Specificity for Fibroblast Growth Factors Determined by Heparan
Sulfate in a Binary Complex with the Receptor Kinase*
Mikio
Kan,
Xiaochong
Wu,
Fen
Wang, and
Wallace L.
McKeehan
From the Department of Biochemistry and Biophysics, Texas A&M
University, and the Center for Cancer Biology and Nutrition, Institute
of Biosciences and Technology, Texas A&M University System Health
Science Center, Houston, Texas 77030-3303
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ABSTRACT |
A divalent cation-dependent
association between heparin or heparan sulfate and the ectodomain of
the FGF receptor kinase (FGFR) restricts FGF-independent
trans-phosphorylation and supports the binding of
activating FGF to self-associated FGFR. Here we show that in contrast
to heparin, cellular heparan sulfate forms a binary complex with FGFR
that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in
liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and
FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4
bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when
recombinant FGFR4 was expressed back in epithelial cells by
transfection, it failed to bind FGF-2 unless heparan sulfate was
depressed by chlorate or heparinase treatment. Isolated heparan sulfate
proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4
restored the specificity for FGF-1 and supported the binding of both
FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF.
In a physiological context, the heparan sulfate subunit may limit the
redundancy among the current 18 FGF polypeptides for the 4 known
FGFR.
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INTRODUCTION |
The FGF1 signal
transduction system is ubiquitous and is a local mediator of
developmental processes in the embryo and homeostasis in the adult (1).
Through associations of both FGF and the FGFR ectodomain with heparan
sulfate chains of pericellular matrix or transmembrane proteoglycans,
the FGFR signaling complex senses perturbation and remodeling of the
pericellular microenvironment (1). Heparan sulfate has been implicated
in stability of FGF (2), access of FGF to the FGFR complex (3),
oligomerization of FGF (4-6) and FGF-FGFR complexes (1, 4-8), and
conformational repression and activation of FGFR oligomers (1, 9, 10). Heparin or heparan sulfate exhibits an FGF-independent, divalent cation-dependent high affinity interaction with
immunoglobulin module II of self-associated oligomers of the FGFR
ectodomain to form a complex that will bind FGF (8-10). Divalent
cations and heparan sulfate cooperate to maintain the dependence of the FGFR complex on FGF (9), presumably by conformational restriction of
the enzyme-substrate relationship between FGFR, which otherwise are
self-activating by trans-phosphorylation (1). A large number of genetically distinct FGF polypeptides have emerged, currently 18, for which there are only four FGFR (1). The high ratio of ligands to
receptors, the co-expression of multiple FGFs within the same tissue or
cells, and the apparent ability of the four FGFR to bind multiple FGFs
when heparin is used as the experimental mimic of heparan sulfate raise
questions about the redundancy and functional relevance of the large
number of FGF homologues. Here, we show that cellular heparan sulfate
in a binary complex with the FGFR ectodomain is an FGFR isotype- and
cell-specific determinant for selection of the FGF that interacts with
the complex. In a physiological tissue context, this may restrict
redundancy of the four FGFR for the large repertoire of FGF ligands.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Preparation of Recombinant Receptors, and
Radioreceptor Assays--
The culture of primary rat hepatocytes
collected by perfusion (11), HepG2 cells (12), and human endothelial
cells has been described (13). Preparation and expression of
recombinant FGFR isoforms in the baculoviral-Sf9 system (10),
preparation of FGFR-specific antibodies (14), and procedures for
preparation and iodination, determination of specific binding, and
covalent affinity cross-linking of radiolabeled FGF to infected
Sf9 cells, mammalian cells, and immobilized purified recombinant
FGFR have also been previously described (9, 10, 15, 16). Recombinant FGFR4 was constructed as follows: P1 (5'-TTGGGAATTCCAGCTTGGGTCCCT-3') and P3 (5'-GCCAGGTATACGGACATCATC-3') were used as sense primers beginning at 42 base pairs upstream of the translational initiation site and 15 base pairs upstream of the coding sequence for the transmembrane domain, respectively, using the polymerase chain reaction. P2 (5'-GGATGGAATTCACTTGCCGGAAGAGCC-3') and P4
(5'-CACAGCCTCGAGCCTTGCTCATGT-3') were used as antisense primers ending
124 base pairs downstream of coding sequence for the transmembrane
region and 18 base pairs downstream of the stop codon. Paired primers
P1P2 and P3P4 were separately applied using HepG2 cell cDNA as
template to amplify 1.3-kilobase extracellular and intracellular
fragments, respectively. The P1P2 fragment was ligated into pBSK
cloning vector at an EcoRI site. The P3P4 fragment coding
for the intracellular domain was then ligated into the pBSK vector with
the extracellular domain fragment at AccI and
XhoI sites to obtain the full-length FGFR4 cDNA.
Sequence was verified and then the FGFR4 cDNA was cloned either
into mammalian expression vector pcDNA1neo (Invitrogen Co.,
SanDiego, CA) at BamHI and XhoI sites for
expression on A431 cells or into transfer plasmid pVL1393 (Invitrogen
Co., San Diego, CA) at SmaI sites for preparation of the baculovirus.
Assay for Activity of Heparan Sulfate by FGF Receptor Kinase
Complementation--
Heparin or heparan sulfate activity was assessed
by binding to recombinant FGFR receptor kinase ectodomain, and then the
ability of the binary complex to support the binding of radiolabeled
FGF was assessed (8, 9, 16). About 5 × 105 Sf9
cells expressing recombinant FGFR were extracted with 200 µl of 1%
Triton X-100 in PBS. Lysates containing approximately 5 µg of FGFR
were mixed with either 4 µg of anti-FGFR1 (17A3) monoclonal antibody
or 16 µl of anti-FGFR4 rabbit antiserum (17) and 80 µl of protein
A-Sepharose beads. The immobilized FGFR was washed with 1 M
NaCl in PBS and then extensively with PBS. After division into 8-12
portions, beads were incubated with standard heparin (Sigma, 174 USP
units/mg) or extracts or partially purified fractions containing
heparan sulfate for 1 h at room temperature in 25 mM
Tris-HCl (pH 7.4), 0.15 M NaCl, 10 mM
MgCl2, and 0.01% CHAPS (binding buffer). After washing
three times with PBS, 125I-FGF (4 ng/ml in 250 µl) was
added for 1 h. Beads were then washed with PBS, 1 mM
disuccinimidyl suberate was added for 15 min, and extracts from the
beads were counted and analyzed by SDS-PAGE and autoradiography.
Scatchard Analysis--
Dissociation constants
(Kd) were determined by Scatchard analyses. Assays
using isolated FGFR were performed under conditions that promote
maximum dependence on external heparin/heparan sulfate for FGF binding
(9). Procedures to determine specific binding and to distinguish
binding to complexes of FGFR and heparin/heparan sulfate from FGF bound
to heparin/heparan sulfate sites that are not in sufficient proximity
to FGFR to yield covalent FGF-FGFR complexes have been described in
detail (9, 15, 16). Immobilized binary FGFR complexes were prepared by
incubation of immobilized FGFR and 1 µg/ml heparin or 2 µg/ml
cellular heparan sulfate followed by removal of unbound carbohydrate.
Analysis was performed in a range of 1.5-10 ng/ml radiolabeled FGF.
Nonspecific binding was less than 10% of total at all concentrations
determined in the presence of 30-fold unlabeled FGF.
Kd and receptor site number were estimated by linear
least squares analysis. Except where indicated, the
Kd values cited in the text are the mean ± S.D. of three separate determinations.
Preparation and Partial Purification of Cellular Heparan Sulfate
Proteoglycan Fragments--
Subconfluent cell cultures described in
the text in 75-cm2 flasks in batches of 10 were treated
with 25 µg/ml trypsin and 0.02% EDTA in PBS (5 ml/flask) for 15 min
at 4 °C. The solution containing material released from the
monolayers was acidified with 0.1% trifluoroacetic acid, dialyzed
against water, and freeze-dried. One flask of cells per batch was
labeled by the addition of 20 µCi/ml 35SO4
overnight prior to extraction for use as a tracer. The freeze-dried powder was reconstituted in PBS and applied to a Biosil SEL-400 HPLC
gel filtration column (exclusion limit, 106 kDa, 300 × 7.8 mm, Bio-Rad). The radioactivity (35S) of each
fraction was monitored by liquid scintillation, and the activity of
each fraction was screened by ability to complement recombinant insect
cell-derived FGFR1 for binding of 125I-FGF-1. In some
cases, fractions were prescreened for binding activity using intact
Sf9 cells expressing the recombinant FGFR prior to extraction
and immobilization. Activity was expressed as a stimulation index,
which was the binding in the presence of fraction divided by that in
absence of the test fraction. Fractions of coincident radioactivity and
FGF binding activity were pooled and applied directly to a Bio-Gel
TSK-DEAE-5-PW anion-exchange HPLC column. Application of a gradient of
NaCl from 0.15 to 1.0 M revealed a peak of coincident
binding activity and 35S radioactivity at 0.5 M
NaCl, which was dialyzed and freeze-dried prior to reconstitution.
Carbohydrate content was measured by the carbozole method and
standardized to the dry weight of heparin. Carbohydrate equivalent to
about 50 µg of heparin containing 10 µg of protein was obtained
from 108 HepG2 cells.
A portion of the reconstituted partially purified material with uronic
acid content equivalent to 2.5 µg of standard heparin was diluted
with 2 ml of 200 mM borate buffer (pH 9.0), mixed with 100 µl of a fresh solution of 5 mg/ml Bolton-Hunter reagent in
Me2SO and incubated for 3 h on ice. The fraction was
then dialyzed against PBS and then iodinated to a specific activity of
1 × 105 cpm/ng with 1 mCi of Na125I using
the Chloramine T method. Free iodine was eliminated by centrifugal
filtration (Millipore Ultra free-MC, molecular weight 30,000 cut-off,
Millipore Products Division, Bedford, MA) with a recovery of
TCA-insoluble material of 40%.
Assay and Partial Purification of Radiolabeled Heparan Sulfate
Proteoglycan by FGFR or FGF Affinity--
An FGFR-affinity matrix for
heparan sulfate was prepared using the membrane-anchored recombinant
FGFR ectodomain expressed in Sf9 cells following extraction and
immobilization on antibody-protein A beads or immobilization of
constructs of the FGFR ectodomain fused to glutathione
S-transferase downstream of the transmembrane domain to
GSH-beads (9). Heparan sulfate was applied in conditions described
above for preparation of FGF-binding binary complexes of FGFR and
heparin/heparan sulfate. Elution was carried out with increasing
concentrations of NaCl or 10 mM EDTA (9).
Noncovalent affinity matrices of FGF-1 and FGF-2 (5 µg) bound to
Cu-chelated beads (500 µl) (Chelating-Sepharose Fast Flow, Amersham
Pharmacia Biotech, Uppsala, Sweden) were prepared in the presence of
100 µg of heparin and 10 mM MgCl2 in PBS
containing 1% Triton X-100 for 2 h at 4 °C. The beads were
washed with 2 M NaCl in PBS and incubated with another 5 µg of FGF, and the procedure was repeated. About 85% of the applied
FGF could be immobilized under these conditions; this percentage was
unaffected by the presence or absence of heparin and the subsequent 2 M NaCl wash. Separate analyses revealed that elution of the
column with 10 mM imidazole removed about 75% of the bound
FGF after multiple runs and that the activity of the recovered FGF was
equal to unbound FGF in receptor binding and cell growth assays. The
binding of radiolabeled heparin or 125I-labeled heparan
sulfate proteoglycan to the affinity matrix was dependent on the
presence of FGF. Nearly 100% of bound heparin/heparan sulfate was
eluted by salt or imidazole. Bound heparin or heparan sulfate was
routinely eluted from FGF-1 columns with 1.0 M NaCl and
from FGF-2 columns with 1.5 M NaCl.
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RESULTS |
Cell Type-dependent Specificity of FGFR4 for
FGF-1--
Cultured rat hepatocytes, which express exclusively FGFR4
(Fig. 1A), exhibit a mitogenic
response to specifically FGF-1 (11, 18). Hepatocytes exhibit about 5000 FGFR sites/cell that specifically bind FGF-1 with Kd
of about 60 pM (11, 18). The mitogenic response to or
binding of FGF-2 in hepatocytes was undetectable (11, 18) (Fig.
1C). In contrast, hepatocyte-like human hepatoblastoma (HepG2) cells ectopically express isoforms of FGFR1, FGFR2, and FGFR3
in addition to FGFR4 (Fig. 1B), bind FGF-2, and exhibit 180,000 FGFR sites/cell that bind FGF-1 (12). However, an analysis with
antiserum specific for FGFR1 and FGFR4 revealed that the FGFR1 fraction
in HepG2 cells bound both FGF-1 and FGF-2, but the FGFR4 fraction bound
only FGF-1 (Fig. 1D). This absolute specificity of FGFR4 for
FGF-1 in liver parenchymal cells was similar to the specificity of
recombinant FGFR4 for FGF-1 reported in monkey kidney epithelial cells
(19). However, it differed from FGFR4 in mouse fibroblasts (20), muscle
cells (21), or leukemia cells (22), which bound both FGF-1 and FGF-2.
Similar to the latter three mammalian cells, a reconstituted complex of
heparin and isolated recombinant FGFR4 derived from insect cells bound FGF-1 and FGF-2 equally (Fig. 2).
Separate Scatchard analyses of the binding of FGF-1 and FGF-2 to binary
complexes prepared with 1 µg/ml heparin yielded apparent
Kd values of 243 ± 30 and 247 ± 28 pM, respectively. This was similar to the recombinant FGFR1-heparin complex, which bound both FGF-1 and FGF-2 with
Kd values of 253 ± 18 pM and
197 ± 61 pM, respectively. Thus both FGFR1 and FGFR4
have an intrinsic ability to bind both FGF-1 and FGF-2, but a factor
that either supports or prevents the binding of FGF-2 to specifically
FGFR4 differs among different types of mammalian cells.

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Fig. 1.
Specific binding of FGF-1 to FGFR4 in liver
parenchymal cells. A and B, analysis of
expression of FGFR. Subconfluent primary cultures of rat hepatocytes
(A) and HepG2 (B) cultures were prepared in
75-cm2 flasks as described (8-10). Total RNA was extracted
with RNAZol (Biotech Labs Inc., Houston, TX). First strand cDNA was
synthesized from the RNA using a random hexamer (Promega Corp.,
Madison, WI) and reverse transcriptase (SuperscriptII, Life
Technologies, Inc.). Polymerase chain reaction was carried out in 100 µl containing 2 µl of 0.1 µg/ml cDNA, 0.5 µl of Tag DNA
polymerase (5 units/ml, Promega), and 1 µl each of 5' and 3' primers
(0.1 µg/ml) of total volume for 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 3 min for 40 cycles. Primers were human
and rat FGFR1 (5'-GGCAAGGAATTCAAACCTGAC-3' and 5'-CATCACGGCTGGTCTCTC
TTC-3'); human FGFR2 (5'-AACGGGAAGGAGTTTAAGGAG and
5'GGAGCTATTTATCCCCGAGTG-3' (IIIb) or 5'-TGGGAGAACTGTCAACCATGC-3'
(IIIc)); rat FGFR2 (5'-TTCATCTGCCTGGTCTTGGTC-3' and 5'
ATGGAGCCGCTTCTCCATCTT-3' (IIIb) or 5'-ATGGAGCCACCTCACTATCGT-3' (IIIc));
human FGFR3 (5'-AACGGCAGGGAGTTCCGCGGC-3' and
5'-CTTGGGGCCCGTGAACACGCAGCC-3' (IIIb) or 5'-GCAGCACCACCAGCCACGGAGAGT-3'
(IIIc)); rat FGFR3 (5'-AATGGCAAAGAATTCCGAGGGCAG-3' and
5'-CTTGGGGCCCGTGAACACGCAGCC-3' (IIIb) or 5'-GCAGCACCACCAGCCACGCAGAGT-3'
(IIIc)); human FGFR4 (5'-GATGGACAGGCCTTTCATGGG-3' and
5'-TGCTGCGGTCCATGTGGGGTCCTC-3'); and rat FGFR4
(5'-GATGGACAGGCCTTGCACGGG-3' and 5'-GGTTGTTGTTGTCCACGTGAG-3').
Products were analyzed with 1% agarose gel electrophoresis in
the presence of ethidium bromide and visualized under UV light.
C and D, FGF-1 and FGF-2 binding to hepatocytes
(C) and HepG2 cells (D). Subconfluent cells in
6-cm2 dishes were incubated with 125I-FGF-1 or
125I-FGF-2 at 4 ng/ml in 1.5 ml of binding buffer
(NaHCO3-free RITC80-7 medium containing 100 µg/ml bovine
serum albumin) for 1 h at room temperature. After incubation, the
cells were washed stepwise with PBS, heparin (250 µg/ml) in PBS, and
PBS and exposed to 1 mM disuccinimidyl suberate for 15 min
at room temperature. Cells were extracted with 0.5 ml of 1% Triton
X-100 in PBS, and the lysate, after clarification, was mixed with 5 µl of the indicated FGFR-specific antisera (8-10) and 20 µl of
protein A beads. The beads were washed sequentially with PBS, 1 M NaCl in PBS, and then PBS followed by extraction with
SDS-PAGE buffer and analysis by SDS-PAGE and autoradiography. The
radiolabeled band at lower apparent molecular mass is a result of
proteolysis of the full-length FGFR at a site near the transmembrane
domain (14).
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Fig. 2.
Binding of both FGF-1 and FGF-2 to complexes
of heparin and isolated FGFR4. The binding of radiolabeled FGF-1
and FGF-2 to binary complexes of recombinant FGFR1 and FGFR4 prepared
by incubation with the indicated amounts of heparin was performed as
described under "Experimental Procedures." Insets show
results of covalent affinity cross-linking analysis at 1 µg/ml
heparin (right lanes). The left lane of each pair
was from assays in which heparin was not added. The data set presented
is representative of three reproductions.
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Selectivity of Recombinant FGFR4 for FGF-1 Is Restored in
Reconstituted Epithelial Cells and Abrogated by Depression of Heparan
Sulfate--
To test the hypothesis that a cell
type-dependent factor confers selectivity of FGFR4 for
FGF-1, we reconstituted FGFR-deficient epithelial cells (A431 from a
squamous cell carcinoma) with 1 × 105 FGFR1 or 5 × 104 FGFR4 and then examined the binding of FGF-1 and
FGF-2 (Fig. 3). Scatchard analysis
indicated that FGF-1 and FGF-2 bound to cells expressing FGFR1 with
Kd values of 104 ± 13 pM and
142 ± 9 pM, respectively, whereas cells expressing
FGFR4 bound only FGF-1 with a Kd of 243 ± 24 pM. The binding of FGF-2 to FGFR4 was below the limits of
detection by both Scatchard analysis and covalent affinity
cross-linking (Fig. 3A).

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Fig. 3.
Analysis of FGF-1 and FGF-2 binding to FGFR4
in reconstituted epithelial cells. A, restoration of
specificity of recombinant FGFR4 for FGF-1. A431 cells (5 × 105) maintained in medium supplemented with 2% fetal calf
serum were switched to serum-free medium prior to addition of 5 µg of
mammalian expression vector pcDNAIneo bearing either FGFR-1 and
FGFR-2 cDNA and 15 µl of LipofectAMINE (Life Technologies, Inc.)
at 37 °C for 24 h. Stable transfected cell lines were selected
in medium supplement with 2% fetal calf serum and 400 µg/ml
Geneticin (Life Technologies, Inc.). Clonal lines were selected by
limiting dilution, and FGFR expression levels were determined by
Scatchard analysis of binding of 125I-FGF-1 and
125I-FGF-2. FGF-1, squares; FGF-2,
circles. B, suppression of cellular heparan
sulfate abrogates specificity of FGFR4 for FGF-1. A431 cells expressing
recombinant FGFR1 or FGFR4 were treated with 10 mM sodium
chlorate (Ch) for 2 days prior to binding assays in
sulfate-free medium containing 2% dialyzed serum and compared with
control cells maintained in sulfate-containing medium. The far
right lane of each panel (Hp) shows cells that were
treated with 0.83 unit/250 µl of heparinase I (Sigma, EC 4.2.2.7) for
2 h at 37 °C prior to analysis.
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To determine whether cellular heparan sulfate played a role in
specificity of the binding of FGF in the reconstituted A431 cells,
we applied sodium chlorate, which inhibits sulfation of heparan
sulfate, or heparinase, which modifies heparan sulfate chains. The
treatment of the A431 cells expressing FGFR4 with sodium chlorate
reduced the binding of FGF to cellular heparan sulfate sites for FGF-1
and FGF-2 to 15 and 35%, respectively, of untreated controls.
Surprisingly, the treatment resulted in the binding of FGF-2 to FGFR4
(Fig. 3B). Similar to chlorate, treatment of cells with
heparinase also resulted in the gain in the binding of FGF-2 in the
cells reconstituted with FGFR4 (Fig. 3B). This implicated
heparan sulfate as the determinant of the cell
type-dependent specificity of FGFR4 for FGF-1.
Parenchymal Cell Heparan Sulfate Complexed to Isolated FGFR4
Restores Specificity for FGF-1--
We then tested whether the
specificity of FGFR4 for FGF-1 was mediated by cellular heparan sulfate
capable of binding to FGFR4 in the absence of cells and the
pericellular matrix. Cell-derived heparan sulfate proteoglycan (HSPG)
was released into the medium by treatment of cells with trypsin and
partially purified by molecular filtration and anion exchange
chromatography as described under "Experimental Procedures." The
activity of HSPG fractions was assessed by their ability to form a
complex with the immobilized FGFR ectodomain after removal of unbound
material and then by the ability of the resultant binary complex to
bind radiolabeled FGF-1 or FGF-2 (8, 9, 16). The activity co-purified
with macromolecules metabolically labeled with sodium
[35S]sulfate, which ran ahead of the bulk of the protein
on gel permeation chromatography (Fig.
4A). Activity and
[35S]sulfate also co-purified during ion exchange
chromatography slightly behind the main absorption peak at A280 (Fig.
4B). The partially purified fraction was radiolabeled with
iodine as described under "Experimental Procedures" and analyzed by
SDS-PAGE and autoradiography (Fig. 4C). On 7.5% gels, about
90% of radiolabeled material failed to penetrate the gel, whereas the
rest of the material migrated in a band with mean apparent mass of
about 75 (± 10) kDa (Fig. 4C, left panel). Treatment with
heparinase resulted in a broad band of labeled material from 45 to 130 kDa. Treatment with Pronase destroyed 100% of the radiolabeled
material. Analysis on 15% gels after treatment with heparinase (Fig.
4C, right panel) revealed that about 70% of the treated
material migrated at apparent mass of 35 kDa that ran off the 7.5%
gels. Separate experiments not shown here revealed that the FGF
complementation activity survived treatment with both Pronase and
chondroitinase A, B, C but was destroyed by treatment with heparinase
or nitrous acid. Inclusion of 8 M urea in the SDS-PAGE gels
did not change the electrophoretic pattern of the indicated bands.

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Fig. 4.
Partial purification of cellular heparan
sulfate proteoglycan fragments. A, separation by size.
An extract of HepG2 cells (1.92 A280 units; 56,149 cpm of
35S) prepared as described under "Experimental
Procedures" was applied to the Biosil SEL-400 column and developed at
a flow rate of 0.5 ml/min. B, anion exchange chromatography.
Fractions under the solid bar from the molecular filtration
chromatography in A were pooled and applied to a TSK-DEAE5PW
column. The pool contained 5.9% of the A280 and 70% of the
35S. After loading and washing with PBS, a NaCl gradient
from 0.15 to 1.0 M, indicated by the solid line,
was applied at a flow rate of 0.5 ml/min. Fractions of 1.0 ml were
collected, a 50-µl portion was counted by liquid scintillation, and a
10-µl portion was assayed for binding activity in the FGFR
complementation assay. Activity was expressed as a stimulation index
(SI), which was the amount of 125I-FGF-1 bound
to FGFR preincubated with a fraction divided by binding to an FGF
incubated with a blank fraction. Fractions under the solid
bar were pooled, de-salted, and used for FGFR complementation
assays. The pool contained 2.6% of the A280 and 30% of the
35S that was applied to the column. C,
iodination and analysis of the partially purified HSPG fractions. A
portion of the pooled fraction that was equivalent to 2.5 µg of
heparin based on uronic acid content from A and B
above was radiolabeled with 125I as described under
"Experimental Procedures" and analyzed by SDS-PAGE on 7.5 and 15%
gels followed by autoradiography. A 6.25-ng sample of the radiolabeled
material (105 cpm) was subjected to treatment with 1.25 units of heparinase (Sigma, EC 4.2.2.7) or 25 µg of Pronase (Roche
Molecular Biochemicals) at 37 °C for 1 h. N, no
treatment; Hp, heparinase; Pr, Pronase.
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The iodinated partially purified heparan sulfate proteoglycan
(125I-HSPG) was employed to further characterize direct
binding to FGFR. Under FGF binding assay conditions, about 200 pg of
the radiolabeled HSPG bound per ng of immobilized FGFR1. One hundred % of the 125I-HSPG that bound to immobilized FGFR1 was
displaced by a 100-fold excess of heparin or treatment with 1 M NaCl. As reported previously, introduction of FGF into
complexes of FGFR and heparin/heparan sulfate increased resistance of
the bound heparin/heparan sulfate to displacement with external
heparin, but not with 0.5 M NaCl (16). Displacement of
125I-HSPG bound to FGFR1 or FGFR4 was undetectable by
either FGF-1 or FGF-2 at concentrations below 10 ng/ml. This was 2.5 times the standard amount of 125I-FGF used in binding
assays and the highest concentration employed in the Scatchard
analyses. Concentrations above 50 ng/ml of either FGF-1 or FGF-2
displaced 50% of the bound HSPG. Preliminary affinity chromatography
experiments using immobilized FGFR1 columns (see under "Experimental
Procedures") indicated that a maximum of 20% of the
125I-HSPG can be retained on FGFR1 or FGFR4 at 0.15 M NaCl and that over 90% of the retained material, which
elutes at 0.65 M NaCl, is in the high molecular weight
fraction shown in Fig. 4A.
Additional experiments using the complementation assay with purified
FGFR1 indicated that over 80% of the activity of the partially
purified HSPG from HepG2 cells that bound to either FGFR1 or FGFR4
eluted from the immobilized FGFR between 0.25 and 0.65 M
NaCl. This represented 7 and 5% of total material bound at 0.15 M NaCl to FGFR1 and FGFR4, respectively. About 65% of the
FGFR1- or FGFR4-bound material that eluted between 0.25 and 0.65 M NaCl bound the other FGFR at 0.25 M NaCl
after removal of the elution salt by dialysis. 75-85% of the
recovered material bound back onto the homologous FGFR. Of the
0.25-0.65 eluate from FGFR1, 10 and 20% bound immobilized FGF-1 and
FGF-2, respectively, at 0.25 M NaCl. About 10 and 46% of
the HSPG from a similar eluate from FGFR4 bound FGF-1 and FGF-2,
respectively. Of the total 125I-HSPG in the partially
purified preparation from HepG2 cells, 2 and 14%, respectively, bound
to affinity columns of FGF-1 and FGF-2. Of the fraction that bound to
immobilized FGF-1 that was eluted by 1.0 M NaCl, 18 and
14% bound to FGFR1 and FGFR4, respectively, at 0.25 M
NaCl. Of the fraction that bound to immobilized FGF-2, 18 and 9%
subsequently bound to FGFR1 and FGFR4, respectively. The results of
purification of the HSPG fraction by combinatorial affinity
chromatography using immobilized FGFR, FGF, and
anti-thrombin2 will be
reported elsewhere.
Scatchard analysis indicated that FGFR1 incubated with 0.2 µg/ml of
the HSPG fraction supported FGF-1 and FGF-2 binding to isolated FGFR1
with a Kd of 60 pM (mean of two
experiments) and 50 pM (mean of two experiments),
respectively. The same fraction supported FGF-1 binding to isolated
FGFR4 with a Kd of 140 pM (mean of two
experiments). However, the binding of FGF-2 was insufficient to assign
a Kd by Scatchard analysis. No radiolabeled FGF-2
could be detected by covalent affinity cross-linking, even when FGFR4
was incubated with more than 2 µg/ml of the HSPG fraction (Fig.
5, A and B). These
results indicated that unlike heparin, cell-derived heparan sulfate
restores the specificity of isolated FGFR4 for FGF-1 to that observed
in the intact cell.

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Fig. 5.
Cellular heparan sulfate proteoglycan
complexed with FGFR determines specificity for FGF. The ability of
FGFR-HSPG complexes to bind FGF-1 and FGF-2 was compared using both
total binding determined by gamma-counter (A and
C) and covalent cross-linking (B and
D). A and B, specific binding of FGF-1
to complexes of isolated liver cell heparan sulfate and recombinant
FGFR4. Recombinant FGFR1 and FGFR2 were expressed in insect cells,
isolated, and immobilized on antibody-protein A beads, and analyses
were performed as described in Fig. 2, except that FGFR complexes were
prepared with the indicated amount of liver cell HSPG prepared as
described under "Experimental Procedures" instead of heparin.
Lanes 1-3 in each set in B show the binding of
the indicated FGF to FGFR with no added HSPG or heparin (N),
to complexes of FGFR and liver cell HSPG and complexes of FGFR and
heparin (H), respectively. B and D,
specific binding of FGF-2 to complexes of isolated endothelial cell
heparan sulfate proteoglycan and recombinant FGFR1 and FGFR4.
C shows the binding of FGF-1 and FGF-2 to FGFR1
(R1) or FGFR4 (R4) after incubation and removal
of complexes from unbound material with no carbohydrate (N),
with 2 µg/ml HSPG isolated from human umbilical vein endothelial
cells (EC), or with 1 µg/ml heparin (H).
D shows the corresponding covalent cross-linking analysis of
the immobilized complexes. Data sets in A and B
and those in C and D are representative of seven
and five reproductions, respectively, in which at least three
independent preparations of HSPG and radiolabeled FGF-1 and FGF-2 were
employed. The specific experiments indicated were performed with the
same radiolabeled preparations of FGF-1 and FGF-2.
|
|
Finally, we isolated HSPG fraction by the same procedure from human
umbilical vein-derived endothelial cells in which FGFR1 is a resident
isoform. In the absence of exogenous heparin, the cell line binds and
responds mitogenically to FGF-2, but only poorly to FGF-1 (13, 18). In
contrast to the HSPG fraction from HepG2 cells, the fraction from the
endothelial cells supported the binding of FGF-2 to both FGFR1 and
FGFR4 when tested in the FGFR complementation assay (Fig. 5,
C and D). In contrast to heparin (Fig. 2) and
liver cell HSPG (Fig. 5, A and B), the
endothelial cell HSPG was deficient in support of the binding of FGF-1
to binary complexes of both FGFR1 and FGFR4.
 |
DISCUSSION |
The FGF signal transduction system is a ubiquitous and local
mediator of embryonic development and homeostasis in adult tissues. It
is a tripartite oligomeric complex composed of the transmembrane tyrosine kinase and heparan sulfate chains from pericellular matrix proteoglycan (1, 8-10). Pericellular matrix heparan sulfate plays
multiple roles in assembly and activation of the FGFR complex. These
roles include stability of FGF (2), access of FGF to the FGFR complex
(3), and oligomerization of the FGFR complex through both the FGFR
ectodomain and FGF (4-10). Heparin and heparan sulfate exhibit a
divalent cation-dependent interaction with apparent Kd of about 10 nM to a specific sequence
domain in Ig module II of the FGFR ectodomain (8-10). This is thought
to restrict the trans-phosphorylation between
self-associated FGFR kinases in the absence of FGF (8-10). In
addition, the bound heparin/heparan sulfate is required and sufficient
for the high affinity interaction of FGF with the binary complex in the
absence of soluble heparin/heparan sulfate (8, 9, 23). These results
support a model in which an activating FGF docks into inactive
oligomeric complexes of FGFR and heparan sulfate chains from the
pericellular matrix. In this report, we utilized this model of the
stepwise assembly of the ternary complex of heparan sulfate-FGF-FGFR to
determine whether the heparan sulfate subunit of the complex
contributes to discrimination between FGFs.
Because there were conflicting reports concerning the discrimination of
FGFR4 relative to FGFR1 for FGF-1 and FGF-2 among cell types (19-22),
we employed FGFR4 and FGFR1 as experimental prototypes. FGFR4 is the
sole isotype of the four known FGFR genes expressed in normal liver
parenchymal cells and in that context, FGFR4 exhibits an absolute
specificity for FGF-1 relative to FGF-2. In contrast, ectopically
expressed FGFR1, which is co-expressed with FGFR4 in differentiated
hepatoma cells, bound FGF-1 and FGF-2 equally. Yet reconstituted
complexes of heparin and recombinant FGFR4, which were removed from
cell membranes, pericellular matrix, and other cellular factors, bound
both FGF-1 and FGF-2 in a manner similar to FGFR1. Expression of
recombinant FGFR4 back into epithelial cells by transfection restored
the specificity of FGFR4 for FGF-1. This suggested that the specificity
was conferred by a cellular co-factor that was present in parenchymal
cell lineages represented by the hepatocytes and HepG2 hepatoma cells,
kidney epithelial cells (19), and the A431 squamous epithelial cells.
The fact that the sulfation-inhibitor chlorate or treatment with
heparinase abrogated the selectivity of FGFR4 for FGF-1 in intact cells
suggested that cellular heparan sulfate was responsible.
Isolated and partially purified extracts containing cellular HSPG from
the surface of the liver parenchymal tumor cell HepG2 substituted for
heparin in formation of binary complexes with isolated recombinant
FGFR1 and FGFR4 from insect cells. Similar to those formed with
heparin, the binary complexes were then capable of binding FGF.
Moreover, the isolated liver cell HSPG fraction restored the
specificity of FGFR4 for FGF-1 to that observed in the liver
parenchymal cells while continuing to support the binding of both FGF-1
and FGF-2 to FGFR1. This confirmed that the liver cells expressed a
distinct HSPG that, when complexed with FGFR4, permits the high
affinity docking of only FGF-1 into the complex. This explained the
specificity of hepatocytes for FGF-1.
Finally, we demonstrated that the discrimination for FGF conferred by
HSPG when complexed to FGFR4 was cell-specific and, therefore, not a
consequence of a general interference of FGFR4-bound HSPG with access
of FGF-2 to the active site or general interference of FGF-2 with the
binding of HSPG to FGFR4. In contrast to the liver cell HSPG, but
similar to heparin, partially purified HSPG from endothelial cells
supported the binding of FGF-2 to reconstituted complexes of both FGFR1
and FGFR4. Unlike liver cell HSPG and heparin, binary complexes of both
FGFR1 and FGFR4 that formed with endothelial cell HSPG failed to
efficiently bind FGF-1. This was consistent with the selective
requirement for exogenous heparin for the binding and mitogenic
response of endothelial cells to FGF-1 (13, 18). The results suggest
that a deficiency in composition of an HSPG subunit of the FGFR complex
may limit the response of some endothelial cells to FGF-2.
Cell or tissue specificity of heparan sulfates in respect to affinity
for and differential effects on activity of FGF-1, FGF-2, and FGF-7 has
been demonstrated (24-30). We conclude that FGFR- and cell
type-specific pericellular matrix heparan sulfates complexed to the
FGFR kinase ectodomain as an integral subunit may limit binding and
activation of the FGFR signaling complex to one or a subset of the FGF
ligands. In tissue and in a physiological context, this may limit the
redundancy of the current 18 FGF polypeptide activators for the four
FGF receptor kinases. Recently, we have demonstrated that only the
fraction of commercial heparin and cellular heparan sulfate that binds
anti-thrombin and inhibits Factor Xa activity can form a binary complex
with the ectodomain of the FGFR kinase, and only the fraction enriched
by recombinant FGFR affinity matrix exhibits anti-coagulant
activities.2 Thus, the requirement for anticoagulant
heparan sulfate and divalent cations (9) distinguishes the requirement
for formation of the binary complex with all four FGFR from the simpler
requirements for the FGFR-independent binding to FGF. Currently, it is
unclear whether FGFR1 and FGFR4 select heparan sulfate chains that
discriminate between FGFs, or the selectivity for FGF is mediated by
the same chain when bound to different FGFR. Although there appears to be a small fraction selected by both FGFR1 and FGFR4 that cannot bind
the other, our preliminary results indicate that most of the liver
cell-derived HSPG that binds either FGFR1 or FGFR4 can bind the other
FGFR. On the one hand, the cross-reactivity may be due to multiple
chains on the same HSPG fragment or multiple, but FGFR-specific,
binding sites within a single chain. On the other hand, the composite
complex formed by the same binding site within the same heparan sulfate
chain in the two different FGFR may form the basis of the specificity
for FGF. Clarification of the structural basis for the results requires
purification of the specific HSPG underlying the activities through
combinatorial affinity chromatography using FGFR, FGF and anti-thrombin
columns, identification of the core protein sequence, and structural
characterization of the heparan sulfate chains.
 |
ACKNOWLEDGEMENTS |
We thank Maki Kan, Kerstin McKeehan, and
Thanh Tran for technical assistance.
 |
FOOTNOTES |
*
This work was supported by United States Public Health
Service Grants DK40739 and DK35310 from the NIDDK, National Institutes of Health, and Grant CA59971 from the NCI, National Institutes of
Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Institute of
Biosciences and Technology, Texas A&M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030-3303. Tel.: 713-677-7522; Fax: 713-677-7512; E-mail: wmckeeha{at}ibt.tamu.edu.
2
W. L. McKeehan, X. Wu, and M. Kan,
manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
FGF, fibroblast
growth factor;
FGFR, FGF receptor kinase;
HSPG, heparan sulfate
proteoglycan;
PBS, phosphate-buffered saline;
Ch, chlorate;
PAGE, polyacrylamide gel electrophoresis;
Sf9, Spodoptera
frugiperda;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
 |
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