(Received for publication, October 20, 1994; and in revised form, December 20, 1994)
From the
We demonstrate that purified fibroblast growth factor (FGF) 3
from Xenopus laevis (XFGF3) activates the mitogen-activated
protein kinase pathway and induces DNA synthesis in quiescent cells. To
characterize the high affinity cell surface receptors that mediate
these responses, the ligand binding domains of different FGF receptors
(FGFR) were expressed on COS-1 cells, and their affinity for XFGF3 was
determined. Unlabeled XFGF3 efficiently competed with I-FGF1 for binding to the IIIb and IIIc isoforms of
FGFR2, giving 50% displacement (ID
) at 0.3-0.8
nM. Higher XFGF3 concentrations were needed to displace
I-FGF1 from FGFR3 and FGFR1 (ID
4 and
21 nM, respectively), indicating that XFGF3 has a lower
affinity for these receptors. No association of XFGF3 with FGFR4 was
found using this assay. FGFR2 isoforms isolated from both mouse and Xenopus showed similar high affinity binding of XFGF3 as
determined by direct binding assays (K
values in the range of 0.2-0.6 nM). These
results indicate that the binding specificity of XFGF3 is different
from that of other FGFs, and identifies FGFR2 as its high affinity
receptor.
The fibroblast growth factor (FGF) ()family is
composed of at least nine members based on amino acid sequence
similarity (1, 2) (reviewed in (3) and (4) ). In cell culture, some FGFs are mitogenic for a broad
spectrum of cell types including epithelial, mesodermal, and neuronal.
Other activities associated with the FGFs include the stimulation of
cell migration, neurotrophic properties, and the induction or
inhibition of cell differentiation, depending on the cell type. They
have been implicated in a number of normal physiological responses,
such as neovascularization, wound repair, as well as inductive and
patterning processes that occur during embryonic and fetal
development(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) (reviewed in (4) ). There is also compelling
evidence to suggest that mesoderm induction in vertebrate embryos
requires the obligatory function of an FGF(17) .
The FGFs
bind to high and low affinity cell surface receptors (for review, see (18, 19, 20) ). The heparan sulfate
proteoglycans constitute the low affinity receptors and facilitate the
interaction of FGFs with the high affinity signaling receptors. To
date, four high affinity receptor genes have been identified in mammals (FGFR1, FGFR2, FGFR3, and FGFR4).
However, identifying the receptor for the different FGF ligands is
complicated by the generation of different receptor isoforms from the
same gene by alternative
splicing(21, 22, 23, 24, 25, 26) .
The basic structure of FGF receptors consists of an extracellular
portion composed of three immunoglobulin-like domains (Ig-loops), a
transmembrane segment, a juxtamembrane region, and a split tyrosine
kinase domain. Some FGF receptor variants lack the first Ig-loop and
may or may not contain a region rich in acidic residues (acid box)
which resides between Ig-loops I and II. The consequences of lacking
the first Ig-loop or acid box are not clear as the truncated receptors
appear to function normally(23, 27) . The three and
two Ig-loop receptors have been termed and
, respectively.
FGF receptor genes 1, 2, and 3, but not 4, encode a choice of exon for
the second half of the third Ig loop which changes ligand binding
specificity(26, 27, 28, 29) . The
receptor isoforms generated by the alternate splice are termed IIIb and
IIIc, respectively. Other vertebrates also appear to encode the
analogous receptor genes with isolates described from chicken,
amphibians, and
fish(22, 30, 31, 32, 33, 34) .
FGF binding causes the activation of an intrinsic receptor tyrosine
kinase activity and autophosphorylation. The receptor phosphotyrosine
residues have been shown in some instances to form src homology 2 (SH2) binding sites that interact with second messenger
generators to propagate an intracellular signal (reviewed in (18) and (19) ).
FGF3 was first identified as the product of a cellular oncogene (formerly int-2) associated with virally induced mouse breast cancers (reviewed in (35) ). The gene is not detectably transcribed in normal mouse mammary tissue, suggesting that inappropriate expression contributes to tumorigenesis. This idea was strengthened by the induction of proliferative abnormalities and tumors in the mammary glands of transgenic mice ectopically expressing FGF3 (36, 37, 38) . Apart from its potential oncogenic properties, considerable interest in FGF3 arises from its suspected role in embryonic and fetal development(6, 12, 14, 39, 40) . The generation of mice with a Fgf-3 null genotype (11) has confirmed the importance of this gene for proper development, since these mice exhibit structural abnormalities of the tail and many have inner ear defects resulting in differing degrees of deafness. However, abnormalities were not found at all sites of Fgf-3 expression, suggesting a degree of signaling redundancy or lack of FGF3 function at some of these sites.
The inefficient
secretion of mouse FGF3 in cell cultures (41, 42) and
its insolubility as a recombinant protein expressed in prokaryotes ()have severely hampered the isolation of active protein.
These problems have now been circumvented by the isolation of the FGF3
homolog from Xenopus laevis (XFGF3) which shows a high level
of amino acid identity to the mouse protein, but is efficiently
secreted. Furthermore, conditioned medium containing this protein is
mitogenic and induces phenotypic transformation in a number of
mammalian cell lines. Here we use purified XFGF3 to identify its high
affinity receptors by specific binding, Scatchard analysis, and
covalent cross-linking. We show that, in quiescent cells expressing the
appropriate receptors, XFGF3 activates the MAP kinase pathway, and
reinitiates DNA synthesis.
The extracellular, transmembrane, and juxtamembrane domain sequence of Xenopus FGFR2 (IIIb) and (IIIc) variants were cloned by reverse transcription of RNA from stage 22/23 Xenopus embryos followed by cDNA amplification using the RT-PCR. For the PCR the 5` primer was CGGAATTCACCATGGGGATGTCCTTAGTGTGGCGT and the 3` primer CTCCCACATGGGATCCTGTGGTAGCTC. A unique BamHI site (underlined) was introduced at the same position as the naturally occurring BamHI site in the mouse FGFR1, which results in the change of a histidine to glutamine. The PCR products were inserted as EcoRI-BamHI fragments in pGEM4 (Promega).
The extracellular, transmembrane, and juxtamembrane domain sequences encoding the mouse FGFR2 (IIIb) and (IIIc) variants were cloned by RT-PCR using total RNA from hindbrain and tail of 9.5- and 12.5-day embryos, respectively. The 5` primer was CGGAATTCCATGGTCAGCTGGGGTCGTTTCATC and the 3` primer was TGCTCTAGATTTGCCCAGCGTCAGCTTATCTCT. The PCR products were digested by EcoRI/BamHI and ligated into pGEM4.
To construct full-length receptors, the extracellular, transmembrane, and juxtamembrane domain sequences of the different receptors were linked to the region encoding the tyrosine-kinase and cytoplasmic tail of mouse FGFR1, via a unique BamHI site. These hybrid cDNAs were subcloned into the EcoRI and XbaI sites of the SV40-based expression vector, pKC3.
All PCRs were performed using the Pyrococcus furiosus DNA polymerase (Stratagene) which has proofreading activity. The plasmid constructs were then sequenced using Sequenase version 2.0 (U. S. Biochemical Corp.).
Figure 1:
Induction of DNA synthesis by XFGF3,
FGF1, and FGF7. Quiescent BALB/MK (Panel a) or NIH3T3 cells (Panel b) were treated with increasing concentrations of XFGF3 (closed circles), FGF1 (open squares), or FGF7 (filled squares), and the stimulation of DNA synthesis was
measured by the incorporation of [H]thymidine as
described under ``Materials and Methods.'' The mean value of
duplicate determinations is shown. Results are representative of at
least two experiments.
An emerging paradigm of ligand-mediated activation for several classes of tyrosine-kinase receptor is the stimulation of the MAP kinase pathway (for review, see (50) ). Two isoforms of these kinases (Erk-1 and Erk-2) are present in most mammalian cells, and, within minutes of ligand/receptor binding, these proteins are activated by phosphorylation on both threonine and tyrosine residues(51, 52) . Since the hyperphosphorylated Erks have a reduced mobility on SDS-PAGE, the activation can be monitored by immunoblot analysis of cell extracts (53) . Treatment of serum-deprived NIH3T3 cells with 50 ng/ml XFGF3 or FGF1 resulted in a rapid and transient phosphorylation of Erk-1 and -2 (p44 and p42, respectively) which was detected within 1 min of ligand addition. The hyperphosphorylated state was maintained for at least 15 min but was no longer detected at 30 min (Fig. 2). The activation of MAP kinases is part of a phosphorylation cascade involving a MAP kinase kinase and a MAP kinase kinase kinase (MAPKKK). The serine/threonine kinase Raf-1 has been shown to function as a MAPKKK (reviewed in (50) ). To determine whether, Raf-1 (p74) was involved in the stimulation of the MAP kinase pathway by XFGF3, an additional blot, prepared using the same extracts, was probed with a specific Raf-1 antiserum. The results show that the reduced mobility, reflecting hyperphosphorylation of Raf-1(54) , became apparent 15 and 30 min after treatment with either FGF1 or XFGF3. Although activation of Raf-1 would be expected to precede that of MAP kinase, the hyperphosphorylation response was delayed compared to that of p44 and p42. However, it is not clear whether the detected phosphorylation of Raf-1 correlates with its activation (see ``Discussion''). Similar results were obtained when BALB/MK cells were used instead of NIH3T3 (data not shown).
Figure 2: FGF1 and XFGF3 induces hyperphosphorylation of Raf-1, Erk-1, and Erk-2. Quiescent NIH3T3 cells were treated with 50 ng/ml FGF1 or XFGF3 for 1-30 min as indicated. Equal amounts of cell extracts were analyzed by immunoblotting using polyclonal antibodies that recognize Raf-1 (Panel a) or Erk-1 and -2 (Panel b).
Figure 3:
Cross-linking of I-XFGF3 to
BALB/MK and NIH3T3 cells. Cell cultures were incubated with 10 ng/ml
I-XFGF3 in the absence or presence of a 20-fold excess of
unlabeled XFGF3 (- or +, respectively). The receptor/ligand
complexes were cross-linked and analyzed by SDS-PAGE as described under
``Materials and Methods.'' The size of the labeled proteins
indicated on the left were estimated relative to rainbow size
markers (Amersham Corp.).
Figure 4:
Competition of FGF1 and XFGF3 for I-FGF1 binding to different FGF receptors. COS-1 cells
expressing different receptors as indicated were incubated with 0.3
nM
I-FGF1 in the presence of increasing
concentrations of XFGF3 (open circles) or FGF1 (filled
circles). Cells were then washed and lysed, and specific binding
was determined as described under ``Materials and Methods.''
The calculated ID
values are presented in Table 1.
Figure 5:
Cross-linking of I-XFGF3 to
COS-1 cells expressing FGFR2 variants. COS-1 cells were transfected
with the indicated receptor cDNA and 48 h later were incubated with 10
ng/ml
I-XFGF3 in the absence or presence a 20-fold excess
of unlabeled XFGF3 (- or +, respectively). The
receptor/ligand complexes were cross-linked and analyzed by SDS-PAGE as
described under ``Materials and
Methods.''
Figure 6:
Alignment of the predicted amino acid
sequences for XFGFR2 (IIIc), XFGFR2 (IIIb
),
and XFGFR2 (IIIb
+ab). a, the arrowhead marks the predicted site for signal peptide cleavage, and the circles indicate the cysteine residues which define the
disulfide links of the extracellular Ig domains. Underlined is
the position of the acid box, and the dashed gap in XFGFR2
(IIIb
+ab) marks the missing sequences of Ig-loop-I absent in
all
-forms. The transmembrane region is under and overlined, while the region of divergence between the IIIc and
IIIb isoforms is boxed. b, an amino acid sequence comparison
of the divergent domains of Ig-loop III for XFGFR2 IIIb and IIIc
isoforms. c and d show a comparison of the IIIb and
IIIc isoforms, respectively, of FGFR2 from frog (Xenopus),
salamander, newt, chicken, mouse, and
human(30, 33, 49, 56, 57, 58, 59, 60) .
The numbers to the right refer to the percentage amino acid
identity in comparison with the Xenopus sequence. A dash indicates a gap, and a dot indicates a conserved
residue.
Figure 7:
Cross-linking of I-XFGF3 and
I-FGF1 to COS-1 cells expressing Xenopus FGF
receptors. a, COS-1 cells were transfected with the indicated
receptor cDNA and 48 h later incubated with 10 ng/ml
I-XFGF3 in the absence or presence a 20-fold excess of
unlabeled XFGF3 (- or +, respectively). The receptor/ligand
complexes were cross-linked and analyzed by SDS-PAGE as described under
``Materials and Methods.'' b, COS-1 cells expressing
the Xenopus receptors, as indicated, were incubated with 10
ng/ml
I-FGF1 in the absence or presence (- or
+, respectively) of a 15-fold excess of unlabeled XFGF3 and
processed as in a. The size of the labeled proteins indicated
on the left were estimated relative to rainbow size markers
(Amersham Corp.).
The affinity of XFGF3 for the IIIb and IIIc variants
of XFGFR2 was determined using I-XFGF3 in direct binding
assays (data not shown). From the Scatchard plots, K
values in the range of 0.4-0.6 nM were calculated
similar to those determined for the mouse FGFR2 homologs (see Table 1). In similar experiments, no binding to XFGFR1
(IIIc
) was detected, and therefore the ability of unlabeled XFGF3
to compete with
I-FGF1 for binding was used as an
alternative means to assess affinity (Fig. 8). Increasing
amounts of XFGF3 or FGF1 in the presence of 0.3 nM
I-FGF1 were added to COS-1 cells expressing XFGFR1
(IIIc
), and the retained radioactivity was measured by
counting. As a positive control, COS-1 cells expressing XFGFR2
(IIIc
) were used in a parallel experiment. An ID
of
23 nM was determined for the binding of XFGF3 to XFGFR1
(IIIc
) compared to 0.8 nM for XFGFR2 (IIIc
),
implying an approximately 29-fold lower affinity (see Table 1).
The ID
value for XFGFR2 (IIIc
) is consistent with the K
of 0.5 nM determined using the direct
binding procedure (Table 1). As expected, efficient competition
by FGF1 occurred on both receptors (Fig. 8).
Figure 8:
Competition of FGF1 and XFGF3 for I-FGF1 binding to Xenopus FGF receptors. COS-1
cells expressing XFGFR1 (IIIc
) (Panel a) or XFGFR2
(IIIc
) (Panel b) were incubated with 0.3 nM
I-FGF1 in the presence of increasing concentrations
of unlabeled XFGF3 (open circles) or FGF1 (filled
circles). Cells were then washed and lysed, and specific binding
was determined as described under ``Materials and Methods.''
The calculated ID
values are presented in Table 1.
Comparison of the predicted amino acid sequences for each of
the different FGF receptors shows a high level of conservation across
several species, which is much more marked than that seen for the
different FGF receptors within a single species(18) .
Therefore, it could be predicted that ligand binding specificity is
similar for the same receptor from different species. Indeed, direct
binding and competition analyses have shown that XFGF3 binds with the
same high affinity to both mouse and Xenopus FGFR2, and it
also interacts with the same low affinity with either mouse or Xenopus FGFR1 (IIIc) (Table 1). In addition, a weak
interaction was also observed with human FGFR3 (IIIc
), while no
binding to mouse FGFR4 could be detected ( Fig. 4and Table 1). Considering the strong sequence conservation between
mammalian and Xenopus receptors (Fig. 6, c and d) (22, 55) , we would predict the same is
true for the Xenopus homologs of FGFR3 (IIIc
) and FGFR4.
The high affinity binding to FGFR2 suggests it is the most likely
partner for XFGF3 at physiological ligand concentrations. In addition,
the affinities of XFGF3 for the a- and b-variants of FGFR2 were found
to be very similar (Table 1). This finding is in contrast to a
report that shows FGF1 affinity for the
-form of FGFR1 being
8-fold higher than its affinity for the
-form(61) . This
discrepancy may be due to the different cell types that were used to
overexpress the receptors, as factors such as the structure of
cell-derived heparan sulfates may modulate ligand binding affinity, and
potentially specificity(62, 63, 64) .
Alternatively, it may be an inherent property of the ligand/receptor
combination and could result from nonidentical ligand binding domains
(see below).
Whereas XFGF3 binds to the IIIb and IIIc variants of FGFR2 with similar affinities, FGF7 interacts exclusively with the IIIb isoform, and FGF2 shows a strong preference for the IIIc isoform (Table 1)(24, 65) . Like XFGF3, FGF1 binds both variants with high affinity, but then it also associates strongly with all the other FGF receptors and their known variants. Recent studies indicate that Ig-loops II and III contribute to the ligand binding site, albeit to different degrees depending on which FGF is involved in the interaction. For example, when individual Ig-loop domains of FGFR2 (IIIb) are fused to an immunoglobulin heavy chain Fc domain, Ig-loop II confers binding to FGF1 but not to FGF7, while Ig-loop III confers the converse specificity. However, in both cases the affinity is lower than that of a construct containing both Ig-loops(66) . The importance of Ig-loop II for FGF binding is further illustrated by analyses of chimeric receptors made between FGFR2 and either FGFR1 or FGFR3; whereas both Ig-loop II and the IIIb domain from FGFR2 are necessary to confer FGF7 binding when transferred to FGFR1, the single IIIb domain from FGFR2 is sufficient to confer this property when transferred to FGFR3(28, 67) . Additional experiments using such constructs should permit precise localization of the sites that interact with the ligand. However, to establish a complete model of ligand/receptor interaction, the regions on the FGF molecules themselves that determine receptor binding specificity need to be defined. Chimeric ligands made between the various FGFs could help to provide such information.
XFGF3 was shown to be mitogenic for BALB/MK and NIH3T3 cells which naturally express the IIIb and IIIc isoforms of FGFR2, respectively (Fig. 1). Furthermore, at XFGF3 concentrations that induce maximal DNA synthesis, hyperphosphorylation of Erk-1 and Erk-2 was detected 1 min after ligand addition (Fig. 2), suggesting efficient activation of the MAP kinase pathway. Phosphorylation of Raf-1 was observed after 15 min, which was surprising since Raf-1 activation is thought to precede that of Erk-1 and Erk-2(50) . However, the functional significance of Raf-1 phosphorylation is not clear, and similar results from other groups have led to the suggestion that Raf-1 phosphorylation serves to inactivate this kinase, thereby facilitating a transient response (68) . Whatever the functional consequences of Raf-1 phosphorylation, these studies provide evidence for the activation of the Raf-1/MAP kinase pathway by FGFs in cells expressing endogenous FGF receptors, confirming previous results obtained with receptor-transfected L6 myoblasts(69, 70) , and Xenopus embryos(71, 72) .
As might be expected for a ligand and its cognate receptor, there is a striking similarity between the expression patterns of XFGF3 and XFGFR2 (55) . Furthermore, in amphibians, the levels of both FGF3 and FGFR2 have been shown to increase upon neural induction(40, 58) . Some insights into the functions of FGFs and their receptors in vivo derive from transgenic animal studies. For example, mice null for Fgf-3 or Fgf-5 were found to have specific but restricted phenotypic alterations(11, 73) . The limited phenotypic consequences of these gene knockouts despite extensive gene expression patterns, suggests there may be some functional complementation. Similar experiments designed to generate mice null for the FGF receptor genes may have more serious consequences since signaling by several FGF-ligands could be affected. An alternative strategy is to target the expression of a dominant negative form of a given receptor exclusively to one tissue/organ at a stage when endogenous expression of this receptor is known to occur. This was recently achieved by Peters et al.(74) who showed that expressing a dominant negative FGFR2 (IIIb) in the lung bud epithelium of transgenic mice results in a complete inhibition of branching morphogenesis, clearly implicating this receptor in lung organogenesis. Since FGF3 disrupts mammary gland development(36, 37, 38) , we are using a similar stategy to determine whether signaling via FGFR2 (IIIb) is necessary for normal breast morphogenesis. Thus, knowledge of FGF and FGFR specificities combined with data on the times and sites of their expression will facilitate the design of experiments to determine the functions of these signaling molecules in animal development.