(Received for publication, May 22, 1995; and in revised form, August 11, 1995)
From the
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.
The fibroblast growth factor (FGF) ()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.
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 -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
-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.
Figure 2:
Purification of HS chains with specificity
for FGF-2. A, [H]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 (
). 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
[
H]thymidine when they were grown in the presence
of FGF-2 (1 ng/ml).
Figure 3:
Dose response of cultured 2.3D cells grown
in the presence of purified HS. 2.3D neuroepithelial cells were labeled
with [H]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.
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 () or absence
(
) of FGFR1-A22K. B, cells were incubated with
increasing concentrations of HS (from 2 to 30 ng/ml) in the presence
(
) 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.
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.
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. ()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)
1,4GlcNSO
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. ()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.