(Received for publication, August 14, 1995; and in revised form, November 8, 1995)
From the
Receptor specificity is an essential mechanism governing the activity of fibroblast growth factors (FGF). To begin to understand the developmental role of FGF-9/glial activating factor, we have cloned and sequenced the murine FGF-9 cDNA and expressed the protein in mammalian cells and in Escherichia coli. We demonstrate that the FGF-9 protein is highly conserved between mouse and human. Receptor specificity was determined by direct binding to soluble and cell surface forms of FGF receptor (FGFR) splice variants and by the mitogenic activity on cells, which express unique FGF receptor splice variants. Our data demonstrate that FGF-9 efficiently activates the ``c'' splice forms of FGFR2 and FGFR3, receptors expressed in potential target cells for FGF-9. Significantly, FGF-9 also binds to and activates the ``b'' splice form of FGFR3, thus becoming the first FGF ligand besides FGF-1 to activate this highly specific member of the FGF receptor family.
Fibroblast growth factors (FGFs) ()are a family of at
least nine related polypeptides that can differentially activate a
family of four related tyrosine kinase
receptors(1, 2, 3) . FGFs are involved in
embryonic and fetal development, neovascularization, wound healing, and
neoplastic transformation. Glial activating factor (GAF), a polypeptide
secreted by the human glioma cell line, NMC-G1, was originally
identified as a trophic factor for primary rat glial cells(4) .
Purified GAF is a potent mitogen for glial cells, rat primary cortical
astrocytes, BALB/c3T3 fibroblasts, and oligodendrocyte-type 2 astrocyte
progenitor cells(4) . GAF has weak mitogenic activity for rat
adrenal pheochromocytoma cells (PC-12) and no activity toward human
umbilical vein endothelial cells(4) . Similarities between GAF
and FGFs are based on their affinity for heparin, heat stability, and
chromatographic behavior. However, target cell specificity indicates
significant differences in activity between GAF and FGF-2. The cloning
and sequencing of the GAF cDNA revealed approximately 30% sequence
homology with members of the FGF family and complete preservation of
conserved amino acids that define the FGF
family(1, 2) . Because of both the biochemical and
sequence similarities, GAF was classified as the ninth member of the
FGF family and will henceforth be referred to as FGF-9. Using a rat
cDNA probe, FGF-9 expression was detected in rat brain and kidney but
not in liver, lung, spleen, thymus, testis, heart, or adrenal
gland(2) . Like FGF-1 and FGF-2, FGF-9 lacks a signal peptide
sequence. Nevertheless, FGF-9 is secreted by the glioma cell line,
NMC-G1, and by transfected COS and Chinese hamster ovary
cells(2) .
FGFs differentially bind to and activate up to four related transmembrane receptors, which in turn mediate a biological response. FGF receptors (FGFRs) are members of the tyrosine kinase receptor superfamily(5) . The extracellular region of the FGFR contains two or three immunoglobulin-like (Ig-like) domains that are differentially expressed as a result of alternative splicing(5) . Additionally, another alternative splicing event can alter the sequence of the carboxyl-terminal half of Ig-like domain III without altering the reading frame of the remainder of the receptor. These two splice forms, referred to as ``b'' and ``c,'' occur for FGFRs 1, 2, and 3 but not 4(6, 7, 8, 9) . The specificity of FGF ligand-receptor interaction involves the region of the FGFR ectodomain encompassing Ig-like domain II and III and is dependent on the alternative splicing event in Ig-like domain III(8, 10, 11) . The proposed sequence of events involved in the activation of FGFRs includes the formation of a complex between FGF, a heparin-like molecule or a heparan sulfate proteoglycan and an FGFR (12, 13, 14, 15) . The initial binding event is followed by receptor dimerization, autophosphorylation, and the subsequent activation of downstream signaling molecules(16, 17, 18, 19, 20) .
In this study, the biochemical characteristics of FGF-9 are further elucidated. We have cloned the murine FGF-9 cDNA and demonstrate that it can transform NIH3T3 fibroblasts. We demonstrate that recombinant murine FGF-9 requires heparin for optimal receptor activation and that FGF-9 preferentially binds to and activates FGFR2c and FGFR3c. Additionally, we demonstrate that unlike FGFs 2-8, FGF-9 is able to bind to and activate FGFR3b.
Soluble FGFR (1c, 2b, 3b, and 3c)-alkaline phosphatase fusion
proteins were made in COS cells as described
previously(8, 18) . Binding components were added at 4
°C in the following order: Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) with 0.1% bovine serum albumin, 50
µl of a 2 slurry of anti-alkaline phosphatase monoclonal
antibodies coupled to Sepharose(25) , 10 µl of 25 µg/ml
heparin, 50 µl of FGFR-alkaline phosphatase-conditioned media
containing specific soluble FGFRs (0.3 optical density
units/min)(18) , non-iodinated FGF as a competitor, and
I-FGF (20,000 cpm) in a total volume of 250 µl. The
reaction was then gently rotated for 90 min at 4 °C. Bound receptor
and FGF were recovered by centrifugation (10 s at 12,000 rpm (4000
g), 4 °C in a microcentrifuge), and washed two
times with 500 µl of ice-cold phosphate-buffered saline.
I-FGF binding was determined by counting the washed tubes
directly in a
counter (Beckman). Binding to cell surface FGF
receptors was performed as for the soluble FGF receptors except that
anti-alkaline phosphatase-Sepharose and FGFR-alkaline phosphatase were
replaced with 3
10
FGFR-expressing BaF3 cells.
20,000-60,000 cpm of iodinated FGF were incubated with cells in
the presence of 2 µg/ml heparin for 2 h at 4 °C. Cells were
washed and counted as described above.
The plasmid pSVFGFR1b was provided by Werner et al.(7) . A 2.9-kb BamHI-SpeI fragment was cloned into the corresponding sites of MIRB. FGFR1c (14) was cloned as a 3.2-kb EcoRI fragment into MIRB by converting a 3`-Asp-718 site into an EcoRI site and then excising with EcoRI. FGFR2b (26) was cloned as a 2.9-kb BamHI fragment into MIRB by converting a 5`-Asp-718 site into a BamHI site and then excising with BamHI. FGFR2c (27) was cloned into MIRB as a 3.6-kb SpeI fragment by converting unique NarI and XbaI sites into SpeI sites.
The FGFR3 cDNAs were reengineered to enhance signaling in BaF3 cells by constructing chimeric cDNAs encoding the extracellular region of FGFR3 fused to the cDNA encoding the tyrosine kinase domain of FGFR1. FGFR31c cells express an FGFR that has the extracellular region from FGFR3c (28) and transmembrane domain and tyrosine kinase domain from FGFR1; FGFR31b cells express an FGFR that has the extracellular region and transmembrane domain derived from FGFR3b (8) and tyrosine kinase domain derived from FGFR1. The details of these chimeric receptors will be described elsewhere.
The mouse FGF-9 cDNA was excised with HindIII and EcoRI, blunted with the Klenow fragment of DNA polymerase, ligated to BamHI linkers, re-excised with BamHI, and then cloned into the BamHI site of the MIRB expression vector. MIRB-FGF-9 was then transfected into mammalian cells.
BaF3 cells
expressing specific FGFRs were washed and resuspended in
Dulbecco's modified Eagle's medium, 10% neonatal bovine
serum, L-glutamine. 22,500 cells were plated per well in a
96-well assay plate in media containing 2 µg/ml heparin, except
where indicated. Test reagents were added to each well for a total
volume of 200 µl per well. The cells were then incubated at 37
°C for 2 days. To each well, 1 µCi of
[H]thymidine was then added in a volume of 50
µl. Cells were harvested after 4-5 h by filtration through
glass fiber paper. Incorporated [
H]thymidine was
counted on a Wallac
plate scintillation counter.
Figure 1: cDNA sequence of FGF-9. The nucleotide and amino acid sequence of human and murine FGF-9 (hFGF-9 and mFGF-9, respectively) is shown. Oligonucleotide primers used to amplify FGF-9 are underlined. Coding sequence begins at human base pair 151 and ends at base pair 774 and is shown above the nucleotide sequence. Differences between the human and murine nucleotide sequence are depicted in bold type in the murine sequence. The two amino acid differences are depicted in bold type with the human amino acid listed before the murine amino acid.
Figure 2: Gel electrophoretic analysis of recombinant FGF-9. Purified recombinant FGF-9 was run on an 18% SDS-polyacrylamide gel and stained with Coomassie Blue. Left, purified recombinant FGF-9; Right, protein molecular mass markers (in kDa). Molecular mass standards used were bovine serum albumin (67 kDa), human growth hormone (21 kDa), FGF-2 (17.5 kDa), lysozyme (14 kDa), and epidermal growth factor (6 kDa).
Figure 3: FGF binding to soluble FGFR-alkaline phosphatase fusion proteins. A, binding of iodinated-FGF-9 to soluble FGFRs 1c, 2b, 3b, and 3c. Shaded bars, iodinated FGF-9; solid bars, iodinated FGF-9 in the presence of 20 nM unlabeled FGF-9. B, binding of iodinated FGF-9 to soluble FGFR3b without competitor(-) or with 20 nM unlabeled FGF-1, 2, or 9 as indicated. C, relative binding of iodinated FGF-1 to soluble FGFRs 1c, 2b, 3b, and 3c. Shaded bars, no competitor; open bars, competition with 80 nM unlabeled FGF-1; solid bars, competition with 80 nM unlabeled FGF-9. Error bars indicate standard deviation.
The observed binding
of FGF-9 to soluble FGFR3b was surprising in that no other FGF ligand,
besides FGF-1, can bind to or activate this receptor(8) . ()Therefore, to further characterize this binding
interaction, we tested the ability of FGF-1, FGF-2, and FGF-9 to
compete with the binding of labeled FGF-9 to FGFR3b. Both FGF-1 and
FGF-9 can efficiently compete for binding to FGFR3b; however, FGF-2 was
only a weak competitor (Fig. 3B). This is consistent
with the inability of FGF-2 to bind to or activate FGFR3b(8) .
FGF-1 binds with high affinity to all known FGFRs(7, 8, 9, 10, 26, 28, 29, 30) . Competition binding experiments demonstrate that FGF-9 cannot compete with labeled FGF-1 for binding to FGFR1c yet does demonstrate an increasing ability to compete with FGF-1 for binding to FGFR2b, FGFR3b, and FGFR3c (Fig. 3C), respectively. These data are consistent with the direct binding experiments in which FGF-9 preferentially binds to FGFR3c and less well to FGFR2b and FGFR3b. The ability of FGF-9 to compete with labeled FGF-1 for receptor binding is poor compared to that of unlabeled FGF-1. However, this observation is consistent with previous results in which FGF-7 could only partially compete with FGF-1 for binding to its primary receptor, FGFR2b(8) .
Figure 4:
Mitogenic activity of recombinant FGFs.
Mitogenic activity ([H]thymidine incorporation)
of BaF3 cells expressing specific FGFR splice forms in the presence of
recombinant FGF-1 (open circles), FGF-7 (open
squares), FGF-8b (open diamonds), and FGF-9 (closed
triangles). Error bars indicate standard deviation. A, FGFR1b; B, FGFR1c; C, FGFR2b; D,
FGFR2c; E, FGFR3b; F,
FGFR3c.
To
assess the relative mitogenic activity on individual FGFR splice
variants, we normalized the mitogenic data in Fig. 4to that of
FGF-1. The relative mitogenic activity for each ligand
([H]thymidine incorporation) at concentrations of
312 and 1250 nM was calculated. These values were then
averaged and plotted in Fig. 5. This analysis clearly
demonstrates that the best receptors for FGF-9 are FGFR2c and FGFR3c.
These FGFRs can be activated by FGF-9 with 89 and 96% of the activity
of FGF-1, respectively. FGFR3b-expressing cells respond to FGF-9 with
42% of the activity of FGF-1, and FGFR1c-expressing cells respond to
FGF-9 with 21% of the activity of FGF-1.
Figure 5: Relative mitogenic activity of FGFs. Mitogenic activity at concentrations of 312 and 1250 pM for each FGF (see Fig. 4) was normalized to that of FGF-1 and then averaged and plotted. Error bars indicate the average of the standard deviations at the two FGF concentrations used. Solid bars, FGF-1; open bars, FGF-7; hatched bars, FGF-8; shaded bars, FGF-9.
Figure 6: Relative mitogenic activity of native FGF-9. The relative mitogenic activity of BaF3 cells expressing FGFR1c, FGFR3b, and FGFR3c is shown in the presence of 100 pM FGF-1 (shaded bars), 10 µl of conditioned media from confluent FGF-9 transformed NIH3T3 cells (solid bars), or control NIH3T3 cell-conditioned media (open bars). The data were normalized to that of FGF-1. Error bars indicate standard deviation.
Figure 7:
Heparin dependence of FGF-1 and FGF-9.
Mitogenic activity ([H]thymidine incorporation)
of FGFR3c-expressing BaF3 cells at varying concentrations of heparin in
the presence of 200 pM FGF-1 (circles) and 200 pM FGF-9 (squares) is shown.
FGFs compose a family of growth factors that play key roles
in a variety of developmental events. Some FGFs are expressed in adult
tissues and may be important for maintaining normal tissue homeostasis.
FGFs are also involved in mediating a physiological response to injury (31) . In adult mice, FGF-9 is expressed in both brain and
kidney(2) . During development, FGF-9 is expressed at low
levels in mid-gestation mouse embryos. ()Thus, it is likely
that FGF-9 plays a role in both developmental events and in normal
adult physiology. Important elements controlling the activity of FGFs
include tissue- and temporal-specific gene expression and specificity
of ligand-receptor interactions. In this study, we have determined the
receptor specificity of FGF-9, thus identifying its potential
physiologically relevant receptors.
Our data indicate that the
preferred receptors for FGF-9 are FGFR2c, FGFR3c, and FGFR4. ()Additionally, the binding and mitogenic data presented
here demonstrate that FGF-9 can also bind to and activate FGFR3b.
Although the activity of FGF-9 toward FGFR3b is only 42% of that of
FGF-1, it is nevertheless significant because no other FGF ligand shows
any activity toward this receptor (8) .
FGFR2 is expressed in glial cells(32) , low grade astrocytomas(33) , and oligodendrocytes present in fiber tracts of the central nervous system(34) . FGFR2 transcripts have been identified in the germinal epithelium of the developing central nervous system and, later in development, in a diffuse pattern consistent with expression in glial cells(35) . FGFR2 is also prominently expressed in epithelial tissues in limb bud, kidney, stomach, and lung(35, 36) . FGFR3 mRNA has been localized to the germinal epithelium of the developing central nervous system, to glial cells (later in development), to the sensory epithelium of the cochlea, to proliferating cartilage of developing bone, and to the lens of the eye(37) . Although the cellular localization of FGFR3b and FGFR3c is not known, RNase protection studies indicate that both splice forms are expressed in kidney(8) . In the case of FGFR2, ``b'' splice forms are restricted to epithelial tissues, and ``c'' splice forms are expressed in mesenchymal tissues (10, 38) .
The identification of FGFR2 and FGFR3 expression in glial cells and the identification of a functional ligand, FGF-9, expressed in the mouse and rat brain suggests that FGF-9, FGFR2, and FGFR3 may participate in either autocrine or paracrine loops in the central nervous system. The discovery of FGF-9 secretion by a glioma cell line (2, 4) also supports the functional pairing of FGF-9 with these receptors and may account for the formation of the tumor that originally gave rise to this cell line. Additional support for FGF-9 forming physiologically relevant ligand-receptor pairs with FGFR2 and FGFR3 comes from gene expression in kidney where FGF-9(2) , FGFR2c, FGFR3, and FGFR4 are all expressed (30, 37, 39) . FGF-9 may therefore play a role in both neural and renal development, tissue homeostasis, and response to injury. Spatial localization of FGF-9, FGFR2, and FGFR3 at the cellular level should help to define the physiological relationship between these signaling molecules.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33535[GenBank].