From the
Two tandem immunoglobulin-like disulfide loops (Loops II and
III) linked by a short connecting sequence in the ectodomain of the
fibroblast growth factor receptor kinase compose the binding sites for
glycosaminoglycan and fibroblast growth factor (FGF) ligands. Alternate
splicing of exons IIIb and IIIc coding for the COOH-terminal half of
Loop III confers high affinity for FGF-7 or FGF-2, respectively, on the
fibroblast growth factor receptor ectodomain without effect on the
binding of FGF-1. Here we show that a 139-amino acid fragment composed
of Loop II, the inter-Loop II/III sequence, and a short segment of the
NH
The heparin-binding fibroblast growth factor (FGF)
The
tyrosine kinase component of the complex consists of the products of
four genes, FGFR1, FGFR2, FGFR3, and FGFR4
(2) . At least 16
single-site splice variations in the FGFR1 and FGFR2 genes form
combinatorial variants in the extracellular and intracellular
juxtamembrane and intracellular kinase and COOH-terminal domains
(2, 8, 9, 10, 11, 12) .
The heparan sulfate- and ligand-binding domain of the FGFR
transmembrane tyrosine kinase is composed of Ig-like disulfide loop
units
(13, 14) and interloop sequences encoded by one
or two exons
(9) . Tandem Loops II and III form the heparan
sulfate- and ligand-binding sites
(5) . A highly conserved
18-amino acid sequence in the NH
Although FGF-1, FGF-2, and FGF-7 are
expressed in many tissues, FGF-7 is a specific product of stromal
fibroblasts in tissues with distinct epithelial and stromal cell
compartments
(11, 18, 19) . Epithelial cells
display the FGFR2(IIIb) isoform, while stromal fibroblasts and other
non-parenchymal cells display the FGFR1(IIIc) isoform
(11, 16, 18) . The stromal cell-specific
expression of FGF-7 and epithelial cell-specific expression of
FGFR2(IIIb) appear to constitute a directionally specific paracrine
communication system from stromal to epithelial cells. Subversion of
the directional specificity occurs in a variety of tumors by a switch
from expression of the FGFR2(IIIb) to the FGFR2(IIIc) isoform and
subsequent activation of the FGF-2 gene, other embryonic isoforms of
the FGF ligand family, and the normally mesenchymal cell-associated
FGFR1(IIIc) isoform of the receptor in epithelial cells
(11, 20, 21) . Since the differential
specificity of the FGFR(IIIb) and FGFR(IIIc) isoforms for FGF ligands
may underlie the autonomous growth and independence of tumor epithelial
cells on stroma, we undertook the current study to define the
ligand-specific structural domains within the extracellular domain of
the FGFR
Mutant cDNAs were generated by overlap extension with pairs of
complementary oligonucleotide primers in the PCR as described
(5, 8, 23) . Mutant DNA fragments (621 bp) for
the FGFR1
Mutants in the fl2
domain of exon IIIb in the FGFR2 portion of the FGFR1
cDNAs coding for
wild-type and mutant secreted truncates of the FGFR1 extracellular
domain (SFR1) were prepared and cloned into transfer vector pVL1393 for
preparation of recombinant baculovirus
(21, 22) .
Partial cDNAs coding for the wild-type and mutant FGFR1
PCR was performed for 40 cycles at 94
°C for 1 min, 55 °C for 2 min, and 72 °C for 3 min.
Reaction mixtures (100 µl) contained 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 1.5 mM MgCl
Recombinant baculovirus bearing cDNA
coding wild-type or mutant FGFR constructions was prepared by
co-infection of Sf9 cells with the pVL1393 vector bearing FGFR cDNAs
and BaculoGold viral DNA (PharMingen, San Diego, CA) by the Lipofectin
method
(21, 22, 24) . The recombinant expression
products were identified and purified by immunochemical methods as
described
(21) . Each recombinant viral stock was standardized
by analysis of the level of expression of antigen determined by
immunoblot
(10) .
For analysis of secreted
recombinant FGFR antigens, the medium of baculovirus-infected Sf9 cell
cultures (5
Receptor antigen
levels from constructions expressed in baculovirus-infected Sf9 cells
were standardized by immunoblot analysis
(5, 21) . For
analysis of secreted antigens in the medium, antigens were enriched
from a 0.20-ml portion of neutralized medium used for binding assays by
treatment with 20 µl of heparin-agarose beads. The beads were
extracted, and immunoblot analysis of the extract was performed
(21) . In the tryptic fragmentation experiments (see
Fig. 3
), portions of receptor antigens immobilized on
heparin-agarose before and after exposure to trypsin were similarly
analyzed.
To determine whether sequence domains between the COOH
terminus of the 30-33-kDa fragment and the beginning of
alternately spliced exons IIIb and IIIc were determinants of specific
ligand binding, we constructed recombinant baculoviruses bearing cDNAs
coding for SFR1 ending at Ile
To determine the minimal requirement for ligand binding,
we then constructed and expressed cDNAs coding for secreted fragments,
SFR1K
Scatchard analyses confirmed that FGF-1
bound to the four immobilized active FGFR1 fragments with a similar
affinity ( K
To determine whether
the binding of FGF-7 might be restored by alteration of specific
residues within the fl1 domain of Loop III, we constructed two mutant
cDNAs coding for the secreted fragment SFR1I
The extracellular domain of the transmembrane tyrosine kinase
component of the heparan sulfate-FGF receptor complex is a member of
the immunoglobulin supergene family of cell surface recognition
proteins
(14) . The glycosaminoglycan-FGF-binding site is within
two tandem Ig units separated by an interloop sequence, the individual
units of which conform to a template based on the well characterized
three-dimensional structures of Ig units
(8, 21) (Fig. 1). We have previously proposed that clusters
of highly conserved sequences that compose the back of Loop II
(A-bl1-B, bl2-D, and E-bl3-F domains), the inter-Loop II/III domain,
and the B-fl1-C domain on the front of Loop III interact to form the
FGF ligand-binding site
(21) (Fig. 1). Here we show that
the minimal ligand-binding site within the two tandem Ig loops is
shared by FGF-1, FGF-2, and FGF-7 and is composed of
NH
Although the active core extending
through lysine 189 binds FGF-1, FGF-2, and FGF-7, addition of the
sequence SDPQP, which composes most of the fl1 domain (SD(P/A)PQPHI)
specifically restricted the binding of FGF-7 with little impact on the
binding of FGF-1 and FGF-2. Residues SD(P/A)PQP are invariant
throughout the four genes and all isoforms of FGFR. Since this is the
same ligand binding profile exhibited by exon IIIc variants of FGFR, we
conclude that exon IIIc sequence domains are passive and do not affect
the ligand binding profile set by subdomains extending through the fl1
sequence. In contrast, exon IIIb actively abrogates the restriction on
the binding of FGF-7 concurrent with a reduction in affinity for FGF-2
by an indirect interaction with the fl1 and potentially other sequence
domains upstream of it. These conclusions differ significantly from
those that suggest that sequence domains encoded in alternately spliced
exons coding for the COOH-terminal half of Loop III interact directly
with different FGF ligands
(26, 27) . In our model of
FGFR
In sum, our results suggest that the
affinity or specificity of an FGF ligand bound to the active site core
(the back face of Loop II, the inter-Loop II/III sequence, the NH
Mounting evidence suggests that the mutually exclusive splicing of
exon IIIb in the FGFR2 gene in epithelial cells underlies the normal
paracrine control of epithelial cell growth and differentiation by
stromal cells, which is mediated by stromal cell-derived FGF-7
(11, 18, 19) . A switch from exclusive
expression of exon IIIb to exclusive expression of exon IIIc concurrent
with overexpression of FGF-2 and embryonic and oncogenic FGF ligands in
epithelial cells correlates with the stromal independence, the lack of
differentiation, and the malignancy of tumor-derived epithelial cells
(11) . The observations presented here should be of utility in
design of antagonists to block interaction of specific FGF ligands with
specific FGFR complexes.
We thank Maki Kan, George McBride, and Kerstin
McKeehan for excellent technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
terminus of Loop III is sufficient and near the minimal
requirement for binding of FGF-1, FGF-2, and FGF-7. Extension of the
fragment by five additional highly conserved residues (SD(P/A)QP)
within a distinct constitutive structural domain (fl1) in Loop III
restricts the binding of FGF-7 without effect on FGF-1 and FGF-2. Since
the presence of exon IIIc in the full-length ectodomain does not change
this ligand binding profile, we suggest that alternately spliced exon
IIIc plays no active role in binding of the three ligands. In contrast,
exon IIIb actively abrogates the restriction on the binding of FGF-7
and concurrently lowers the affinity for FGF-2.
(
)
family consists of nine genetically distinct polypeptides
that elicit receptor-mediated effects on growth, function, and
differentiation
(1, 2) . The FGF receptor (FGFR) is a
trimolecular complex of the glycosaminoglycan portion of a heparan
sulfate proteoglycan, an FGF ligand, and the ectodomain of a tyrosine
kinase transmembrane glycoprotein
(3, 4) , each
component of which exhibits an interactive domain for the two other
components of the complex
(5, 6) . Although the
functional role of the direct interaction of heparan sulfate with the
receptor kinase ectodomain is unclear, we show in the accompanying
paper
(7) that the affinity of the interaction of heparin with
it varies with the structure of the ectodomain and correlates with the
affinity of the glycosaminoglycan-ectodomain complex for ligand.
terminus of Loop II of all
FGFR kinase isoforms composes a heparan sulfate-binding domain that is
obligatory for ligand binding
(5) . Although coding sequences
for Loop II, the inter-Loop II/III sequence, and the
NH
-terminal half of Loop III are not alternately spliced,
the COOH terminus of Loop III in the FGFR1 and FGFR2 genes varies as a
result of alternate splicing
(2) . Alternate splicing of exons
IIIb and IIIc in the FGFR2 gene dramatically alters specificity for FGF
ligands
(15, 16, 17) . While both FGFR2(IIIb)
and FGFR2(IIIc) bind FGF-1 with equal affinity, FGFR2(IIIb) binds FGF-7
and exhibits a lower affinity for FGF-2. Both FGFR1(IIIc) and
FGFR2(IIIc) isoforms bind FGF-2 with high affinity, but they reject
FGF-7 at all concentrations.
tyrosine kinase component of the FGFR complex.
Construction of FGFR cDNAs
In the
oligonucleotide sequences below, residues in linker and restriction
sites are underscored, mutant residues are in boldface, and residues
not in the FGFR cDNAs are in lowercase. Where possible, restriction
sites (silent mutations) were introduced into oligonucleotide primers
to aid in identification of cloned cDNAs. DNAs were purified by agarose
gel electrophoresis, excised, and electroeluted. Orientation of cloned
constructions was determined by restriction enzyme analysis. Wild-type
cDNAs coding for the extracellular domain of human FGFR1 extending
through the transmembrane and 18 residues into the intracellular
juxtamembrane sequence were cloned and prepared as described
(8) . The cDNAs for the extracellular domain of the rat
FGFR2
(IIIb) (GenBank
number L19104) and
FGFR2
(IIIb) (GenBank
number L19107) isoforms
extending from the translational initiation site through the
transmembrane domain and 20 residues into the intracellular
juxtamembrane domain were generated in the polymerase chain reaction
(PCR) with human sense primer pB1 (gcctgaattcCACATGGAGATATGGAAGAGGAC)
and antisense primer pB2 (gcctgaattcAGCTTGTGCACAGCCGGCTGGCT) with rat
prostate cDNA template
(11, 12) . The cDNA coding for
the intracellular kinase domain of FGFR2
(12) (GenBank
number L19106) was ligated to the FGFR2
(IIIb) to generate
the cDNA coding for the full-length FGFR2
(IIIb) type 1 kinase
(12) . The FGFR2 portion of the chimeric human FGFR1 and rat
FGFR2 cDNAs consisted of FGFR2 residues corresponding to
FGFR1
(IIIc) residues Glu
-Leu
.
The FGFR2 segment was generated in the PCR with sense primer pB3
(TGGATCAAACATGTCGAAAAGAAC) containing a TaqI restriction site
and antisense primer pB2, which contained an EcoRI site using
the FGFR2
(IIIb) cDNA template. After digestion with TaqI
and EcoRI, the 367-base pair (bp) fragment from the PCR was
ligated at the TaqI site of both wild-type and mutant
(described below) human FGFR1
cDNA fragments (621 bp) prepared by
digestion with PstI and TaqI. The FGFR1 portion began
13 bp upstream of the translation initiation site and extended through
the coding sequence for residue Ile
of FGFR1
.
(P194L,Q195Q,P196L) and FGFR1
(S192I,D193L)
constructions were generated in the same PCR from the FGFR1
cDNA
template with sense primer pR1a (AAAGAAACAGATAACACCAAA) and antisense
primer pR1b (GTTACCCGCCAAGCACGTATA) and a pair of asymmetric mutant
primers (sense: mpFL1a GTGTACAGTGACCTGCAGCTGCACATC;
antisense: mpFL1b GATGTGCGGCTGCGGGACAATGTACAC). A
PstI site was incorporated into the mpFL1a primer, which was
used to discriminate clones of the FGFR1
(S192I,D193L) from the
FGFR1
(P194L,Q195Q,P196L) cDNA. The 275-bp core of the mutant DNA
fragments from the PCR was prepared by digestion with BstXI
and TaqI and ligated at the BstXI site with wild-type
FGFR1
cDNA (401 bp) on the 5` side and at the TaqI site
with wild-type FGFR1
(IIIc) or wild-type and mutant
FGFR2
(IIIb) cDNAs on the 3` side. Mutant cDNA fragments in which
His
or His
was substituted with
phenylalanine were also generated in the same PCR from FGFR1
cDNA
template with sense primer pR1a and antisense primer pR1b and a pair of
asymmetric mutant primers (sense (mpHa),
CAGCCGTTCATCCAGTGGCTAAAGCACATCGAG; antisense (mpHb),
CTCGATGAACTTTAGCCACTGGATGTGCGGCTG). After digestion with
BstXI and TaqI, the resultant core 275-bp mutant
fragments were inserted between flanking wild-type FGFR1
cDNAs at
the BstXI and TaqI sites.
/R2(IIIb)
chimeric construction (described above) were generated with 5` sense
primer mpFL1a complementary to the FGFR1 sequence and 3` antisense
primer pB2 complementary to the FGFR2(IIIb) sequence as flanking
primers in the presence of specific mutant primers. The
FGFR1
/R2(IIIb)(H313T,S314A,+KE) mutant cDNA was generated
with sense mutant primer mpFL2a
(GGGATTAATAGTTCCAATAAGGAAGCAGAA) coding for the Lys-Glu
insert and antisense mutant primer mpFL2b
(ATTGGAACTATTAATCCCCGCGGTCTT) coding for the
H313T,S314A substitution were used in the PCR with the
FGFR1
/R2(IIIb) cDNA template. To generate the individual mutant
FGFR1
/R2(IIIb)(H313T,S314A) and FGFR1
/R2(IIIb)(+KE)
cDNAs, sense mutant primer mpFL2a and an antisense mutant primer mpFL2c
(TGCATTGGAACTATTAATCCC) coding for the Lys-Glu deletion in exon
IIIb were employed in the PCR with the mutant FGFR1
/R2(IIIb)
(H313T,S314A,+KE) cDNA template. After digestion with
TaqI and EcoRI, the mutant DNA fragments were ligated
with the TaqI site of the FGFR1
cDNA.
extracellular domains through Ile
were prepared by
treatment of the FGFR1
cDNA with TaqI followed by an
end-fill with Klenow enzyme and a digestion with XbaI. They
were then cloned into the pBluescript SK vector at XbaI and
SmaI sites. After partial digestion with EcoRI, the
679-bp cDNA was cloned into the EcoRI sites of the pVL1393
vector. The ligation resulted in an expression product ending with
Ile
of the FGFR1
sequence with a tail of 14
unrelated amino acid residues due to an in-frame ligation with the
transfer vector. SFR1 cDNA constructions that coded for secreted
extracellular domains truncated at Pro
(SFR1P
), Tyr
(SFR1Y
),
Lys
(SFR1K
), and Lys
(SFR1K
) were constructed by generation of DNA
fragments containing a stop codon in the antisense mutation primers,
which resulted in the desired truncation. Mutation antisense primers
pSFP
(TAGGCACTGCAGGTGCTACGGCTG), pSFY
(GTGCGGCTGCAGGTCTCAGTACAC), pSFK
(GGGCTGCAGGTCACTGTACTACTT), and pSFK
(ACCCACCTGCAGTCATTTGTTGGC) containing the stop codons and
a PstI restriction site were used with sense primer pR1a and
FGFR1
cDNA template. After digestion with PstI and
BstXI, the cDNA fragments were ligated with the
XbaI- BstXI FGFR1 cDNA fragment described above at the
BstXI site and cloned into the pVL1393 vector at XbaI
and PstI sites.
, 0.2
mM each of deoxynucleotide triphosphates, 1 µg of
template, and 15 pM of each primer. cDNAs were cloned into
pBluescript SK vector for restriction enzyme and sequence analysis. The
complete sequence of mutant cDNA fragments generated by the PCR and the
sequence across ligation sites with wild-type and mutant FGFR cDNAs
were determined. cDNAs coding for transmembrane forms of FGFR
extracellular domains were partially digested with EcoRI and
cloned into mammalian expression vector P91023B for transient
expression in mammalian cell hosts
(5, 7, 8, 21) . cDNAs coding for SFR1
were cloned into baculoviral transfer vector pVL1393 (InVitrogen, San
Diego, CA) for preparation of recombinant baculovirus and expression in
Spodoptera frugiperda (Sf9) insect cells
(21, 22) .
Expression of Recombinant FGFR Variants
Wild-type
and mutant FGFR cDNAs in mammalian expression vector P91023B were
transiently transfected into monkey kidney cells (COS-7) in replicate
monolayer cultures (10cells in 60-mm tissue culture
dishes) by the DEAE-dextran method as described
(8, 21, 23) . After exposure to individual
plasmids bearing cDNA constructions indicated under
``Results'' for 5 h, transfected cells were harvested by
trypsin from replicate 60-mm dishes, pooled, and then divided equally
among 60-mm dishes and cultured for 2 days prior to analysis of binding
and covalent cross-linking of
I-labeled FGF-1
(
I-FGF-1),
I-FGF-2, and
I-FGF-7. For concurrent analysis of expression of surface
antigen and ligand binding without covalent cross-linking, transfected
cells harvested from a 60-mm dish were distributed into 24-well plates
and cultured for 2 days prior to the analysis. In a typical experiment,
experimental groups consisted of cells transfected with three or four
different cDNA constructions.
Binding and Cross-linking of
FGF-1, FGF-2, and FGF-7 were iodinated, activated by
reduction, and purified to a specific activity of 2-5 I-FGF
Ligands
10
cpm/ng, and analysis of binding and covalent affinity
cross-linking of
I-FGF were performed as described
(5, 10, 21, 23, 24, 25) .
Binding mixtures contained 2 ng/ml labeled ligand and 2 µg/ml
heparin. Prior to analysis of ligand-labeled receptors by
SDS-polyacrylamide gel electrophoresis and autoradiography
(5, 7, 8) , radioactivity of extracts was
counted by
-counter. Specific binding was determined, and
specificity of ligand-labeled bands was confirmed by addition of a
100-fold excess of unlabeled ligand. Since FGF-1 bound to products of
all constructions that exhibited specific ligand binding, it served as
a qualitative standard for comparison of FGF-2 and FGF-7 binding.
Levels of receptor antigen present in binding assays were standardized
by the immunoassays described below.
10
cells in 25-cm
culture
flasks) was changed to 3 ml of Grace's medium containing 1 mg/ml
bovine serum albumin and 2 µg/ml heparin 12 h after infection, and
then culture was continued for 24 h. Portions (0.60 ml) of neutralized
medium (pH 7.2) were immobilized on anti-FGFR1 antiserum A50
(21) bound to 60 µl of protein A-agarose beads for 1 h at
room temperature or 4 h at 4 °C; the immunocomplexes were washed
once with 0.50 ml of PBS, washed once with PBS containing 1 M
NaCl, washed again with PBS, and then suspended in 0.6 ml of binding
assay buffer. Three equal portions of the same preparation were used
for comparison of the binding of
I-FGF-1,
I-FGF-2, and
I-FGF-7
(5) . Harvested
soluble receptor fragments were used immediately in binding assays.
Products stored in culture medium or as purified immunoprecipitates
exhibited a time-dependent decrease in ligand affinity indicated by
non-linear Scatchard plots and an increase in heparin-independent and
nonspecific binding followed by loss of total ligand binding activity.
Scatchard and Displacement Analysis of
For Scatchard analysis and
competition assays, 1.5 ml of medium from infected cells was
immobilized on protein A-agarose beads as above, and the
immunocomplexes were divided equally for binding assays. The binding
was carried out in 0.4 ml of binding buffer. For Scatchard analysis,
the binding buffer contained various concentrations of
I-FGF-1 Binding
I-FGF-1 and 2 µg/ml heparin. For competition assays,
the binding buffer contained 2 ng/ml
I-FGF-1, 2 µg/ml
heparin, and various concentrations of unlabeled FGF-1, FGF-2, and
FGF-7. After incubation at room temperature for 2 h, the beads were
washed with PBS twice and heparin-PBS once, and the bound radioactivity
was quantitated by
-counter.
Analysis of Recombinant FGFR Antigens
FGFR
antigens displayed on the surface of transfected COS-7 cells were
analyzed by double antibody radioimmunoassay as described
(5, 21) . Triplicate wells from the same 24-well
replicates used for ligand binding were analyzed.
Figure 3:
Binding of FGF ligands to fragments of the
FGFR ectodomain. A, FGF binding to tryptic fragments. The
lysate (0.40 ml in 0.5% Triton X-100 and PBS) of 5 10
Sf9 cells infected with recombinant baculovirus bearing the cDNA
coding for full-length FGFR1
(IIIc) or FGFR2
(IIIb) was diluted
to 2 ml with PBS containing 1 mg/ml bovine serum albumin and was
immobilized on 120 µl of heparin-agarose beads by incubation at 4
°C for 2 h. After a wash with PBS, the beads were divided into two
portions. Aliquots of one portion ( part a) were
incubated in PBS for 1 h at 4 °C prior to performance of ligand
binding assays. Aliquots of the second portion ( part b) were incubated with 0.2 ml of 10 µg/ml trypsin in
PBS for 1 h at 4 °C as described (5). The beads were then washed
with 0.2 mM phenylmethylsulfonyl fluoride in PBS twice and
then used in ligand binding assays. FGF binding was determined as
described under ``Experimental Procedures.'' Bands between
the 66- and 97-kDa markers in part a are the
truncated ectodomain resulting from an endogenous protease (5, 21).
B, FGF binding to recombinant fragments truncated at specific
residues. Secreted recombinant fragments ending at the indicated
residues were expressed in baculovirus-infected Sf9 cells and
immobilized to antibody-agarose beads, and covalent affinity
cross-linking of
I-FGF-1,
I-FGF-2, and
I-FGF-7 to each construction was determined. The SF9 lane indicates an immunocomplex prepared from the medium
of uninfected Sf9 cells. Sf9 cells infected with wild-type baculovirus
yielded the same result. The three upper panels are autoradiographs, and the lower panel is an
immunoblot analysis of a portion of each of the same samples used in
the binding analysis. C, FGF binding to secreted SFR1I
fragments with the indicated mutations in the fl1 sequence.
PQP
LQL, P194L,Q195Q,P196L; SD
IL,
S192I,D193L.
Hypothetical Model of the FGFR
To visualize the FGFR ectodomain in a three-dimensional
context and to guide structure-function analysis, structural domains
were assigned to the sequence of tandem Ig Loops II and III of
FGFR Extracellular
Domain
using a template of the well defined structural domains
exhibited by the constant loop in immunoglobulins for which the
three-dimensional structure is well characterized
(13, 14, 21) (Fig. 1 A). The
constant Ig homology unit is composed of two
-sheets composed of
four (A, B, D, and E) and three (C, F, and G) antiparallel
-strands connected by a disulfide.
-Strands are connected by
front (fl) and back (bl) interstrand loops designated according to
whether they are on the front or back ends of the cylindrical Ig unit.
Consistent with the idea that interaction of the back of Loop II and
the front of Loop III may form the binding site for the
glycosaminoglycan-FGF complex, the interstrand loops and flanking
strand sequences on the back face of Ig Loop II and the front face of
Loop III as well as the inter-Loop II/III sequence are highly conserved
across isoforms of the four FGFR genes in all species
(21) . The
A-bl1-B domain in Loop II is the major heparan sulfate-binding domain
(5) . The variant COOH-terminal domain of Loop III encoded by
alternately spliced exons begins with the fl2 domain at the COOH
terminus of the D strand. Important residues within the B-fl1-C and the
fl2-E domains of Loop III that are discussed in the text are indicated
in Fig. 1 A and in boldface in
Fig. 1B.
Figure 1:
Model of
the FGFR complex. A, sequence domains of Ig Loops II and
III and the interloop sequence of FGFR1
were assigned by combined
Chou-Fasman, Garnier, and EMBL PHD methods using a template constructed
from the x-ray coordinates for a constant Ig loop unit from the light
chain of IgG1 (21). The putative disulfides between strands B and F
that connect the two
-sheets within each unit are indicated in
black. The Ig units, which are actually cylinders whose back
and front faces are composed of interactive structural domains, are
flattened out and separated for clarity. The
heparan sulfate( HS)-binding domain (A-bl1-B) in Loop II and
the fl1 domain of Loop III are indicated in black. Key
residues discussed in the text are indicated. Numbering of residues is
from the translational initiation site of FGFR1
(8). The open triangle and hatched area within strand
E indicate location of the variant TA/HS and KE residues in exons IIIb
and IIIc, respectively. The
-carbon backbone of FGF-2 is from
Eriksson et al. (29). SO indicates the location of a
bound sulfate ion in crystals of FGF-2, which is thought to be the
heparan sulfate-binding pocket (29). The interstrand 9/10 domain of
FGF-2 is shaded. B, sequence of B-fl1-C
( top) and fl2-E-bl3 ( bottom) domains of FGFR.
Conserved residues in all variants and species are indicated with
closed circles (21). Serine/threonine and
valine/isoleucine/leucine residues were scored as conserved. Key
residues discussed in the text are in reverse print.
Alternately Spliced Exons IIIb and IIIc Modify the
Affinity for FGF-2 and FGF-7
cDNAs encoding extracellular
domains of FGFR1 and FGFR2
beginning from the translational
initiation site extending through the transmembrane domain and
18-20 residues into the intracellular juxtamembrane domain,
respectively, were constructed, cloned into mammalian expression vector
P91023B, and transiently transfected into monkey kidney COS cells
(5, 7, 8, 21, 22, 23) .
Binding and covalent cross-linking of
I-FGF-1,
I-FGF-2, or
I-FGF-7 to the
membrane-anchored recombinant extracellular domains on the cell surface
was distinguished from endogenous full-length FGFR isoforms by size on
SDS-polyacrylamide gel electrophoresis after autoradiography. The
results confirmed that FGF-1 and FGF-2 but not FGF-7 bound to
FGFR1(IIIc) (Fig. 2). In contrast, both FGF-1 and FGF-7 bound to
FGFR2(IIIb) while the binding of FGF-2 was significantly reduced. A
chimeric construction FGFR1/R2(IIIb) in which the exon IIIc sequence
and 19 residues upstream of it in FGFR1 were substituted with exon IIIb
and homologous upstream residues from FGFR2 exhibited a similar ligand
binding profile to the FGFR2(IIIb) variant (Fig. 2). This
indicated that the determinants for differences in binding of FGF-2 and
FGF-7 were encoded in the portion of the FGFR2 gene containing exon
IIIb independent of whether the NH
-terminal fragment was
from FGFR1 or FGFR2. Throughout this study, we detected no significant
difference in the FGFR2(IIIb) and chimeric FGFR1/R2(IIIb) product in
respect to affinity for the three FGF ligands.
Figure 2:
Impact of alternately spliced exons IIIb
and IIIc on binding of FGF-1, FGF-2, and FGF-7. COS-7 monkey kidney
cells were transfected with cDNAs coding for the membrane-anchored
ectodomains of the indicated isoforms followed by analysis of covalent
affinity cross-linking of the I-labeled ligands to the
products expressed on the cell surface. The antisense lane indicates a sample from cells transfected with an FGFR1
(IIIc)
cDNA cloned in the antisense orientation. R1(IIIc) and R1/R2(IIIb)
refer to the 2-loop
isoforms, while R2(IIIb) was the 3-loop
variant.
The Conserved fl1 Domain of Ig Loop III Restricts FGF-7
Binding in the Absence of Alternately Spliced Exons IIIb and
IIIc
Heparin interacts with and protects 30- and 33-kilodalton
(kDa) fragments of the extracellular domain of recombinant
baculovirus-infected insect cell-derived FGFR1(IIIc) from
degradation by trypsin
(8) . Immunochemical and biochemical
analysis of the fragments indicated that they began at residues
Glu
and Pro
, respectively, in the NH
terminus of FGFR1
and spanned Loop II, the inter-Loop II/III
sequence, and the NH
terminus of Loop III through tryptic
cut sites at Lys
, Lys
, or Lys
(Fig. 1). A complex of the 30-33-kDa fragment and
heparin still bound FGF-1 and FGF-2, similar to the parent FGFR1(IIIc)
molecule
(8) (Fig. 3 A). Surprisingly, the
fragment acquired the ability to bind FGF-7, which was restricted in
the intact FGFR1(IIIc) isoform. A similar treatment of the FGFR2(IIIb)
isoform, which binds FGF-7 but has low affinity for FGF-2, yielded a
fragment that appeared to bind FGF-1, FGF-2, and FGF-7 to nearly equal
extents (Fig. 3 A). Competition experiments indicated
that FGF-2 and FGF-7 competed with
I-FGF-1 binding to the
tryptic fragments from either isoform to an extent equal to or better
than unlabeled FGF-1. A 3-fold excess of unlabeled FGF-1, FGF-2, and
FGF-7 reduced labeled FGF-1 binding by 60, 90, and 85%, respectively,
with standard errors of 20-30% among triplicates (not shown). The
results suggested that (i) FGF-1, FGF-2, FGF-7, and heparin share a
binding site that resides in the 30-33-kDa fragment and (ii) the
tryptic fragments independent of parent isoform have lost specificity
for FGF ligands. Therefore, structural domains downstream of the
tryptic cleavage site confer the specificity for FGF ligands. In the
exon IIIc isoform, the COOH-terminal domains restrict the binding of
FGF-7 while downstream domains in the IIIb isoform lower the affinity
for FGF-2.
, Pro
,
Tyr
, Lys
, and Lys
(Fig. 1). SFR1I
extended through residue
Ile
in strand C of Loop III. Ile
is 15
residues downstream of Lys
, a candidate for the COOH
terminus of the 30-33-kDa tryptic fragments
(5) , and is
19 residues upstream of the beginning of the alternate exons IIIb.
Immobilization of the SFR1I
fragment from the medium of
baculovirus-infected Sf9 cells on antibody-protein A-agarose beads
followed by analysis of
I-FGF binding revealed that a
complex of heparin and SFR1I
bound FGF-1 and FGF-2 but
failed to bind FGF-7 (Fig. 3 B). The secreted product
SFR1P
ending at proline 196 also bound FGF-1 and FGF-2
but not FGF-7 (Fig. 3 B). However, truncation of the
secreted product (SFR1Y
) to tyrosine 191 restored FGF-7
binding. This suggested that in the absence of downstream sequence
domains, five residues or fewer within the constitutive fl1 domain
restrict FGF-7 binding with little or no effect on the binding of FGF-1
and FGF-2.
and SFR1K
, which ended at lysine 189
and lysine 176, respectively. Both lysine residues were potential COOH
termini of the 30-33-kDa fragment that resulted from trypsin
treatment of full-length FGFR1
(IIIc) in the presence of heparin
(5) (Fig. 3 A). Similar to the 30-33-kDa
tryptic fragment, the SFR1K
product bound FGF-1, FGF-2,
and FGF-7 (Fig. 3 B). Reduction of the ectodomain to
Lys
eliminated the binding of all three FGF ligands
(Fig. 3 B).
= 400-1000
pM) (see Fig. 5 A), which is of the same order
exhibited by the full-length membrane-anchored receptor in intact cells
(2, 8, 10, 20) . In agreement with the
results of the covalent affinity cross-linking analysis, quantitative
displacement analysis of
I-FGF-1 demonstrated that
unlabeled FGF-1, FGF-2, and FGF-7 competed with
I-FGF-1
binding to SFR1K
with similar efficiencies. In contrast,
unlabeled FGF-7 competed with the binding of
I-FGF-1 to
SFR1P
and SFR1I
much less effectively than
FGF-1 and FGF-2, which is the characteristic pattern exhibited by the
intact FGFR1(IIIc) variant (Fig. 4 B).
Figure 5:
FGF binding to membrane-anchored FGFR with
mutations in the fl1 domain. A and B, the indicated
variants of the full-length FGFR ectodomain extending through the
transmembrane domain were expressed on the surface of transfected COS-7
cells, and FGF binding was then analyzed by covalent affinity
cross-linking. The mutants in A were in the FGFR1(IIIc)
isoform. Binding to the FGFR2
(IIIb) isoform was included as a
positive control for FGF-7 binding assays. Mutants in B were
in the fl1 domain of the FGFR1 portion of the FGFR1
/R2(IIIb)
chimera. Antisense indicates cells transfected with a vector
bearing the FGFR1
(IIIc) A or FGFR2
(IIIb) B cDNAs cloned into the vector in the antisense direction.
C, both FGF binding and expression of cell surface receptor
antigen were measured on COS-7 cells that were transfected with the
indicated FGFR1
cDNAs. Solid bars, FGF-1;
hatched bars, FGF-2. Surface antigen was determined with
anti-FGFR1 antiserum A50 and a secondary
I-labeled donkey
anti-rabbit IgG (5). Data are the mean of duplicates. D, FGF
binding to the indicated constructions of FGFR1
(IIIc) containing
histidine-to-phenylalanine mutations was determined as in A.
Mutants were in the FGFR1
(IIIc) isoform. Binding to COS cells
transfected with the FGFR1
/R2(IIIb) chimera was included as a
positive control for FGF-7 binding ( lower panel).
PQP
LQL, P194L,Q195Q,P196L; SD
IL,
S192I,D193L.
Figure 4:
Quantitative analysis of ligand binding to
recombinant fragments of the FGFR1 ectodomain. Secreted recombinant
fragments ending at the indicated residues were expressed in
baculovirus-infected Sf9 cells and immobilized to antibody-agarose
beads. The immunocomplexes were divided into equal portions and used
for the following analyses. A, Scatchard analysis of FGF-1
binding. B, displacement analysis of FGF-1 binding by FGF-1,
FGF-2, and FGF-7. Unlabeled FGF-1 ( diamonds), FGF-2
( squares), and FGF-7 ( circles) were added to assays
containing the indicated fragments of the ectodomain to result in the
indicated ratios of unlabeled FGF to I-FGF-1 (2 ng/ml).
100% binding was about 40,000 cpm. Data are the mean of
duplicates.
These combined
results suggested that (i) the 30-33-kDa fragment that is
protected against tryptic degradation by heparin and binds FGF-1,
FGF-2, and FGF-7 most likely results from tryptic cleavage at lysine
189; (ii) a complex of glycosaminoglycan and the 30-33-kDa
fragment is the minimal structure required to bind FGF ligands in
general; (iii) the putative fl1 domain, which is highly conserved and
constitutive in all FGFR isoforms, restricts the binding of FGF-7 to
the base ligand-binding site in the FGFR fragment; and (iv) structural
domains encoded in variant exon IIIb abrogate the restriction placed on
FGF-7 binding and lower the affinity for FGF-2.
in which the
two proline residues and the serine-aspartate (SD) residues within the
SDPQPHI sequence were substituted with bulky hydrophobic leucine or
isoleucine residues. Similar to the wild-type SFR1I
construction, both mutant expression products bound FGF-1 and
FGF-2 but not FGF-7 (Fig. 3 C). This indicated that the
restrictive role of the fl1 domain on FGF-7 binding in the absence of
alternate exons IIIb and IIIc is not dependent on specific sequences.
In contrast, the same substitutions in the fl1 sequence significantly
affected ligand binding in the presence of the complete COOH-terminal
domain of Loop III when the full-length extracellular domain was
expressed as a transmembrane product. The S192I,D193L substitution
impaired binding of all three FGF ligands in both exon IIIc and IIIb
isoforms (Fig. 5, A and B). The
P194L,Q195Q,P196L substitution abrogated the binding of FGF-2 to the
FGFR1(IIIc) isoform and the binding of FGF-7 to the FGFR1/R2(IIIb)
isoform without effect on FGF-1 binding (Fig. 5, A and
B). Analysis of expression of cell surface antigen on the same
transfected cells indicated that the lack of FGF binding was not due to
inadequate expression of the mutant constructions
(Fig. 5 C). In contrast, mutants in which histidine 197
within the fl1 domain and histidine 203 within strand C of FGFR1(IIIc)
(Fig. 1) were substituted with phenylalanine bound both FGF-1 and
FGF-2 with equal efficiency with no change in the rejection of FGF-7
(Fig. 5 D). Results not shown here indicate that the
impact of several of the mutations in the full-length ectodomain
described here is not dependent on whether ligand binding was performed
on membrane-anchored or detergent-solubilized expression products.
These results suggest that, although alterations in specific residues
within the fl1 domain of Loop III do not alter the affinity of FGF-2
and FGF-7 for the shared active site core, specific residues within the
sequence may be important in the mechanism of how exons IIIb and IIIc
impact ligand specificity in the context of the full-length ectodomain.
In ascending order, FGF-1, FGF-2, and FGF-7 are sensitive to changes in
structural domains downstream of the common base binding site.
Dipeptide Residues in the fl2 and E Domain of Alternate
Exon IIIb Indirectly Modify FGF-2 and FGF-7 Binding
The exon
IIIb sequence in both the FGFR1 and FGFR2 genes is distinguished from
exon IIIc and homologous sequences in the FGFR3 and FGFR4 genes by
histidine-serine (HS) residues at the NHterminus of the
fl2 domain combined with the absence of lysine-glutamate (KE) residues
within the E strand of Loop III (Fig. 1). To determine whether
the HS and lack of the KE dipeptide sequences were determinants of the
permissive effects of exon IIIb on FGF-7 binding, we substituted the HS
residues with TA residues and inserted the KE sequence into the
chimeric FGFR1/FGFR2(IIIb) construction, which permits FGF-7 binding
(Fig. 3). Both substitutions significantly reduced the binding of
FGF-7 without effect on FGF-1 (Fig. 6). However, an apparent
increase in FGF-2 binding to the FGFR1/R2IIIb(H313T,S314A) construction
indicated that the HS residues at the NH
terminus of exon
IIIb may have greater impact on the reduction of the affinity for FGF-2
than the absence of the KE residues.
Figure 6:
Impact of dipeptide residues in the fl2-E
domain of exon IIIb on the binding of FGF-7. FGF binding to the
indicated mutants in the sequence of exon IIIb in the FGFR2 portion of
the chimeric FGFR1/R2(IIIb) is shown. cDNA constructions coding
for the full-length membrane-anchored ectodomain were expressed on the
surface of transfected COS-7 cells. HS
TA,
H313T,S314A.
-terminal Loop II, the sequence that connects Loops II
and III, and a short segment of the NH
terminus of Loop III
that ends between lysine residues 176 and 189. The active core of the
receptor is precisely the fragment of the ectodomain that is resistant
to degradation by trypsin when complexed with heparin or heparan
sulfate. This is consistent with the integral and obligatory role of
the glycosaminoglycan in formation of the active FGFR complex
(5) . The conserved A-bl1-B domain within Loop II has been
identified as an obligatory binding site for heparan sulfate
glycosaminoglycan
(5) .
(Fig. 1), the fl2 domain, which is the NH
terminus of alternate exons IIIb and IIIc, is in sufficient
proximity to interact with the invariant fl1 domain of Loop III. The
fl2 domain of exon IIIb is distinguished from that of exon IIIc by the
presence of HS residues instead of (T/A)A residues and the absence of
KE residues within the E strand (Fig. 1). Substitution of the HS
residues of exon IIIb with the TA residues of exon IIIc and insertion
of the KE sequence of exon IIIc indicated that both dipeptide sequence
domains in exon IIIb are necessary to permit high affinity FGF-7
binding, while the HS residues appear to have the most impact on the
lowered affinity for FGF-2.
terminus of Loop III, and glycosaminoglycan) is determined by how
the other side of the ligand interacts with the highly conserved
invariant fl1 domain on the front face of Loop III. Variant domains
encoded in the COOH-terminal half of Loop III either play no role in
ligand binding ( e.g. exon IIIc) or in the case of exon IIIb
indirectly interact with constitutive upstream domains to alter
affinity for both FGF-2 and FGF-7. The requirement for a precise
relationship between sequence domains in the COOH-terminal half of Loop
III, the fl1 domain of Loop III, and the core ligand-binding site is
consistent with the fact that several mutations of residues within or
downstream of the fl1 domain in the membrane-anchored full-length
ectodomain interfere with the binding of different FGF ligands to
different extents. We reported previously that substitution of the
COOH-terminal cysteine 252 with serine in Loop III impaired both FGF-1
and FGF-2 binding in both the FGFR1
(IIIc) and FGFR1
(IIIc)
isoforms
(23) . The S192I,D193L mutation in the fl1 domain shown
in the current study had a similar effect, while the P194L,Q195Q,P196L
substitution interfered with both FGF-2 and FGF-7 binding without
effect on FGF-1. X-ray crystallographic analysis of FGF-1 and FGF-2
revealed that FGF exhibits dual structural domains separated by 25
Å on opposite faces of the ligand, which consist of the
glycosaminoglycan-binding domain formed by the NH
- and
COOH-terminal amino acid sequences on one side (Fig. 1 A)
and a distinct solvent-accessible loop between
-strands 9 and 10
within the core sequence domain on the other side. The latter domain
varies among the nine genetically distinct FGF ligands
(28, 29, 30) . This is compatible with our model
of the FGFR ectodomain, which includes a common 139-residue base
FGF-binding site (Loop II, inter-Loop II/III, A-bl1-B domain of Loop
III) whose central feature is the Loop II glycosaminoglycan-binding
domain A-bl1-B, which anchors the FGF-glycosaminoglycan complex to the
receptor (Fig. 1 A). Interaction of the spatially
distinct FGF type-specific interstrand 9/10 domain with domains on the
front face of Loop III is potentially a distinct second determinant of
specificity and affinity of the FGF-FGFR interaction (Fig. 1).
Two recent reports that show evidence for two interactive domains on
FGF-2 for the heparin-FGFR1 complex
(31) and show that the
interstrand 9/10 domain appears to be the basis of the low affinity of
FGF-2 for the FGFR2(IIIb) ectodomain
(32) support this model.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.