diferential effects of heparin saccharides on the

HS requirement for different FGF/FGFR complexes

1

DIFERENTIAL EFFECTS OF HEPARIN SACCHARIDES ON THE FORMATION OF SPECIFIC

FGF AND FGF- RECEPTOR COMPLEXES

Classification: Cell Biology

Running Title: HS requirements for different FGF/FGFR complexes.

Olga Ostrovsky+, Bluma Berman+, John Gallagher#, Barbara Mulloy>, David G. Fernig♪, Maryse Delehedde♪, and Dina Ron+∗∗∗∗

+ Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel;

# Department of Medical Oncology, Cristie CRC Research Center, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK

> Laboratory for Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts, EN6 3QG, UK

♪ School of Biological Sciences, Life Science Building, University of Liverpool,

Crown Street, Liverpool, L69 7ZB, UK

∗∗∗∗ To whom correspondence should be addressed. Fax number: 972-4-8225153; Telephone

number: 972-4-8294217. e-mail: dinar@ techunix.technion.ac.il

Key Words: FGF-1, FGF-7, heparin, KGF-receptor, FGFR2 IIIb, FGFR1, FGFR4, heparan-sulfate.

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 19, 2001 as Manuscript M108540200 by guest on January 30, 2018

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Summary

Heparan-sulfates (HS) play an important role in the control of cell growth and differentiation

by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue

that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate

the interaction of FGFs with their receptors, FGFRs, and are thought to play a critical role in

receptor dimerization. Biochemical and crystallographic studies, conducted mainly with

FGF2/FGF1 and FGF-receptors 1 and 2, suggests that an octasaccharide is the minimal length

required for FGF and FGFR induced dimerization and subsequent activation. In addition, 6O-

sulfate groups are thought to be essential for binding of HS to FGFR and for receptor

dimerization. We show here that oligosaccharides shorter than 8 sugar units support

activation of FGFR2 IIIb by FGF-1, and interaction of FGFR4 with FGF-1. In contrast, only

relatively long oligosaccharides supported receptor binding and activation in the FGF-

1/FGFR1, or FGF-7/FGFR2 IIIb setting. In addition, both 6O- and 2O-desulfated heparin

activated FGF-1 signaling via FGFR2 IIIb while neither one stimulated FGF-1 signaling via

FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required

for activating FGFs is dictated by the specific FGF and FGFR combination. These different

requirements may reflect the differences in the mode by which a given FGFR interacts with

the various FGFs.

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Abbreviations AP: Alkaline-Phosphatase

DP: Degree of polymerization

DSS: Disuccinimidyl suberate

FGF: Fibroblast growth factor

FGFR: Fibroblast growth factor receptor

GAGs: Glycosaminoglycans

HSPG: Heparan sulfate proteoglycan

HS: Heparan Sulfate

PBS: Dulbecco's phosphate-buffered saline


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Introduction

Fibroblast growth factors (FGFs) represent a family of over 20 members sharing common

structural features (1,2). FGFs regulate the growth, differentiation, survival and migration of

a wide variety of cell types, and have been implicated in a number of diseases including

cancer, rheumatoid arthritis and diabetic retinopathy (1). Acidic- FGF (aFGF or FGF-1) and

basic- FGF (bFGF or FGF-2) were the first to be isolated. They act on a wide spectrum of

tissues and cell types and play an important role in a multitude of physiological and

pathological processes including embryonic development, neuronal survival, angiogenesis,

wound repair and tumor growth (1). The keratinocyte growth factor (KGF or FGF-7) is

unique among its family members, since unlike the other FGFs that have broad cell type

specificity, FGF-7 acts predominantly on cells of epithelial origin to elicit a variety of

responses including cell proliferation and cell migration (3). FGF-7 is implicated in tissue

development and repair, as well as in cancer (4-7).

FGFs act primarily by binding and activating tyrosine-kinase receptors encoded by four

genes (FGFR1-FGFR4) (2). Further diversity is provided by an alternative splicing

mechanism that generates receptor isoforms with altered ligand binding properties (2,8).

FGF-1 interacts with the four known FGFRs and their isoforms that have been characterized

so far, whereas FGF-7 interacts only with a splice variant of FGFR2 known as the KGFR or

FGFR2 IIIb isoform (9-11). The FGFR2 IIIb is expressed predominantly in cells of epithelial

origin (12), and it binds FGF-1 with an affinity similar to that observed for KGF (9,13).

Binding of FGFs to their receptors induces FGFR dimerization, a step that is essential for

subsequent receptor activation and triggering of downstream intracellular signaling events

(14).


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In addition to FGFRs, FGFs interact with the sulfated domains of heparan sulfate (HS)

moieties of cell surface and extracellular matrix associated heparan sulfate proteoglycans

(HSPGs) (15-17). FGFs can also interact with heparin, a highly sulfated polysaccharide that

shares structural similarity with heparan sulfates and can therefore mimic their action (16).

HS protect FGFs from heat inactivation and proteolytic degradation and also provide an

extracellular reservoir from which FGFs can be rapidly released (17). In the absence of HS,

FGFs can activate their receptors but only at very high concentrations, and in most cell

systems studied FGFs do not induce cell growth in HS deficient cells (18-20). Addition of HS

to physiological concentrations of FGFs can increase their efficacy by 2-3 orders of

magnitude and the effect on bioactivity correlates well with effects on primary receptor

binding (18,19). HS can also dictate specificity of FGF/FGFR binding depending, most

likely, on the fine structure of their sulfated domains (19,21).

It has been suggested that this dependence of the growth-stimulatory activity of FGFs on

HS is due to the polysaccharide facilitating binding of FGFs to the FGFRs, most likely by

stabilizing ligand-receptor complexes (22). It was also proposed that HS are essential for

FGFR dimerization. According to this model, binding of HS to FGF induces oligomerization

of the ligand, which in turn, enables receptor dimerization and subsequent activation of the

intracellular tyrosine kinase (23). Biochemical and structural studies demonstrated that HS

indeed induce FGF oligomerization (24,25). However, the concentrations of FGF and HS

required are far higher than concentrations regarded as being physiologically relevant and

only a minor fraction of the total FGF is oligomerized (19). By contrast, at physiological

concentrations of FGF, and heparin concentrations required for full biological activity, the

primary form of FGF is monomeric (19). The FGF induced oligomerization model gained

further support from the findings that at least an octasaccharide is required to activate FGF-2

(25-27). Crystallographic studies showed that a hexasaccharide is unable to dimerize FGF-1,


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and recent resolution of the crystal structure of FGF-1/FGFR2/ heparin complex suggested

that a glycosaminoglycan (GAG) shorter than an octasaccharide would not be able to cross-

link ligand and receptor (28,29). Contrary to these studies, it was reported that synthetic di-

and trisaccharides can activate FGF-2 signaling, but this observation has not been

independently confirmed (30). In addition, other studies suggested that a monomer of FGF2,

or FGF-7 could form an active signaling complex (31-34).

The majority of these studies focused on FGF-2 and FGF-1, and the HS or heparin

structure required for productive interaction of these growth factors with the IIIc isoform of

FGFR1 or FGFR2. Little is known about the structural requirements of HS and heparin for

other FGF/FGFR interactions. In the present work we studied the effect of heparin

oligosaccharides of variable size, and selectively desulfated heparins, on stimulating FGF-1

and FGF-7 activities via FGFR2 IIIb, and FGF-1 with other FGFRs. We show here, that

FGF-1 and FGF-7 vary considerably in the saccharide-structures required for supporting their

ability to bind and activate FGFR2 IIIb. Moreover, we show that the minimal saccharide

length required for productive interaction of FGF-1 with its different high affinity receptors

can be shorter than an octasaccharide depending on the FGFR type.


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Experimental Procedures

Materials - Human recombinant FGF-7 and FGF-1 were produced in bacteria and purified as

previously described (11,35). Bovine brain FGF-1 was purchased from R&D. Bovine-lung

derived heparin was from Sigma. Heparin-Sepharose was from Pharmacia. Carrier-free

Na125I was purchased from Du Pont- New England Nuclear, Microtiter ELISA plates were

from Corning (Cat. # 25805-96). Tissue culture medium, sera, and cell culture supplements

were from Gibco-BRL. Disuccinimidyl suberate (DSS) was obtained from Pierce Chemical

Co. All other chemicals were purchased from Sigma.

Cells – Rat myoblast cells (L6E9) were grown in DMEM containing 10% FCS as previously

described (11). The parental lymphocytic cell line BaF3, BaF3 expressing FGFR1 (25) or

FGFR2 IIIb (BaF/KGFR cells) (19), were grown in RPMI 1640 supplemented with 10% FCS

and 10% IL-3 conditioned medium from WEHI-3B cells. The cells expressing the FGFRs

were also grown in RPMI plus 10 ng/ml recombinant FGF-1 and 5 µg/ml heparin. In the

latter case, cells were extensively washed and transferred to RPMI containing IL3 at least 8-

10 days prior to their use in experiments.

Expression of human FGFR4 in BaF3 Cells - Cells were transfected by electroporation

(960 µF, 240V) with pCEV plasmid bearing the human FGFR4 gene (11) and selected in

BaF3 growth medium containing G418 (500 µg/ml). These cells were grown in RPMI plus

10% FCS, WEHI-3B conditioned medium and G418.

Radioiodination of FGFs - FGFs were radioiodinated using chloramine-T and separated

from free iodine by heparin-Sepharose affinity chromatography as previously described (11).

Specific activities of iodinated FGFs were in the range of 1-2.5x 105 cpm/ng.


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Receptor binding assays and cross-linking - Binding assays and cross-linking of ligand

receptor complexes in BaF3/KGFR cells were performed essentially as described (19).

Briefly, 2.4x106 BaF/KGFR cells in 0.4 ml binding buffer containing 8 ng/ml of

radioiodinated growth factors were incubated for 2 h at 4oC on an end-over-end mixer. Cells

were collected by centrifugation, washed with PBS and subjected to cross-linking.

Determination of specific binding and cross-linking were done as previously described (19).

Ligand-receptor complexes were resolved on 6% SDS-PAGE. Equal amounts of total cell

lysates were loaded onto each lane. Binding to low affinity sites on L6E9 cells and

determination of specific binding were described previously in details (36). Briefly, cells

were incubated with 1-2 ng/ml radioiodinated growth factors for 2 hrs on ice, unbound

growth factors were washed out, and bound material was extracted by salt solution at neutral

pH.

Biosensor competitive binding assays- Streptavidin was immobilized on planar aminosilane

surfaces according to the recommendations of the manufacturer and measurements were

carried out in an IAsys Auto+ resonant mirror biosensor (Affinity Sensors, Cambridge, UK)

at 20°C. Heparin-derivatized aminosilane surfaces (37) and a customized program for the

Auto+ instrument (available from the manufacturer, Affinity Sensors) were used in these

experiments. Controls showed that in PBST (PBS supplemented with 0.02 % (v/v) Tween

20), FGF-7 did not bind to this surface (result not shown). Examination of resonance scans

showed that at all times during binding reactions, FGF-7 was distributed uniformly on the

sensor surface and therefore was not microaggregated (results not shown). The cuvette was

equilibrated in 40 µl PBST and then 5 µl of the relevant dilution of heparin or chemically-

modified heparin in PBST was added, followed by 5 µl 10 µg/ml FGF-7 to initiate binding.

The binding reaction was continued for 5 min. The surface was washed three times with 50

µl PBST and 2 min later was regenerated with 2 M NaCl. The heparin-binding capacity of


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FGF-7 remained intact in the course of the experiment, since no change was detected in the

amount of FGF-7 bound to immobilized heparin in the absence of competing polysaccharide

at the start and the end of the experiments (result not shown). The extent of binding was

calculated by fitting the association curve to a single site binding model using the non-linear

curve fitting FastFit software (Affinity Sensors), as describe previously (38).

Proliferation assay- BaF/KGFR cells were washed 3 times with PBS, seeded into 24 well

microtiter plates (5x104 cells/well) in RPMI containing 10% FCS and the desired

concentrations of growth factors, heparin or oligosaccharides. Fresh growth factor and other

supplements were added every other day, and viable cells were counted on day 4 or 5 of

incubation. Each data point was performed in duplicates or triplicates and each experiment

was repeated at least 3 times. The variation between different experiments did not exceed

10%.

Preparation of heparin fragments- Heparin oligosaccharides were prepared from low

molecular weight heparin (Innohep) produced by partial heparinase cleavage: the saccharides

were separated by high resolution gel filtration on Bio Gel P 10 essentially as described

previously for heparan sulfate saccharides (39). We also used heparin fragments (kindly

provided by Dr. Israel Vlodavsky, at the Hebrew University, Israel) produced following

alkaline treatment of heparin methyl ester (β-elimination) as previously described (40).

Fragments from both types of preparations have similar effects.

Preparation of desulfated heparin-The parent material for both modified heparins was

bovine lung heparin remaining from the 2nd International Standard Heparin, a sample which

is unusually homogeneous and highly N-and O-sulfated. 2-O- desulfated heparin was

prepared by the method of Jaseja et al., (41). Briefly, about 100 mg bovine lung heparin was

taken up in 10 ml 1.0 M NaOH solution (pH > 12.2), frozen and lyophilized. The residue was

taken up in 10 ml water, neutralized with dilute HCl, dialyzed against at least five changes of


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demonized water, and evaporated under reduced pressure. The product was converted to the

sodium salt on a column (1 x 10 cm) of Dowex 50 resin in the Na+ form. 6-O-desulfated

heparin was prepared by solvolysis of the pyridinium salt in n-methylpyrrolidinone-water

(42) About 100 mg bovine lung heparin was passed several times through Dowex 50 H+

form, neutralized with pyridine and evaporated to dryness under reduced pressure. Solvolysis

was carried out in water:N-methyl pyrrolidinone (10 ml) at 90 º for 16 hours. The cooled

reaction mixture was diluted with water and neutralized with NaOH to pH 9, concentrated by

evaporation, and dialyzed against at least five changes of deionized water before

lyophilization. These conditions give a degree of N-desulfation besides 6-O-desulfation, so

re-N-sulfation was carried out on the 6-O-desulfated product using trimethylamine-SO3

complex (43). The structure and homogeneity of the parent heparin and the modified heparins

was assessed by 1H NMR spectroscopy at 500 MHz (spectra recorded at 30 ºC for samples in

D2O, using a Varian Unity 500 spectrometer running under the manufacturer�s software). 2-

O- and 6-O- desulfation were found to be complete to the limit of detection, which is below

5%. The degree of N-sulfation of all the samples was found to be over 95%. Loss of 2-O-

sulfate in the solvolytically 6-O-desulfated sample was below the limit of detection.


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Results

The effect of heparin-derived oligosaccharides on the bio-potency of FGF-1 and FGF-7. In order to reveal structural characteristics of heparin that are required for promoting the

growth-stimulatory activity of FGF-1 and FGF-7, we have utilized the BaF3 cell system.

These cells are negative for both HSPGs and FGFRs, and were engineered to individually

express FGFR2 IIIb, FGFR1 or FGFR4 [(19,44), and present study]. All three receptors bind

FGF-1 with similar high affinity, whereas only FGFR2 IIIb can interact with FGF-7

(9,10,25). The efficiency of cellular responses of the BaF3 transfectants to FGFs is dependent

on the presence of exogenous heparin or activating HS species (19,25,45). Therefore, this

well-characterized model system is ideal for studying structural-HS requirement for FGFR

mediated signaling.

We first evaluated the concentration of native heparin that supports maximal biological

response of FGFR2 IIIb to FGF-1 and FGF-7. The two growth factors activate this receptor

subtype with a similar high efficiency (9,13). Maximal response to 10 ng/ml of growth factor

was obtained in the presence of 1-5 µg/ml of full-length heparin (see Figure 1A). Based on

these results, we initially assessed the ability of 5 µg/ml of heparin-derived oligosaccharides

ranging in size from DP2 to DP18 to support the growth-stimulatory responses to FGF-1 and

FGF-7. As shown in Figure 1B, heparin fragments shorter that DP10 did not efficiently

promote biological activity of FGF-7, whereas heparin fragments from two to eighteen sugar

units (DP2 to DP18) enabled FGF-1 to stimulate cell proliferation, but with different

efficiencies. For example, DP4 and DP6 promoted about 2- and 3 folds increase in cell

number in the presence of FGF-1, respectively, and the stimulatory capacity of the DP10 and

longer saccharides was comparable to that of full-length heparin (6-7 fold increase in cell

number). In the case of FGF-7, activity comparable to that of heparin was observed only with


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16 sugar units. Similar results were obtained using two separate preparations of heparin

fragments (data not shown).

To further confirm the marked difference observed between the stimulatory activity of

small size heparin fragments on the bio-potency of FGF-1 and FGF-7, we compared the

efficiency of DP4, DP6 and DP16 oligosaccharides in stimulating the biological activity of

each growth factor in BaF/ KGFR cells. All three fragments activated FGF-1, but with

different efficiencies, as expected from the previous bioassay. The tetrasaccharide, at the

highest concentration examined (20 µg/ml), induced a response that was about 60 % of that

observed with the DP16. The hexasaccharide induced maximal response similar to that

observed with the DP16 oligosaccharide at the concentrations that were examined, but its

ED50 was about 6-fold higher (1µg/ml as compared to 0.15 µg/ml for the DP16) (Fig. 2A).

The DP16 stimulated the biological activity of FGF-7 as efficiently as native heparin whereas

the DP4 and the DP6 failed to stimulate FGF-7 activity (Fig. 2B, and data not shown).

The effect of heparin-derived oligosaccharides on binding of FGF-1 and FGF-7 to FGFR2 IIIb.

HS and heparin stabilize FGFs and protect them from proteolytic degradation (17). Since

the proliferation assay described in the previous section requires long-term incubation, a

possible explanation for the lower potency of short saccharides could be inability to protect

the growth factors during the prolonged incubation at 37oC. Alternatively, the lower potency

could result from genuine effects on ligand/receptor interaction. In the later situation, we

expect a direct correlation between effects of oligosaccharides on receptor binding and

cellular responses to the growth factors. To address these questions, we initially examined the

effect of a single concentration of each of the heparin fragments on the binding of FGF-1 and

FGF-7 to BaF/KGFR cells. Since we observed a perfect correlation between the effect of

oligosaccharides on biological activity (see figure 1B) and receptor binding (data not shown),

we assessed more precisely the relative potencies of the various heparin fragments on ligand-


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receptor interaction. 125I-FGF-1 and 125I-FGF-7 were allowed to bind to BaF/KGFR cells in

the presence of increasing concentrations of DP4-DP18, and ligand receptor complexes were

visualized following covalent cross-linking. All the saccharides stimulated binding of FGF-1

to FGFR2 IIIb whereas only relatively long saccharides stimulated FGF-7 binding.

Representative examples are shown in figures 3 and 4 (DP4, DP6 and DP16 for FGF-1 and

Dp4, DP12 and DP16 for FGF-7). As shown in figure 3, the tetrasaccharide potentiated FGF-

1 binding even at the lowest concentration that was examined (50 ng/ml, see panel A).

Quantification of the intensity of radioactivity indicated that fragments of ≥ 10

monosaccharide units are essentially equivalent in their ability to promote FGF-1 receptor

binding, which was similar to the potency of full-length heparin (Figure 3, and data not

shown). The efficiency of stimulation by DP4, DP6 and DP8 oligosaccharides was lower

compared with the longer saccharides (IC50 of 0.6 and 0.25 and 0.05-0.1 µg/ml, respectively).

A slow migrating complex with a molecular weight compatible with that of a receptor dimer

was observed even with the tetrasaccharide (figure 3A-C, and data not shown).

In accordance with their effect on the response of BaF/KGFR to FGF-7, heparin

fragments shorter than ≤ DP8 failed to promote binding, DP10-DP12 exhibited weak

stimulatory activity whereas fragments ≥ DP14 units were nearly similar to that of full length

heparin in promoting binding (Fig. 4A-C, and data not shown). At concentrations ≥25 µg/ml,

these oligosaccharides gradually inhibited the binding of FGF-7 to its receptor, as previously

observed in experiments with full length heparin (19,36,46). Altogether, these results

reemphasized the pronounced difference observed between the stimulatory activity of small

size heparin fragments on the bio-potency of FGF-1 and FGF-7, and also show that these

effects are already manifested at the level of primary interaction between the FGFs and the

FGFR.


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Binding efficiency of oligoasaccharides to FGF-1 and FGF-7 correlates with the growth-stimulatory response

To gain further insight to the mechanism underlying the difference in stimulatory

activity of the short heparin fragments on the activity of FGF-7 and FGF-1, we examined the

affinity of each growth factor for DP4, DP6, DP8 and DP18 oligosaccharides. To this end,

the ability of these oligosaccharides to compete for the binding of 125I-FGF-7 and 125I-FGF-1

to cell associated heparan sulfate proteoglycans (HSPGs) in L6E9 cells was examined. These

cells do not express detectable levels of FGFRs and do not respond to FGFs, but express

HSPGs that can support cellular responses to FGFs following transfection of various FGFRs

including FGFR1, FGFR2 IIIb and FGFR4 (4,11). Binding of FGF-1 and FGF-7 to low

affinity sites on these cells was previously characterized (36,45). The DP18 oligosaccharide

competed equally well for binding of each of the growth factors to L6E9 associated HSPGs

(Fig 5A and B). By contrast, the shorter saccharides did not efficiently compete for the

binding of FGF-7 to these HSPGs (Fig. 5B). With respect to FGF-1, the competition

efficiency correlated with the fragment size (50% inhibition at 0.2, 0.5 and 5 µg/ml of octa-,

hexa- and tetrasaccharide, respectively).

Selectively O-desulfated heparin bind differently to FGF-7 and FGF-1 and exert different effects on their interaction with FGFR2 IIIb.

To further characterize the properties of the HS binding sites for FGF-7 and FGF-1, and

the HS-structural requirements for productive interaction with FGFR2 IIIb, we studied the

binding efficiency of 2-O- and 6-O- desulfated (2-O-DS and 6-O-DS) heparin to FGF-1 and

FGF-7. In addition, we studied their ability to support receptor binding and biological

activity of each growth factor. Binding efficiency to each growth factor was tested in binding

competition experiments using L6E9 cells, as described in the previous section. As shown in

Fig 6A, both 2-O-DS and 6-O-DS competed equally well for binding of 125I-FGF-7 to cell-

associated HS and with about 3-fold lower efficiency compared with native heparin (half

maximal displacement at 0.25 and 0.08 µg/ml, respectively). In contrast, 2-O-DS heparin,


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could barely compete for the binding of 125I-FGF-1 to L6E9 HSPG at the concentrations that

were examined, whereas 6-O-DS heparin competed with an efficiency that was about 4-fold

lower compared with native heparin (Figure 6B). Previous studies clearly indicated that all

the low affinity sites for FGF-1 on L6E9 cells are HS (36,45). In the case of FGF-7, binding

to low affinity sites is only slightly affected by heparinase or chlorate treatment albeit that

bound FGF-7 could be dissociated from these sites by salt extraction, or in the presence of

heparin (45). To confirm that the selectively O-DS heparins interfere with binding of FGF-7

to HS, we performed a cell-free binding competition assay using heparin that was

immobilized on an aminosilane biosensor surface ( see �Experimental Procedures�). As

shown in Figure 6C, the competition pattern of the 2-O- and 6-O-DS heparins for binding of

FGF-7 to the immobilized heparin was similar to that observed with the L6E9 cells. Thus,

this experiment unequivocally demonstrates that FGF-7 interacts with heparin in the absence

of the 2-O- or the 6-O- sulfates, albeit less efficiently than with native heparin. In addition,

the present findings may provide an explanation to the previously observed lack of effect of

chlorate treatment on binding of FGF-7 to low affinity sites on cells (36).

We next examined the ability of the selectively desulfated heparin to support binding of

each growth factor to FGFR2 IIIb. When evaluated by efficiency of ligand-receptor cross-

linking, both 2-O-DS and 6-O-DS heparin stimulated the binding of 125I-FGF-1 to FGFR2

IIIb, but less effectively than heparin (Fig. 7A and 7B). However, neither of the selectively

O-desulfated heparins could stimulate binding of FGF-7 to FGFR2 IIIb (Fig.7C). Moreover

these selectively desulfated heparins also failed to promote binding of FGF-1 to FGFR1 (Fig.

7D).

Biological activity of selectively O-desulfated heparin In accordance with the results of the binding assays, both the 6-O and the 2-O- desulfated

heparin potentiated FGF-1 induced BaF/KGFR (FGFR 2 IIIb) proliferation (Fig. 8A).

Addition of either of the molecules to sub-optimal concentrations of heparin had no effect on


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biological activity compared with heparin alone (Fig. 8B). Neither one of the selectively O-

desulfated heparin molecules stimulated the activity of FGF-7. The desulfated heparins

showed only weak antagonistic activity, even when added at more than a 100-fold excess

over a suboptimal concentration of heparin. Thus, 30 and 13 percent inhibition was observed

in the presence of 0.25 µg/ml native heparin and 30 µg/ml of either the 2-O- or the 6-O-

desulfated heparin (Fig. 8C).

The stimulatory capacity of short heparin fragments depends on the specific combination of FGF/FGFR complexes.

It was reported that heparin fragments shorter than 8-10 saccharide units failed to

support productive interaction of FGF-1 with FGFR1 (47). Thus, the ability of shorter

saccharides to stimulate interaction of FGF-1 with FGFR2 IIIb may be unique to this

interaction. To address this question we compared the effect of heparin fragments ranging in

size from DP2 to DP14 on binding of FGF-1 to FGFR4, FGFR1 and FGFR2 IIIb. Ligand

receptor complexes were visualized following cross-linking. Similar to the results obtained

with FGFR2 IIIb, short heparin fragments (DP2 to DP6) potentiated binding of FGF-1 to

FGFR4 (compare figure 9, panels A and C). By contrast, binding of FGF-1 to FGFR1 or its

ability to stimulate proliferation of BaF/FGFR1 cells required fragments containing ≥ 12

monosaccharides (Figure 9B, and data not shown), in agreements with effects of heparin

fragments on biological activity of FGF-1 in cells that naturally express FGFR1 (47). These

findings clearly reveal that the minimal number of saccharides that can activate an FGF

depends on the specific FGF/FGFR combination.


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Discussion

In the present work we investigated the influence of heparin and heparin saccharides on

growth factor and receptor selectivity. We compared the effect of length and sulfate

composition of heparin on the productive interaction of two distinct FGFs with the same

FGFR, and their effect on interaction of the same FGF with three different FGFRs. The study

was performed with FGF-7 and FGF-1. These two FGFs are ideal for investigating such

questions because FGF-7 exhibits a unique FGFR specificity whereas FGF-1 is a universal

FGFR ligand (9-11,48).

We found that FGF-1 and FGF-7 markedly differ in their HS requirements for interaction

and activation of FGFR2 IIIb. Firstly, sugar units as short as a disaccharide could activate

FGF-1 signaling via FGFR2 IIIb while the activation of FGF-7 required quite long

saccharides. The potency of each of the short saccharides correlated with their relative

affinity for FGF-1 and FGF-7. Secondly, these two growth factors differ in their mode of

interaction with cell associated HS. FGF-7 interacted similarly with both the 2-O- and the 6-

O-DS heparins whereas FGF-1 exhibited appreciable binding to the 6-O-DS heparin and little

or no binding to the 2-O-DS heparin. Thirdly, the O-desulfated heparins displayed different

effects on receptor binding and biological activity of each growth factor. Both types of

desulfated heparin molecules potentiated receptor binding and biological activity of FGF-1

but failed to activate FGF-7. As expected from their promoting effect, neither of the

desulfated molecules could antagonize the promoting effect of heparin on FGF-1 activity. In

the case of FGF-7, the 6-O-DS heparin exhibited weak inhibitory activity at very high

concentrations. However, at such high concentrations, native heparin also inhibits the

biological activity of FGF-7 (19,36,45,46).


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The effect of short heparin fragments on biological activity of FGF-1 differed

depending on the FGFR type. Thus, similar to the situation observed for FGFR2 IIIb,

interaction of FGF-1 with FGFR4 is supported by short saccharides. By contrast, potentiation

of FGF-1/FGFR1 interaction was observed only with relatively long saccharides. Efficient

binding of FGF-1 to FGFR1 differed also with respect to sulfate requirements because neither

2-O- nor 6-O- DS supported interaction, in agreement with their reported effect on FGF-1

activity in cells expressing FGFR1 (26). These observations show that structural requirements

in the polysaccharide co receptor for FGF activation differ not only between distinct FGFs,

but also for a given FGF depending on the FGFR that interacts with the growth factor.

Co-crystallization of FGF-1 with heparin, in the absence or presence of FGFR, and

biochemical studies, suggest that FGF-1 interacts with both, the 2-O- and the 6-O- sulfate

groups (28,29,47). In addition, it was recently demonstrated that a sequence motif of IdoA,

2S-GlcNS, 6S-IdoA, 2S centrally positioned in a DP8 S-domain from HS, is essential for

high affinity binding of FGF-1 (49). The currently observed stimulatory effect of 2-O-DS or

6O-D-S heparin on the interaction of FGF-1 with FGFR2 IIIb is consistent with the existence

of binding sites for each of the sulfate groups on FGF-1. Furthermore, our binding

competition studies suggest that the affinity of 2O- sulfate groups to FGF-1 is significantly

higher than that of the 6O-sulfate group (see fig. 6). However, this difference in affinity is not

translated to differences in their stimulatory activity at least when heparin is used as the FGF

cofactor. It seems that both, 6-O and 2-O sulfates, are required to achieve a maximal

biological response because the level of stimulation by each one was lower than that of native

heparin. Binding of 6-O and 2-O sulfates to FGF-1 can also explain why 6-O-DS heparin did

not antagonize the stimulatory effect of native heparin on FGF-1/FGFR2 IIIb (present work)

and also FGF-1/FGFR1 interaction, albeit that in the later case, 6-O-DS has no stimulatory


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activity (26). In this respect FGF-1 differs from FGF-2 where 6-O-DS acts as strong

antagonist of the stimulatory effect of heparin of FGF-2 activity via FGFR1 (26).

To explain the antagonistic effect of 6-O- DS heparin on FGF-2 activity it was suggested

that that 6O-sulfate group is required for another interaction, with the FGFR being the most

probable candidate (26). The resolved structure of the FGFR2/FGF-1/heparin complex also

points to the involvement of the 6-O sulfate group in receptor interaction, but the biological

significance of this interaction has yet to be confirmed (29). Here we show that 6-O-sulfation

in heparin is not critical for productive interaction between FGF-1/FGFR2 IIIb, but it is

required to increase efficacy of the growth factor. Because the affinity of 6-O- DS heparin for

FGF-1 is rather low, we assume that binding of this sulfate group to another molecule could

contribute cooperatively to enhance ligand-receptor interaction. Interaction with another

molecule may also explain the observed effects with FGF-7 because sulfation in both

positions is required for FGF-7 activity. Previous studies suggest that heparin at stimulatory

concentrations does not bind FGFR2 IIIb [(50) and Ron D. unpublished results]. In addition,

high affinity binding of both FGF-1 and FGF-7 to soluble FGFR2 IIIb is not dependent on

heparin while efficient binding to the native receptor requires heparin (19,50). These

observations suggest that the other interactions may take place not with FGFR2 IIIb but

rather with a different cell surface molecule that imposes a restriction on ligand/receptor

interaction, and sulfation may be required to alleviate this restriction. However, we cannot

rule out the possibility that FGFR2 IIIb may have low affinity for HS/heparin in solution, and

binding of heparin to the growth factor increases affinity of the glycosaminoglycan for the

receptor.

The question as to whether two FGF molecules bridged by HS are required to induce

FGFR dimerization is rather controversial. On the one hand, the recently resolved crystal

structures of several FGF/FGFR complexes suggest that two molecules of FGF are required


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for receptor dimerization even in the absence of heparin (29,51,52). The resolved structure of

FGF-1/FGFR2/heparin complex revealed that heparin dimerizes the two FGF molecules and

also makes contacts with one of the receptors (29). Based on this structure it was suggested

that at least an octasaccharide would be required for these interactions. The finding that a

hexasaccharide cannot induce FGF-1 dimerization in solution supports this proposal (28). On

the other hand there exist, a large body of findings that are not compatible with this model.

These include the present findings that FGF-1 signaling via FGFR2 IIIb and its binding to

FGFR4 can be activated by short saccharides, and previous reports suggesting that a single

FGF molecule is sufficient for FGFR dimerization (31-34,53). Moreover, FGF-1 or FGF-7

dimers are not observed using nearly physiological concentrations of growth factors and

stimulatory concentrations of heparin (19). How can these differences be reconciled? One

possibility is that short saccharides may still induce FGF dimerization. In general, long

saccharides dimerize FGFs via binding to the major heparin-binding pocket in each molecule.

Short saccharides (DP2-DP6) are not long enough to span the distance between the two

binding pockets (54). To explain how synthetic di- and trisaccharides could activate FGF-2,

it was proposed that FGF-2 dimers can be generated by self association and the synthetic

saccharides stabilize the self associated dimers by binding to a minor heparin-binding site

located in each molecule in the plane where self association take place (54). However, self-

association of FGF-1 has not been observed (28). An alternative possibility would be that

FGFs could activate their receptor via structurally distinct mechanisms. Another possibility is

that FGFRs are activated via one mechanism, and the discrepancy between biological and

structural data is that there may be a difference between the biologically active signaling

complexes and the complexes revealed in the crystallographic data.


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The present findings that signaling induced by the same FGF via different FGFRs can

have distinct structural requirement in HS, lends further support to the importance of HS

diversity in regulating FGF activities. How this regulation is achieved is not clear yet.

Previous studies, together with observations that effects of HS on ligand/receptor binding

correlate with effects on biological activity, suggest that HS can modulate kinetic parameters

of FGF/FGFR interactions [(18,19,22), and present study]. We propose that the efficiency of

signaling by FGFRs is also depended on structural considerations, and the differential HS-

requirements are needed to assist FGF/FGFR complexes in assuming the best configuration

for efficient receptor activation. Such a possibility is supported by studies in other systems.

For example, it has been shown that an erithropoietin (EPO) mimicking peptide can bind and

induces EPO-receptor dimerization but is unable to activate the receptor. The difference

between this peptide, and an agonist peptide, is the relative orientation of each receptor unit

in the complex (55,56). In the FGF family, a small subset of receptors mediate signaling via

more than 20 FGF members, and interactions between a given FGF and different FGFRs or

different FGFs with the same FGFR are not entirely identical. For instance, FGF-1 and FGF-

7 bind and activate the FGFR2 IIIb with a similar potency (10,13,19), yet the two growth

factors differ significantly in their mode of interaction with the third IgG like domain of the

receptor (57,58). In the case of FGF-7, but not FGF-1, interaction of the β4-β5 loop with the

receptor is critical for efficient activity (59). Thus, it may be conceivable that HS can

compensate for such differences by assisting FGF and FGFRs to adopt proper orientation in

the signaling complex. Proper orientation is necessary in order to bring together the

intracellular domains of the receptor dimers for transphosphorylation. Thus, the differential

HS requirements could reflect the difference between the various FGF/FGFR interactions.

Comparison of the ternary structure of the same FGF in complex with the distinct FGFRs and


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biologically active heparin fragments as well as the same FGFR with each of its various

ligands and heparin is likely to shed light on this issue.


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Acknowledgments This work was supported by grants from the Israel Science Foundation (to Dina Ron), by The

Cancer Research Campaign (to John Gallagher), and by the North West Cancer Research

Fund and the Cancer and Polio Research Fund (to David G. Fernig and Maryse Delehedde).


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Figure Legends

Figure 1.

A, The effect of heparin on biological activity of FGF-1 and FGF-7. BaF/KGFR cells were

washed with PBS and seeded at a density of 5X104 cells/well in 24 well microtiter plates in

growth medium lacking IL3. Then, the indicated concentrations of heparin were added along

with 10 ng/ml of FGF-1 or FGF-7. Fresh growth factor and heparin were added on day 3 and

viable cells were counted on day 5 of incubation. B, Differential stimulation of the

biological activity of FGF-1 and FGF-7 by short heparin oligosaccharides. FGFs were

added at 10 ng/ml to BaF/KGFR cells in the absence or presence of 5 µg/ml of heparin

fragments. The assay was performed as described in panel A. S denote saccharide, and H

denote heparin.

Figure 2.

The efficiency by which heparin oligosaccharides affect the biological activity of FGF-1

(panel A) and FGF-7 (panel B). Proliferation was assayed in BaF/KGFR cells in the

presence of 10 ng/ml growth factors and increasing concentrations of DP4, DP6 and DP16.

The number of cells/well in the positive control (1 µg/ml heparin) was 500,000 and 488,000

for FGF-1 and FGF-7, respectively.

Figure 3.

Effect of short versus long heparin fragments on receptor binding efficiency of FGF-1.

Radio-iodinated FGF-1 was added at 8 ng/ml to BaF/KGFR cells in the absence or presence

of increasing concentrations of DP4, DP6 and DP16 oligosaccharides. Binding and cross-

linking were performed as described in �Experimental Procedures� � and Berman et-al., (19).

Ligand receptor complexes were resolved by SDS-PAGE and visualized by autoradiography.


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C: lane1, FGF-1 alone; lanes 2-6, FGF-1 plus 0.05, 0.5, 1, 5 and 10 µg/ml, respectively, of

DP4 (panel A), DP6 (panel B) and DP16 (panel C) oligosaccharides. Panel A, lane 7, FGF-1

plus 1 ug/ml heparin. The specific activity of the radioiodinated FGF-1 was 226,000 cpm/ng .

The position of molecular weight markers is indicated on the left. The position of monomer

(M) and dimer (D) ligand-receptor complexes are indicated on the right.

Figure 4.

Effect of short, medium and long heparin fragments on receptor binding efficiency of

FGF-7. Radio-iodinated FGF-7 was added at 8 ng/ml to BaF/KGFR cells in the absence or

presence of increasing concentrations of 4, 12 and 16 saccharides. Panels A-C: lane1, FGF-7

alone; lanes 2-7, FGF-7 plus 0.05, 0.5, 1,10, 25, 50 µg/ml, respectively, of the DP4 (panel A),

DP12 (panel B) and DP16 (panel C) saccharide units; lane 8, FGF-7 plus 1 µg/ml full length

heparin. The specific activity of radioiodinated FGF-7 was 144,000 cpm/ng .

Figure 5.

Comparison of the growth factor binding affinity of short and long heparin fragments.

Binding of 125I-FGF-1 (panel A) and 125I-FGF-7 (panel B) (10 ng/ml each) to low affinity

sites on L6E9 cells was competed by the above indicated concentrations of tetra-, hexa-, octa

and 18 saccharides. The binding assay was carried out as described under � Experimental

Procedures� and ref. 36. Specific binding in the absence of competitor was 72,000 and 52,000

cpm for FGF-7 and FGF-1, respectively.

Figure 6. Binding of selectively O-desulfated heparin to FGF-1 and FGF-7. 125I-FGF-7 (panel A) or

125I-FGF-1 (panel B) were bound to L6E9 cells in the absence or presence of the indicated

concentrations of 2O- and 6O- desulfated heparin (2O-DS and 6O-DS, respectively) or

native heparin (Hep).Panel C, Binding of FGF-7 to heparin immobilized on an aminosilane


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surface was competed by increasing concentrations of 2O-DS, 6O-Ds and native heparin.

Maximal binding of FGF-7 in the absence of competing oligosaccharides was 56 arc s (600

arc s corresponds to 1 ng protein/mm2 of the cuvette surface). Errors for individual datum

points were less than 1 % of the mean and are omitted for clarity. Similar results were

obtained in two separate experiments on two independently prepared sensor surfaces.

Figure 7.

Both 2O- and 6O- desulfated heparin increase binding of FGF-1 but not of FGF-7 to

FGFR2 IIIb. Binding of radioiodinated FGF-1 or FGF-7 to BaF/KGFR cells and covalent

cross-linking was carried out as described under experimental procedures. A, Binding of 125I-

FGF-1 to BaF/KGFR cells in the absence (lane 1) or presence of 1 µg/ml heparin (lane 2) or

0.25 (lane 3), 0.5 (lane 4), 1 (lane 5) or 5 µg/ml (lane 6) of 2O- desulfated heparin. Lanes 7-9,

binding was carried out in the presence of 0.25, 0.5 and 5 µg/ml 6O- desulfated heparin,

respectively. B, Quantification of the data presented in panel A. Quantification was done

using TINA software following phosphoimageing of the dried gel. C, Binding of 125I- FGF-7

to BaF/KGFR without (lane 1) and with 1 µg/ml heparin (lane 2), 5 and 10 µg/ml of 2O-

desulfated heparin (lanes 3 and 4, respectively), 5 and 10 µg/ml of 6O- desulfated heparin

(lanes 5 and 6). D, Binding of 125I- FGF-1 to BaF/FGFR1 cells without (lane 1) and with 1

µg/ml heparin (lane 2), 5 µg/ml 2O-DS (lane 3), 5 and 10 µg/ml of 6O-DS (lanes 4 and 5,

respectively).

Figure 8.

Effect of selectively O-desulfated heparin on biological activity of FGF-1 and FGF-7.

Proliferation assay in BaF/KGFR cells was performed as described in the legend to figure 1.

FGF-1 (panels A and B) and FGF-7 (panel C) were added at concentration of 10 ng/ml, either

with or without 0.25 µg/ml of native heparin (Hep). Selectively O- desulfated (6O-DS or 2O-


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DS) heparin was added at the above indicated concentrations. SDSH denotes selectively O-

desulfated heparin.

Figure 9.

Binding of FGF-1 to different FGFRs is differentially modulated by heparin

oligosaccharides. BaF-3 cells expressing FGFR2 IIIb, FGFR1or FGFR4 were bound to 8 ng/

ml 125I-FGF-1 in the absence (lane 1 in all panels) or the presence of 5 µg/ml heparin

fragments. Panel A: binding to FGFR2 IIIb expressing cells in the presence of 2-12 sugar

units, respectively (lanes 2-7). Panel B: binding to FGFR1 expressing cells in the presence of

4-16 saccharides, respectively (lanes 2-8). Panel C: binding to FGFR4 expressing cells in the

presence of 2-12 saccharides, respectively (lanes 2-7). Binding and covalent cross-linking

were performed as described under �Experimental Procedures�. The position of molecular

weight markers is indicated on the left. The position of monomer (M) and dimers (D) ligand-

receptor complexes are indicated on the right.


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Fragment size

Fold

incr

ease

0

1

2

3

4

5

6

7FGF-1FGF-7

2s 4s 6s 8s 10s H12s

14s

16s-2s 4s 6s 8s 10s H12s

14s

16s-

B

.

Heparin(ng/ml)10 100 1000

Fold

incr

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0

1

2

3

4

5

6

7

8

FGF-7FGF-1

A

Figure 1


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Figure 2

ug/ml

0.1 1 10 100

Pro

lifer

atio

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Con

trol)

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20

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60

80

1004616

A

ug/ml

0.1 1 10 100

Pro

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Maryse Delehedde and Dina RonOlga Ostrovsky, Bluma Berman, John Gallagher, Barbara Mulloy, David G. Fernig,

FGF-receptor complexesDifferential effects of heparin saccharides on the formation of specific FGF and

published online November 19, 2001J. Biol. Chem.

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diferential effects of heparin saccharides on the

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