vacuo using the MM+ force field with Hyperchem molecular modeling software (Hypercube, Waterloo, Canada).

Purification and Chromatography of Heparan Sulfate Side Chains

Isolated [^3H]glucosamine-labeled HS chains from the 2.3D neuroepithelial cell line were prepared from proteoglycan isolates by Pronase digestion as described previously(29) . At each stage of the purification procedure, appropriate pools of analogous unlabeled material were collected and tested for their mitogenic ability to promote the incorporation of [^3H]thymidine into 2.3D cells(30) . Side chains, which averaged 20 kDa in size(29) , were further fractionated in denaturing solutions on the basis of their relative affinity for recombinant human FGF-2. The preparation of the FGF-2 affinity matrix and its running conditions were essentially as those described by Ishihara et al.(33) .

Preparation of FGF-2 Affinity Matrix

Human recombinant FGF-2 (5 mg) and N-acetylated heparin (20 mg) in 30 ml of coupling buffer (0.1 M NaHCO(3), 0.5 M NaCl, pH 8.2) were added to 3 g of CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.). The mixture was gently rocked overnight at 4 °C, and the remaining active groups were blocked by the addition of 50 ml of 0.1 M Tris, pH 8.0. The FGF-2-coupled beads were then extensively washed with 2 M NaCl in 10 mM Tris, pH 7.3, 2 mM EDTA in 10 mM Tris, pH 7.3, and finally equilibrated with 0.2 M NaCl in 10 mM Tris, pH 7.3. The column was stored at 4 °C in 0.2 M NaCl in 10 mM Tris, pH 7.3, supplemented with 0.02% azide and 100 µg/ml porcine heparin (Sigma).

Affinity Chromatography of HS

The FGF-2-coupled Sepharose was packed into a small column (0.8 times 2 cm) and equilibrated with 0.2 M NaCl in 10 mM Tris, pH 7.3. The ^3H-labeled HS chains (5 times 10^5 cpm) were dissolved in 100 µl of the equilibration buffer and loaded onto the small column, which was then washed with 3 ml of the same buffer. The bound fraction was eluted with 0.15 to 2 M linear gradients of NaCl (1 ml/min), reapplied to the column, eluted again with a linear NaCl gradient, and the bound fractions, pooled, desalted, and tested for mitogenic activity(33) .

Size-exclusion Chromatography of Bioactive HS

Appropriate bioactive pools recovered from affinity chromatography were further analyzed by chromatography on Sepharose CL-6B (1 times 100 cm) according to our previously described methods(29) . Fractions were analyzed for radioactivity by liquid scintillation counting. The elution volume of peaks emerging from the column were calibrated against those of known glycosaminoglycan standards (molecular mass range from 1.3 to 50 kDa). Peaks of unlabeled material were tested in the mitogenic assay. In some cases, the material emerging from FGF-2 affinity chromatography was characterized by first incubating them for 16 h at 37 °C with 2 units of Flavobacterium heparitinase, with 1 mM phenylmethylsulfonyl fluoride, 25 mM sodium phosphate, pH 7.0(34) . Highly mitogenic subpools were desalted, lyophilized, resuspended in water, and filtered through 0.22-µm cellulose acetate filters (Costar, Cambridge MA).

Mitogenesis Assay

2.3D cells were seeded at an initial density of 10,000 cells/well in 24-well tissue culture plates or 2,000 cells/96-well plate (Falcon) and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum (29) . Cells were plated in the presence of FGF-2 or FGF-1 while HS was added 1 h after plating, and the peptides were added 2 h after plating. Dose-response curves generated with these reagents were completed according to our previously published methods(30) . The sequence of the FGF receptor-blocking peptides and their amino acid locations (in parentheses) within the complete receptor protein sequence were FGFR1 peptide(152-175): APYWTSPEKMEKKLHAVPAAKTVK (FGFR1-A22K), FGFR2 peptide(153-176): APYWTNTEKMEKRLHAVPAANTVK (FGFR2-A22K), FGFR3 peptide(150-173): APYWTRPERMDKKLLAVPAANTVR (FGFR3-A22R), and the FGFR4 peptide(146-169): APYWTHPQRMEKKLHAVPAGNTVR (FGFR4-A22R). A peptide homologous to an amino acid sequence in mouse Ca/calmodulin-dependent protein kinase, LKKFNARRKLKGAILTTMLA(35) , was used as a control peptide. Plating efficiencies were not affected by the presence of any of the peptides (data not shown). The treated 2.3D cells were grown for 24 h before the addition of [^3H]thymidine(30) . At the end of the incubation period, the cells were rinsed three times with phosphate-buffered saline (pH 7.4) and then ruptured with lysis solution (3% (v/v) Triton X-100, 0.02% (w/v) EDTA, and 0.02 M NH(4)OH). The lysis solution was collected in a 1.5-ml tube and levels of [^3H]thymidine determined by liquid scintillation counting.

RT-PCR Analysis of FGFR Expression in 2.3D Cells

Reverse primers specific for exons comprising the C-terminal half of Ig-like loop III in the various FGFRs were R1/4, R2/4, R3/4, and R4/4 for isoforms FGFR1, 2, 3, and 4 IIIc, respectively; R1/3 and R3/3 for isoforms FGFR1 and 3 IIIb, respectively; and also R1/2 in the case of exon a` of FGFR1. These primers were used as 3` primers in combination with oligo(dT) to prime cDNA synthesis of mRNA isolated from the 2.3D cell line (see ``Results''). The methodology for both cDNA synthesis and the subsequent PCR were as described in the RNA-PCR kit (Perkin-Elmer, Applied Biosystems, Melbourne Australia) and Nurcombe et al.(29) . The expression of various isoforms was assessed by a PCR assay wherein an isoform-specific primer pair and an isoform specifically primed cDNA template were present; e.g. to assess if FGFR1 IIIc was present, cDNA synthesis was primed with oligo(dT) and reverse primer R1/4, subsequent addition of the forward primer R1/1 followed with PCR for 30 cycles of 1` at 95 °C, 1` at 60 °C, and 1` at 72 °C resulted in the amplification of a specific FGFR1 IIIc fragment of 350 base pairs. Duplicate PCR assays of template from duplicate mRNA preparations were conducted in the manner described for all known isoforms of the FGFRs. The products diagnostic for these specific isoforms and the primer pairs for the PCR amplification of these products were as follows: FGFR1 IIIa`, primer pairs R1/1 and R1/2; FGFR1 IIIb, R1/1 and R1/3; FGFR1 IIIc, R1/1 and R1/4; FGFR2 IIIc, R2/1 and R2/4; FGFR3 IIIb, R3/1 and R3/3; FGFR3 IIIc, R3/1 and R3/4; and FGFR4 IIIc, R4/1 and R4/4. The product sizes are given under ``Results.'' All oligonucleotides were subjected to EMBL and GenBank data base searches to ensure homology to a specific receptor. Primers were designed across introns to ensure that products of expected size were derived from mRNA only. PCR products were run on gels, Southern blotted, and hybridized with labeled oligonucleotides internal to the expected product, R1/5 for FGFR1 III, a`, b, and c; R2/5 for FGFR2 III, b and c; R3/6 for FGFR3 IIIb; R3/7 for FGFR3 IIIc; and R4/7 for FGFR4 IIIc as described in Nurcombe et al.(29) . The products were also subcloned into pGem3zf (Promega), and both strands were sequenced to further verify identity. The oligonucleotides and the reference for the source from which they were derived were as follows: for FGFR1, 1/1, CTTGACGTCGTGGAACGATCT(4) ; 1/2, GCCAAGAAAGGAGGTTAAGAGTAC (15) ; 1/3, CTGGTTAGCTTCACTAATAT(15) ; 1/4, TTCCAGAACGGTCAACCATGCAGA (4) ; 1/5, GGCCAGACAACTTGCCGTATGTCC(4) ; 1/6, CGGGAATTAATAGCTCGGAT(15) ; 1/7; ACTGCTGGAGTTAATACCACCGAC(4) ; for FGFR2. 2/1, CCCATCCTCCAAGCTGGACTGCCT(36) ; 2/4, CTCCTTCTCTCTCACAGGCGCTGG(36) ; 2/5, TGATGGGCTGCCCTACCTCAAGGT(36) ; 2/6, AGCCAGCACTTCTGCATTGGAGCT(36) ; for FGFR3, 3/1, GACAGACACACGGATGTGCTGGA(11) ; 3/3, GTGAACACGCAGCAAAAGGCTTT (13) ; 3/4, AGCACCACCAGCCACGCAGAGTGA(11) ; 3/6, TCCTGGATCAGTGAGATTGTGGAG(13) ; 3/7, TGCAGGCGCTAACACCACCGACAA(11) ; and for FGFR4, 4/1, TACAGCTATCTCCTGGATGTGCTG(8) ; 4/4, GAAACCGTCGGCGCCGAAGCTGCT(8) ; 4/7, GAAGACCTCACGTGGACAACAGCA(8) .


RESULTS

Molecular Modeling of FGFR1 Heparin-binding Domain

In order to examine the possible constraints on the size of heparan sulfate domains that bind to the FGFR, we employed a computer modeling approach to examine the structure of the second Ig loop which includes the heparin-binding domain(25) . Despite considerable differences in their amino acid sequences, the secondary and tertiary structure of loop domains in members of the immunoglobulin superfamily are very similar (31) . The final modeled structure of the second immunoglobulin loop domain of FGFR1 is presented in Fig. 1, A and B. The structure consists of six anti-parallel beta-strands stabilized by a disulfide bond between cysteine 178 and cysteine 230. The loop domain comprises two regions, a hydrophobic domain containing the N-terminal portion of the immunoglobulin loop and a second region containing a cluster of basic lysine and histidine residues at positions 160, 163, 164, 166, 172, 201, and 225, all of which are orientated on the outer surface of the beta-strands. All of the basic residues (with the exception of lysine 207 and arginine 209) were found within this basic domain. The maximum distance between any two positively charged groups in the basic cluster (i.e. N of lysines 198 and 160) was 30 Å. A heparin hexasaccharide spans approximately the same distance (Fig. 1C). With this theoretical constraint in mind, we next set out to further characterize the interaction between the 2.3D HS and the FGFR with which it might be interacting, since the sequences in the four different FGF receptors in this region have quite distinct differences in their basic residues (see Fig. 1D), suggesting the possibility of unique binding sites for specific HS domains.


Figure 1: Molecular modeling of the FGFR1 heparin-binding site. Molecular modeling of the second immunoglobulin loop region (residues 152-230) of FGFR1. The model shows the characteristic anti-parallel beta-strand structure typical of immunoglobulin loops. Basic residues are shown in bold. Note the cluster of basic residues which extends around the outer surface of the beta-barrel. A and B show two different orientations of the same structure rotated 90° to each other around the z axis. C shows the structure of heparin on the same scale. D shows the linear sequence that has been modeled in A and B.



Fractionation of FGF-2-specific HS

Heparan sulfate side chains derived from the 2.3D HSPG were fractionated on an FGF-2 affinity column using continuous gradients of NaCl (Fig. 2A). About 30% of the initially loaded HS fragments were released at NaCl concentrations below 1.0 M. Approximately 70% of the initially loaded HS fragments were released in a single peak between 1.0 and 2.0 M NaCl, indicating that a significant fraction of the 2.3D HS possessed a high affinity for FGF-2. Fractions incorporating the peak were pooled for further analysis by molecular sieving over Sepharose CL-6B (Fig. 2B). The two large peaks emerging from this column could both be degraded almost to completion with Flavobacterium heparitinase, confirming their identity as true HS. The recovery of ^3H-labeled material after affinity chromatography and then molecular sieving was approximately 45%. Dose-response curves generated with HS material from the peaks recovered after gel chromatography using the 2.3D neuroepithelial cell bioassay demonstrated that peak I, which averaged 18-20 kDa in size, but not peak II, was capable of maximally potentiating mitogenesis (Fig. 2C). When cells were grown in submaximal concentrations of FGF-2 (1 ng/ml), it was found that HS from peak I could maximally trigger cell division when it was present at concentrations above 1 µg/ml. From then on a concentration of 2 µg/ml of peak I HS was used in all cell cultures, unless otherwise indicated.


Figure 2: Purification of HS chains with specificity for FGF-2. A, [^3H]glucosamine-labeled chains from 2.3D cell-conditioned medium were first purified over Q-Sepharose and then subjected to FGF-2 affinity chromatography. Bound saccharide was released with a linear NaCl gradient (0.15-2 M) and rerun over the column, and the fractions were released in the peak above 1 M (fractions 33-38, indicated by the arrow), and pooled for further analysis. B, pooled fractions were then chromatographed over Sepharose CL-6B columns, and the indicated fractions from the two peaks (I and II) were pooled. In some experiments the HS was predigested with Flavobacterium heparitinase (circle). C, lyophilized HS material from peaks I and II of HS chains were then tested for their ability to promote mitogenesis in cultured 2.3D cells exposed to [^3H]thymidine when they were grown in the presence of FGF-2 (1 ng/ml).



Dose Response of FGF-2

Dose-response experiments to determine the optimum concentration of FGF-2 needed to perform subsequent peptide-blocking experiments were done. 2.3D cells were maintained in medium containing 2 µg/ml HS and exposed to increasing doses of FGF-2 (Fig. 3). Maximal activity was reached between 1 and 10 ng/ml FGF-2, with inhibition of growth seen at higher concentrations. Therefore, cells were maintained in 5 ng/ml FGF-2.


Figure 3: Dose response of cultured 2.3D cells grown in the presence of purified HS. 2.3D neuroepithelial cells were labeled with [^3H]thymidine and maintained in the presence of purified HS (2 µg/ml). FGF-2 at the indicated concentrations was added to the cultures, and thymidine incorporation was monitored after 24 h.



Inhibition of HS-FGF-FGFR Interaction by FGFR-specific Peptides

In order to test whether the putative heparin-binding site within FGFRs confers any specificity to the mitogenic response, peptides homologous to the region thought to be the heparin-binding domain from all four known FGFRs were synthesized. The 2.3D cells were maintained in FGF-2 (5 ng/ml) and FGF-2-specific HS (2 µg/ml) and challenged with increasing concentrations of each of the synthetic peptides. The peptides were added after cells had settled onto culture plastic, 2 h after the addition of FGF-2, and 1 h after the addition of HS. Counts of cells revealed that addition of the peptides did not alter plating efficiency or the adherence of the cells to the substrate in any way. The results indicate that, of the four different FGFR-specific peptides, only the peptide corresponding to the sequence present in FGFR1 was capable of inhibiting the mitogenic response (Fig. 4A). Although a small amount of thymidine incorporation could occasionally be triggered by the addition of the FGFR1 peptide alone (Fig. 4, treatment 1) this small increase was never seen consistently. It is also important to note that the blocking seen with the FGFR1 peptide was seen even as FGF-2 concentrations were increased to 100 ng/ml or decreased down to 0.1 ng/ml. This indicates that the peptide was working through a blocking action and not by potentiating FGF-2 into suboptimal higher concentration dose ranges (as seen in Fig. 3). The control peptide from calmodulin, which contains a high charge density by virtue of its large number of basic residues, had little blocking effect on 2.3D proliferation in response to FGF-2 and HS. It was interesting to note that HS from 2.3D-conditioned medium which did not bind to FGF-2 affinity columns promoted very little cellular proliferation in the presence of FGF-2, and the little activity triggered could not be blocked by any of the peptides, even when used at 600 µg/ml (data not shown). Further control experiments were performed to show that the blocking effects of the FGFR1 peptide were not likely to be due to any inherent activity specific to it. When cells were grown in FGF-1 and HS and then challenged with each of the receptor peptides, both the FGFR1-specific and the FGFR3-specific peptides were capable of interfering with the mitogenic response (Fig. 4B). This demonstrates that the FGFR3 peptide is also capable of blocking a functional mitogenic response.


Figure 4: Competitive inhibition with receptor specific peptides. A, the mitogenic activity of cells incubated with optimal concentrations of HS and FGF-2 and challenged with increasing concentrations of FGFR type-specific peptides: FGFR1-A22K (), FGFR2-A22K ( ), FGFR3-A22R (&cjs2106;), FGFR4-A22K ( ) and the control Ca/calmodulin-dependent protein kinase peptide ( ). The treatments were (1) peptides alone, 600 ng/ml; (2) peptides plus FGF-2, no HS; (3) no peptides, FGF-2 and HS; (4) FGF-2 and HS with peptides at 0.6 ng/ml; (5) at 6 ng/ml; (6) at 60 ng/ml; (7) at 600 ng/ml. The experiment here is one of six independently conducted experiments in 24-well plates, each with quadruple replicates. B, further analogous control experiments were conducted with FGF-1 (10 ng/ml) and the receptor peptides FGFR1-A22K (solid) and FGFR3-A22R (hatched). The treatments were (1) no FGF-1, (2) FGF-1, (3) at 6 ng/ml, (4) at 60 ng/ml, and (5) at 600 ng/ml. The experiment here is one of two independently conducted experiments in 96-well plates each with quadruplicate replicates.



To test whether the FGFR1 peptide competes with the FGFR or FGF for binding to HS we examined the ability of the peptide to inhibit the activation of the FGFR by varying the concentration of HS or FGF (Fig. 5). In the first experiment, the cells were incubated at a constant concentration of HS with increasing concentrations of FGF (Fig. 5A). When this experiment was performed, the FGFR1 peptide did not inhibit activation. In the second experiment cells were incubated with the FGFR1 peptide in the presence of a constant concentration of FGF but increasing concentrations of HS (Fig. 5B). Under these conditions a 10-fold higher concentration of HS was required to obtain a maximal mitogenic response. The results confirm the hypothesis that the peptide competes with the receptor for a site on the HS (Fig. 6), as increasing amounts of HS are needed to overcome the inhibition by the FGFR1-specific peptide but not increasing amounts of FGF.


Figure 5: Shifting the dose-response relationship with 2.3D cells. The mitogenic activity of cells incubated in the presence or absence of a specific receptor peptide (60 ng/ml). A, cells were incubated with increasing concentrations of FGF-2 (from 0.1 to 10 ng/ml) in the presence (circle) or absence () of FGFR1-A22K. B, cells were incubated with increasing concentrations of HS (from 2 to 30 ng/ml) in the presence (circle) or absence () of FGFR1-A22K. Error bars represent mean ± S.D. from quadruple replicates.




Figure 6: Binding interactions between HS, FGF, and FGF receptor (FGFR). This figure shows that the FGFR1 peptide competitively inhibited the binding of the HS to the FGFR. The FGFR1 peptide did not inhibit the binding of the FGF to the FGF receptor.



RT-PCR Analysis of FGFR Expression

Southern analysis of the DNA products generated by RT-PCR of RNA from the 2.3D cell line showed amplified products at 200 bp (Fig. 7B, lane 1) and 348 bp (lane 5) representative of FGFR1 IIIa` and IIIc. A product representative of FGFR1 IIIb was not present (lane 3). This shows that two particular isoforms of FGFR1 are expressed in the same cell line. Similarly for FGFR3, amplified products at 350 bp representative of FGFR3 IIIb (lane 7) and IIIc (lane 9) were obtained. These results show that at least two isoforms of FGFR3 are also expressed in the 2.3D cell line. RNase protection analysis and semiquantitative PCR experiments show that the relative amounts of each of these isoforms, FGFR1 IIIc:FGFR3 IIIb:FGFR3 IIIc, present in the cell line are 30:5:1 (results not shown). Similar experiments using primers for FGFR2 and FGFR4 were negative, suggesting that these receptors are not expressed in this cell line.


Figure 7: RT-PCR analysis of expression of FGFRs in the 2.3D cell line. A, design of RT-PCR analysis of expression of the FGFRs. Triangular symbols indicate position of introns; direction and position of forward and reverse primers for each FGFR isoform are indicated by arrows; approximate sizes of amplified products are indicated. B, Southern blot of RT-PCR analysis of FGFR1 and FGFR3 expression in the 2.3D cell line. Amplified products and primers used to generate them are as follows: lane 1, FGFR1 IIIa` (R1/1 and R1/2); lane 2, control, no template (R1/1 and R1/2); lane 3, FGFR1 IIIb (R1/1 and R1/3); lane 4, no template; lane 5, FGFR1 IIIc (R1/1 and R1/4); lane 6, no template; lane 7, FGFR3 IIIb (R3/1 and R3/3); lane 8, no template; lane 9, FGFR3 IIIc (R3/1 and R3/4); lane 10, no template. Lanes 1-6 were probed with oligonucleotide R1/5, lanes 7 and 8 with R3/6, and lanes 9 and 10 with R3/7. Results for FGFR2 and FGFR4 were negative and are not presented. The results presented were reproducibly obtained from duplicate RT-PCR assays on duplicate mRNA preparations.




DISCUSSION

The results of this study indicate that there is a specific interaction between HS side chains with the heparin-binding domain of the FGFRs. 2.3D HS side chains with selective affinity for FGF-2 were also capable of interacting specifically with a region in FGFR1 that lies between domain I and domain II, confirming previous studies (25) which suggest a heparin-binding site in the receptor for signal transduction.

A number of studies have demonstrated that members of the FGF family interact with cells through a dual receptor system, encompassing a low-medium affinity set of interactions with membrane-associated or extracellular matrix molecules, and high affinity interactions with a signal-transducing receptor tyrosine kinase(23, 24, 37) . It has yet to be determined, however, whether these associations are sequential, specific, or involve the formation of a ternary complex. The discovery of an apparent heparin-binding consensus sequence between Ig domains I and II on FGFR1(25) , and the effect of its deletion on subsequent mitogenesis, strongly suggests the latter. Closer examination of this region on each FGF receptor type offers some clues as to which residues may be essential to any specific interactions between FGF receptors and heparan sulfate. This specificity may not hold true for the interaction between heparin and FGF receptors, due to the inherent structural homogeneity of the heparin molecule. This is not the case for heparan sulfate, which consists of discrete sulfated and unsulfated domains, only some of which may possess binding selectivity(21) .

Despite differences in their amino acid sequences, the members of the immunoglobulin superfamily show a considerable conservation of three-dimensional structure(31) . For example, the three-dimensional structure Cu,Zn superoxide dismutase is strikingly similar to that of the immunoglobulins(38) , despite the fact that there is very little amino acid sequence homology. Therefore, it is likely that models of the three-dimensional structure of other members of the immunoglobulin superfamily (e.g. FGFR1), modeled on the immunoglobulins, should provide a good first approximation of the native structure in the immunoglobulin loop regions, even though the overall amino acid sequence homology may be quite low.

The structure of the second immunoglobulin loop of FGFR1 containing a predicted heparin-binding domain was modeled using the homologous region of the Fc chain of Fab New(32) . Two major conclusions can be drawn from this model. First, the orientation of the basic residues in the model suggests that more than one surface of the loop could be used to form a heparin-binding domain. Molecular studies by Kan et al. (25) have shown that a region between residues 160 and 177 in the N-terminal portion of the second immunoglobulin loop is required for FGF-stimulated cell growth. Analysis of our modeled structure suggested that this region comprises one domain which forms part of a much larger cluster of basic residues including several other basic residues in the C-terminal region of the second loop (lysines 198 and 225). Residues 160-177 line up to one particular face of the Ig loop in an exposed position; this finding led us to test peptides against this region before other candidates in competition binding studies to block mitogenesis. The model predicts that other basic residues in the loop may also be important for activation of the receptor by heparan sulfate. However, as residues 160-177 form part of this basic cluster it is not surprising that blockade of this region has profound effects on the mitogenic response to FGF (see also (25) ). The second major conclusion is that the maximum separation of positively charged residues in the basic cluster is approximately 30 Å. This indicates that the entire basic domain could not accommodate more than 3-4 disaccharide units of heparan sulfate since the minimum-sized fragment which should bind to the heparin-binding domain of the receptor should not be greater than 30 Å (Fig. 1C). This conclusion is supported by the work of Thompson et al.(39) . Recently Pantoliano et al. (40) conducted an extensive modeling experiment in which they explored the binding interactions between FGF-2, FGFR1, and heparin by a combination of isothermal titrating calorimetry, ultracentrifugation, and molecular modeling. The results indicated that FGF-2 and HS participate in a concerted bridge mechanism for the FGFR1, partly because the thermodynamic driving force most closely favored a series of allosteric multivalent binding reactions involving the cooperative coupling of heparin-binding reactions on FGFR1 and FGF-2. Our modeling confirmed that heparins of a certain minimal size were needed to span spatially separate domains specific for FGF-2 and the second extracellular Ig loop of FGFR1. They calculated that heparins of approximately 60 Å, such as a decasaccharide, would theoretically be large enough to bridge both FGF and FGFR1, whereas heparins of hexasaccharide size, approximately 30 Å, were able to tie up a site on FGF or on FGFR1 only in a nonproductive complex that precludes the second receptor-binding event and consequently inhibiting signal transduction. Results in support of this notion of an optimal minimal size of heparin fragment for FGF activation have been published by Guimond et al.(41) , where they found that a dodecasaccharide length was fully capable of stimulating mitogenesis to control levels. Initial results from our laboratory suggest that an octadecasaccharide bears all the activity of the native 2.3D HS chain, but when broken down further, mitogenic activity is lost. (^2)In apparent contradiction to the data suggesting that glycosaminoglycans greater than 10 saccharides provide optimal length for FGF activation, recent data from Ornitz et al. (42) and Lafont et al.(43) suggests that di- and trisaccharide fragments of synthetic HS polymers can stimulate mitogenesis to a similar level to that achieved by full-length heparin. These data can be absorbed into the current hypothesis. It has been suggested that these fragments may be sufficiently small to bind separately and individually to the FGF- and the FGFR-HS-binding sites and thereby induce the conformational changes required for interaction and activation of FGF mitogenic cascade. Presumably these fragments are also sufficiently small so as to not occlude the occupation of one HS-binding site through binding to the other. These results, although significant, may not necessarily reflect the in vivo situation, whereas the results described in this study show, subtle differences in sequence and composition of the HS chains generate a mode of selectivity and specificity in HS-FGF-FGFR interactions.

Coupled with the data of Tyrrell et al. (44) and Ishihara et al.(33) , the results of this study strongly suggest that specific oligosaccharide structures within HS are required for the activation process. It was recently demonstrated that a high affinity FGF-2-binding fragment of HS obtained from a fibroblast cell-surface HSPG contained a very specific tetradecasaccharide motif which contained a stretch of five consecutive L-iduronic acid (2-OSO(3))alpha1,4GlcNSO(3) disaccharide units(45) . These fragments had similar mitogenic activity to the entire HS chain (46) . HS chains with high intrinsic sulfur content and an ability to bind FGF-2 are not sufficient in themselves to activate the growth factor, suggesting again that specific structural sequences are required(46, 47) . Aviezer et al. (48) concluded that subtle mixtures of inhibitory and activating HS were present both on the cell surface and in the extracellular matrix which regulated FGF-2 binding to receptors and thus biological activity. It is clear that further comparative studies on glycan sequences required for the activation of other HS-dependent growth factors are required, including sequences for the same growth factor from differing tissues. Such interactions might add another layer of flexibility for cellular responses to changing epigenetic conditions.

Control and specificity of a response to FGF-2 in developing neural cells may also depend on the high affinity receptor type found on the cell surface. To determine the validity of this hypothesis, we tested specific synthetic peptides made to the putative HS-binding domains of the four FGFRs for their ability to block the FGF-2 response. If the receptor requires binding of a specific HS for an FGF-2 mitogenic response to occur, competitive inhibition with a peptide homologous to the HS-binding region on the receptor should block the entire mitogenic cascade. This appears to be the case.

Synthetic peptides homologous to a 24-amino acid stretch of each of the four receptors, including the region identified as a heparin-binding site (25) on FGFR1, were prepared. Increasing concentrations of the FGFR1-A22K, but not FGFR2-A22K, FGFR3-A22R, or FGFR4-A22R peptide were capable of depressing the potentiation response. The control experiments (Fig. 5) support the hypothesis that the peptides did not directly inhibit the binding of FGF-2 to the HS but were rather interfering with the binding of the HS to FGFR1. The FGFR1-specific peptides had no effect on the dose-response of the cells to FGF-2, but rather acted to shift the dose-response of the cells for HS. These results lend further support the idea that the peptides are specifically interacting with the HS and not interfering with FGF-2 (Fig. 6). Further controls demonstrated that the FGFR3-specific peptide is capable of blocking the mitogenic response of cells to FGF. FGFR3-A22R peptide was capable of interfering with the growth of cells exposed to FGF-1 and HS (Fig. 4B). This also indicates that FGF-1 is capable of using multiple FGF receptors in different contexts. (^3)Although it could be argued that it is unlikely that synthetic peptides mimic the highly ordered confirmation of the FGF receptor, the molecular modeling suggests that the basic residues are on one side of the Ig loop. HS in solution is a highly linear molecule by virtue of its charge density so it is not unreasonable to expect that a short peptide in solution could interfere with it at its native receptor-binding site.

As the results from the PCR experiments clearly demonstrate, FGFR1 and FGFR3 are both expressed in the 2.3D cells (see also (4) ). The inability of FGFR3-A22R to interfere with the mitogenic response clearly demonstrates that, within the developing nervous system, 2.3D HS shows selectivity for both particular FGFs and particular FGFRs. The results thus support the idea of highly specific and highly regulated sugar sequences that directly activate particular growth factors through cross-linking with particular receptors. Although in vitro experiments have shown FGFs are capable of binding more than one receptor type, our work suggests that in vivo there exist mechanisms such as the expression of highly specific sugar moieties which tightly regulate FGF action.


FOOTNOTES

*
This work was supported in part by the National Health and Medical Research Council of Australia, the Australian Research Council and the A.L.S.-Motor Neurone Disease Research Society Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an Overseas Postgraduate Research Award and a Melbourne University Postgraduate Scholarship.

To whom correspondence should be addressed. Tel.: 61-3-9344-5795; Fax: 61-3-9347-4190.

(^1)
The abbreviations used are: FGF, fibroblast growth factor; FGF-2, basic FGF; FGF-1, acidic FGF; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); FGFR1-A22K, FGFR1 specific peptide; FGFR2-A22K, FGFR2 specific peptide; FGFR3-A22R, FGFR3 specific peptide; FGFR4-A22K, FGFR4 specific peptide.

(^2)
Y. G. Brickman, M.D. Ford, D. H. Small, P. F. Bartlett, and V. Nurcombe, unpublished data.

(^3)
Y. G. Brickman, M. D. Ford, and V. Nurcombe, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. M. Bernfield, Dr. A. Lander, and Dr. J. K. Heath for advice, and Dr. J. Turnbull for reading the manuscript.


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