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(Received for publication, August 24, 1995) From the
The fibroblast growth factor receptors (FGFRs) are a family of
ligand-activated, membrane-spanning tyrosine kinases. Mutations in
several human FGFR genes have been identified as playing a role in
certain disorders of bone growth and development. One of these, Crouzon
syndrome, an autosomal dominant disorder causing craniosynostosis, has
been associated with mutations in the human FGFR-2 gene. We report here
that microinjection of Xenopus embryos with RNA encoding an
FGFR-2 protein bearing a Cys
The fibroblast growth factors (FGFs) ( Within the last year, several mutations have been identified in FGFR
genes that appear to be the cause of several human disorders of bone
growth and development(2) . One of these, Crouzon syndrome, is
characterized by craniosynostosis, an abnormality of skull development
in which the sutures of the growing bones fuse
prematurely(17) . A variety of mutations in exons IgIIIa and
IgIIIc of FGFR-2 have been identified in Crouzon
syndrome(17, 18, 19) . These mutations may
either directly (Cys
Several mutations have been identified in the IgIII domain of
human FGFR-2 in individuals with Crouzon
syndrome(17, 18, 31) . The most frequently
identified mutation is Cys
Figure 1:
Elongation of
ectodermal explants expressing FGFR-2CS. Animal caps were dissected
from stage 8-9 embryos that were either uninjected (a),
uninjected and treated with 200 ng/ml FGF-1 (b), injected into
both blastomeres at the two-cell stage with 50 pg of FGFR-2 RNA (c), 50 pg of FGFR-2CS RNA (d), 50 pg of FGFR-2CS,
and 100 pg of C4-Raf RNA (e) or 50 pg of FGFR-2CS and 100 pg
of N17-Ras RNA (f). Shown are animal caps incubated until
control sibling embryos reached stage 16.
We also assessed mesoderm induction by
assaying for the expression of mesoderm-specific molecular markers. Xbra, the Xenopus homolog of brachyury, is expressed
broadly in the presumptive mesoderm of gastrulating embryos and has
been shown to be a useful molecular marker for mesoderm induction by
growth factors such as FGF(35) . Wild-type FGFR-2 or FGFR-2CS
RNAs were injected into the animal pole of both blastomeres of two-cell
stage embryos. Animal caps were dissected from blastula stage embryos
(stages 8-9) and cultured until sibling controls reached the
indicated stages (stage 10.5 or 18). RNA was isolated and analyzed by
RNA gel blot hybridization. Fig. 2A shows that Xbra mRNA is expressed in animal caps in a dose-dependent manner
following embryo injection with FGFR-2CS RNA. Injection of as little as
20 pg of FGFR-2CS RNA was sufficient to induce expression of Xbra mRNA, and the level detected was similar to that induced by FGF.
No Xbra mRNA expression was detected in caps injected with a
similar amount of wild-type FGFR-2 RNA or in uninjected control animal
caps. Xbra transcripts were not detected in animal caps
co-expressing FGFR-2CS and either a dominant negative Ras or a dominant
negative Raf.
Figure 2:
Induction of molecular markers of mesoderm
formation by FGFR-2CS. Embryos at the two-cell stage were injected with
either FGFR-2 or FGFR-2CS RNA in the indicated amounts. For experiments
involving dominant negative Raf and Ras mutants, 50 pg of FGFR-2CS RNA
was co-injected with 100 pg of C4-Raf RNA or N17-Ras RNA. A,
animal caps were dissected at stage 8-9 and harvested at either
stage 10.5 or 18 as indicated. RNA was isolated and Xbra mRNA
expression analyzed by RNA gel blot hybridization. The blot was
rehybridized with an 18 S rRNA oligonucleotide probe to serve as an RNA
loading control. B, embryos were injected and animal caps
dissected as described in A. Animal caps were harvested when
sibling control embryos reached stage 22-24, and RNA was isolated
and analyzed by RNA gel blot hybridization for muscle
Muscle-specific A
noteworthy feature of most Crouzon syndrome mutations identified thus
far is the creation or loss of cysteine residues in the IgIII domain of
FGFR-2(2) . Both Cys
Figure 3:
Biochemical analysis of FGFR-2 and
FGFR-2CS expressed in Xenopus oocytes. A, oocytes
were either left uninjected or injected with FGFR-2 RNA or FGFR-2CS
RNA, and lysates were prepared and subjected to SDS-polyacrylamide gel
electrophoresis under reducing and nonreducing conditions as described
under ``Materials and Methods.'' Expression of FGFR-2 and
FGFR-2CS was analyzed by immunoblot analysis with a polyclonal antibody
against FGFR. B, samples were prepared as described above for A and subjected to immunoblotting with a monoclonal antibody
to phosphotyrosine (pTyr, PY20). C, lysates from
injected oocytes were subjected to immunoprecipitation with an
anti-FGFR antibody, and in vitro kinase assays were performed
as described under ``Materials and Methods.'' Electrophoresis
conditions are indicated beneath each panel. The molecular
mass markers (in kilodaltons) are shown to the right.
The IgIII domain of FGFR-2 has been shown to be
important for ligand binding(6, 7) . The Cys
Figure 4:
Cross-linking of
These data establish
that creation of a free, exposed cysteine residue in the IgIII domain
of FGFR-2 results in the formation of an intermolecular disulfide bond
and ligand-independent activation of this receptor. These results
indicate a potential mechanism for the dominant phenotypic effects
observed in Crouzon syndrome and other craniosynostoses involving
mutations in FGFR genes(2) . It is likely that most of these
FGFR mutations are activating ones, and the heterogeneity of clinical
features observed in these syndromes may reflect the degree to which
individual mutations activate the receptor. Alternatively, the
variation in phenotype among individuals with identical point mutations
may indicate that other genes may be involved that modify the effects
of mutated FGFRs. A systematic analysis of each FGFR mutation, as
described here, may shed light on subtle functional differences between
different mutations, which may account for phenotypic variability. The Xenopus system has proven to be a very useful model for
the functional analysis of mutant signal transduction molecules in
vivo(41) . In particular, much has been learned about FGF
receptor structure and function employing this
system(12, 30, 42) . Although the induction
of mesoderm in Xenopus animal caps by a constitutively
activated FGFR-2 does not explain all of the events that lead to
craniosynostosis, it does provide an excellent assay system to
determine the functional consequences of the mutations associated with
these syndromes. Particularly advantageous is the ability to
demonstrate a dose-response relationship between the amount of RNA
injected and the observed biological effects (see Fig. 2). This
type of analysis may be particularly important since all of the FGFR
mutations identified thus far are heterozygous, indicating that
homozygous mutants may be lethal, and thus level of expression may be
linked to severity of disease. While the full mechanistic details on
the etiology of Crouzon syndrome remain to be established, our results
demonstrate that a mutation associated with this syndrome results in
constitutive activation of FGFR-2 with potentially adverse biological
consequences for vertebrate development.
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26037-26040
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Tyr mutation
(FGFR-2CS) found in Crouzon syndrome results in fibroblast growth
factor (FGF)-independent induction of mesoderm in animal pole explants.
Wild-type FGFR-2 did not induce mesoderm when injected at similar
doses. The effects of the mutant receptor were blocked by co-expression
of dominant negative mutants of either Raf or Ras. Analysis of the
mutant receptor protein expressed in Xenopus oocytes indicates
that it forms covalent homodimers, does not bind radiolabeled FGF, and
has increased tyrosine phosphorylation. These results indicate that
FGFR-2CS forms an intermolecular disulfide bond resulting in receptor
dimerization and ligand-independent activation that may play a role in
the etiology of Crouzon syndrome.
)are a family
of polypeptide mitogens that currently consists of nine
members(1, 2) . The FGFs mediate a variety of
biological processes including angiogenesis, wound healing, migration,
mitogenesis, neuronal survival, and mesoderm
induction(2, 3) . These biological effects are
mediated via binding to four members of a family of high affinity
membrane-spanning tyrosine kinase receptors(2, 3) .
The FGFs have also been shown to bind to lower affinity cell surface
heparan sulfate proteoglycans(1, 2) . The prototype
FGF receptor (FGFR) is comprised of an extracellular domain made up of
three immunoglobulin (Ig)-like domains designated IgI-IgIII, a
hydrophobic membrane-spanning region, and a cytoplasmic tyrosine kinase
domain(2, 3) . The amino acid sequences of individual
members of the FGFR family are highly conserved among vertebrate
species(2) . The IgIII domain of FGFR1-3 is encoded by
three exons and is generated by alternative splicing of IgIIIa with one
of two alternative exons designated IgIIIb and
IgIIIc(4, 5) . This alternative splicing generates
receptor isoforms with varying ligand binding
specificities(6, 7, 8) . Like the FGFs
themselves, the FGFRs have unique but overlapping spatiotemporal
patterns of expression during vertebrate
development(9, 10, 11) . The unique patterns
of expression of both FGFs and their receptors during vertebrate
development suggest that each may have a specialized function. Recent
experimental evidence indicates that when FGFR function is disrupted by
genetic manipulation, major defects in embryonic development
occur(12, 13, 14, 15, 16) .
Tyr/Cys
Arg/Cys
Ser/Cys
Phe;
Cys
Phe; Tyr
Cys; Ser
Cys; Ser
Cys) or indirectly
(Ser
Pro; Gln
Pro;
Tyr
His) result in the creation of a free cysteine
residue that could result in covalent dimerization resulting in
ligand-independent activation of the mutant receptor(2) . Here
we report that mutation of Cys
Tyr of Xenopus FGFR-2, analogous to the Cys
Tyr mutation
most commonly found in Crouzon syndrome, promotes activation of the
mutant receptor in the absence of ligand.
Materials
C4-Raf cDNA and N17-Ras cDNA were
gifts of Dr. U. Rapp (National Cancer Institute) and Dr. T. Sargent
(NIH), respectively. Affinity-purified rabbit antibodies to FGFR were
prepared as described previously(20) . A murine monoclonal
antibody to phosphotyrosine, PY20, was obtained from Transduction
Laboratories. Recombinant FGF-1 was a gift from Dr. W. Burgess (Holland
Laboratory).In Vitro Mutagenesis
A BamHI fragment
encoding the entire open reading frame of Xenopus FGFR-2 (10) was subcloned into pTZ19U (Bio-Rad). Mutagenesis of
Cys to Tyr in Xenopus FGFR-2 was performed by
the method of Kunkel et al.(21) using the mutagenic
primer 5`-TCCAGCTATATAAGTATATTCCCC-3` to yield FGFR-2CS. The presence
of the Cys
Tyr mutation and the absence of other
mutations were confirmed by sequence analysis.
Plasmid Construction
All constructs for in
vitro transcription were cloned into the BglII site of
the SP64T or SP64T3 vectors (gifts of Dr. D. Melton, Harvard
University). Synthesis of capped mRNA for microinjection was performed
with SP6 RNA polymerase using a Message Machine kit (Ambion).Embryo Injections
Eggs were collected from Xenopus laevis females and fertilized in vitro as
described previously(22) . Embryos were dejellied 30-60
min after fertilization with 2% cysteine, pH 8.0, and maintained at 17
°C. At the two-cell stage, embryos were transferred to 1 
MMR (5 mM HEPES, pH 7.8, 100 mM NaCl, 2 mM KCl, 1 mM MgSO
, 2 mM
CaCl
, and 0.1 mM EDTA) containing 50 µg/ml
gentamicin and 5% Ficoll. Each blastomere of two-cell embryos was
injected in the animal pole with 5-10 nl of the indicated amount
of RNA.Animal Cap Assays and RNA Gel Blot Analysis
Animal
pole ectoderm (animal caps) was dissected from stage 8-9 embryos (23) and incubated in 0.5 MMR containing 1 mg/ml bovine
serum albumin (BSA) and 50 µg/ml gentamicin in the presence or
absence of 200 ng/ml recombinant FGF-1 at 22 °C. Animal caps were
collected at stage 10.5 or 18 for Xbra mRNA analysis and stage
22-24 for muscle 
-actin mRNA analysis unless otherwise
noted. Total RNA was isolated and analyzed by RNA gel blot as
previously described(24, 25, 26) . To control
for RNA loading, blots were hybridized to a Xenopus 18 S rRNA
oligonucleotide probe(26) .Oocyte Injections and Immunoblot Analysis
Oocytes
were collected and staged according to established
procedures(27) . Oocytes were defolliculated by mild
collagenase treatment and maintained in 1 MBS (27) containing 1 mg/ml BSA and 50 µg/ml gentamicin at 18
°C. Oocytes were injected with 10-20 nl of RNA at the
indicated concentrations and cultured as described above for 1-2
days before immunoblot or cross-linking analysis. Oocytes were
extracted in cold lysis buffer (20 mM HEPES, pH 7.4, 150
mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl, 1.0 mM EGTA, and 0.1 mM
NaVO) extracted with 1 volume of Freon to remove yolk
proteins and centrifuged for 10 min at 10,000 g. For
immunoblot analysis, equal amounts of protein were mixed with an equal
volume of 2 SDS sample buffer and proteins separated by
electrophoresis through 6% SDS-polyacrylamide gels. Proteins were
electroblotted onto nitrocellulose, and immunoblotting procedures were
carried out essentially as described(28) . Bound primary
antibodies were visualized with horseradish peroxidase-conjugated
secondary antibodies and enhanced chemiluminescence procedures (DuPont
NEN).In Vitro Kinase Assay
Oocyte lysates were prepared
as described above, and equal amounts of protein were
immunoprecipitated with an FGFR antibody(20) . Immune complexes
were immobilized onto Protein G-Sepharose beads (Pharmacia Biotech
Inc.), washed extensively with lysis buffer and kinase reactions
performed as described(20) . Samples were separated on 6%
SDS-polyacrylamide gels, and tyrosine-phosphorylated proteins were
visualized by autoradiography (20) .FGF Receptor Cross-linking
Oocytes were injected
with 5 ng of either FGFR-2 or FGFR-2CS RNA and cultured for 2 days
prior to cross-linking analysis. Thirty oocytes expressing either
FGFR-2 or FGFR-2CS were incubated in 0.5 ml of 1 MBS containing
1 mg/ml BSA and 10 units/ml heparin (Upjohn) (binding buffer) in the
presence of 20 ng/ml I-FGF-1 (8-10
10 cpm/ng) prepared as described previously (29) . Oocytes
were incubated at 4 °C for 1 h followed by extensive washing with
binding buffer. Ligand-receptor complexes were cross-linked with
disuccinimidyl suberate (0.3 mM; Pierce) for 15 min at 4
°C as described (29) . Oocyte lysates were prepared as
described above, and ligand-receptor complexes were partially purified
by wheat germ agglutinin-agarose adsorption(30) , followed by
analysis on 7.5% SDS-polyacrylamide gels. Cross-linked complexes were
visualized by autoradiography.
Tyr, resulting in the
generation of a free cysteine residue that may then be available to
form intermolecular disulfide bonds. To test this possibility we
created a mutation in a Xenopus FGFR-2 cDNA (Cys
Tyr) analogous to the human Cys
Tyr
mutation. Fifty picograms of RNA transcribed from wild-type FGFR-2 or
FGFR-2CS plasmids were injected into both blastomeres of two-cell stage Xenopus embryos. At the blastula stage, animal pole ectoderm
(animal caps) was dissected, cultured, and assayed for
elongation(32) . By late neurula stages animal caps from
embryos injected with FGFR-2CS RNA (Fig. 1d) elongated
in a manner similar to control caps treated with FGF-1 (Fig. 1b). Animal caps injected with similar amounts of
wild-type FGFR-2 RNA (Fig. 1c) remained spherical and
resembled uninjected control animal caps (Fig. 1a).
Injection of greater than 200 pg of FGFR-2CS RNA results in cleavage
arrest in blastula stage embryos. (
)FGF-mediated mesoderm
induction and animal cap elongation have been shown previously to be
blocked by the expression of a dominant negative Raf (C4-Raf) or
dominant negative Ras (N17-Ras)(33, 34) . Consistent
with this observation, co-expression of either a dominant negative Raf (Fig. 1e) or dominant negative Ras (Fig. 1f) with FGFR-2CS inhibited FGF-independent
animal cap elongation.
-actin mRNA
expression (arrow). Cytoskeletal actin transcripts (upper
two bands) serve as an internal control for RNA
loading.
-actin mRNA, a late marker for
mesoderm formation(36) , is also induced in a dose-dependent
manner by expression of FGFR-2CS as assessed by RNA gel blot analysis (Fig. 2B). Muscle -actin mRNA is not expressed in
uninjected control animal caps (data not shown) or in caps injected
with a similar amount of wild-type FGFR-2 RNA. The expression of muscle
-actin mRNA is blocked in animal caps co-expressing FGFR-2CS and
either a dominant negative Ras or a dominant negative Raf. Thus, by
both morphological criteria and by the expression of early and late
molecular markers of mesoderm, FGFR-2CS, but not wild-type FGFR-2, has
the ability to induce mesoderm in the absence of exogenous FGF. and Cys
are
predicted to form a disulfide bond essential to formation of the Ig
domain. These two cysteine residues are conserved throughout the FGFR
family . Mutation of either of these two residues could result in
destabilization of the structure of the Ig domain and the creation of a
free cysteine residue. The creation of a free cysteine residue either
directly or indirectly predicts a possible mechanism for the
FGF-independent mesoderm-inducing effects of FGFR-2CS: FGFR
dimerization and ligand-independent activation by formation of an
intermolecular disulfide bond. To examine this possibility, we
microinjected wild-type FGFR-2 or FGFR-2CS RNA into Xenopus oocytes. Lysates from uninjected or injected oocytes were analyzed
by reducing or nonreducing SDS-polyacrylamide gel electrophoresis and
immunoblotting with an FGFR antibody(20) . Under reducing
conditions, both FGFR-2 and FGFR-2CS migrated as monomeric forms of 110
and 125 kDa, whereas under nonreducing conditions FGFR-2CS displayed an
additional species at
260-280 kDa (Fig. 3A),
consistent with the size of a disulfide-linked homodimer. These same
protein samples were then subjected to immunoblot analysis with a
monoclonal antibody to phosphotyrosine. The pattern of phosphotyrosine
immunoreactivity was similar to that observed with the FGFR antibody.
However, although similar amounts of receptor protein were present in
each sample (Fig. 3A), FGFR-2CS displayed higher
amounts of Tyr(P) (Fig. 3B). In addition, under
non-reducing conditions, the
260-280-kDa species seen only
in FGFR-2CS lysates displayed significant Tyr(P) immunoreactivity. To
confirm these results, immune complex kinase assays were performed.
While both FGFR-2 and FGFR-2CS displayed in vitro tyrosine
kinase activity, FGFR-2CS consistently displayed higher activity than
FGFR-2 (Fig. 3C). When immune complex kinase assay
samples were analyzed under non-reducing conditions, a high amount of
the kinase activity exists as a
260-280-kDa species
confirming those results seen by immunoblotting with antibodies to FGFR
and Tyr(P). These data indicate that a large amount of FGFR-2CS exists
as a covalent dimer with elevated tyrosine kinase activity resulting in
constitutive activation of the mutant receptor. Such a mechanism has
been described for mutagenized epidermal growth factor
receptor(37) , erythropoietin receptor(38) , and for
the MEN-2A mutations of the RET receptor-like tyrosine
kinase(39) .
Tyr mutation of FGFR-2CS, in addition to generating a free
cysteine residue capable of forming intermolecular disulfide bonds, may
also disrupt the tertiary structure of IgIII resulting in diminished
ligand binding. To test this possibility, ligand binding and
cross-linking experiments were performed on Xenopus oocytes
microinjected with either wild-type FGFR-2 or FGFR-2CS RNA. Oocytes
injected with wild-type FGFR-2 RNA exhibited a major
I-labeled cross-linked band of 150 kDa (Fig. 4).
Oocytes injected with FGFR-2CS RNA exhibited no increase in
I-FGF-1 binding relative to control oocytes (Fig. 4). These data indicate that mutation of Cys
results in a loss of ligand binding, and thus activation of the
mutant receptor is indeed ligand-independent. These data are consistent
with mutation of an equivalent residue in FGFR-1, which has also been
shown to abrogate ligand binding(40) .
I-FGF-1 to
FGFR-2 and FGFR-2CS. Oocytes injected with either FGFR-2 or FGFR-2CS
RNA were incubated with
I-FGF-1 and subjected to
cross-linking, electrophoresis, and autoradiography as described under
``Materials and Methods.'' The migration of the molecular
mass markers (in kilodaltons) is shown to the right.
))
We thank J. Winkles for critical review of the
manuscript and K. Wawazinski for expert secretarial help. K. M. N.
would like to thank T. Sargent, R. Grainger, and S. Guadagno for
helpful advice during the course of this work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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