(Received for publication, July 26, 1995; and in revised form, September 27, 1995)
From the
Basic fibroblast growth factor (FGF) and keratinocyte growth
factor (KGF) are structurally related fibroblast growth factors, yet
they exhibit distinct receptor binding specificity. Basic FGF binds
with high affinity to FGFR1, FGFR2, and FGFR4, whereas KGF does not
interact with these receptors and can only bind an isoform of FGFR2
known as the KGFR. Basic FGF binds KGFR but with lower affinity than
KGF. In order to identify domains that confer this specificity, four
reciprocal chimeras were generated between the two growth factors and
were analyzed for receptor recognition and biological activity. The
chimeras are designated BK1
(bFGF:KGF
), BK2
(bFGF
:KGF
), KB1
(KGF
:bFGF
), and KB2
(KGF
:bFGF
). The two BK
chimera similarly interacted with FGFR1 and FGFR4 but differed from
each other with respect to KGFR recognition. BK1 displayed a slightly
better affinity for KGFR than BK2 and induced a higher level of DNA
synthesis in keratinocytes compared with bFGF and BK2. A neutralizing
monoclonal antibody directed against bFGF specifically neutralized the
biological activity of the BK chimeras. The reciprocal chimeras, KB1
and KB2, exhibited KGF-like receptor binding and activation properties.
However, KB2 displayed higher affinity for KGFR and was significantly
more potent mitogen than KB1. Altogether, our results suggest that the
amino-terminal part of KGF and bFGF plays an important role in
determining their receptor binding specificity. In addition, the
results point to the contribution of a segment from the middle part of
KGF (residues 91-110) for recognition and activation of the KGFR,
as the two chimeras containing these residues (BK1 and KB2) displayed
an enhanced interaction with the KGFR.
Basic fibroblast growth factor (bFGF) ()(1) and the keratinocyte growth factor (KGF) (2) belong to the fibroblast growth factor (FGF) family of
which nine members have been identified (for review, see (3) and (4) ). Unlike bFGF that acts on a wide variety
of cells types, the activity of KGF is restricted to cells of
epithelial origin (5) .
FGFs elicits their biological activities by interacting with cell surface tyrosine kinase receptors. Four closely related receptors, designated FGFR1-FGFR4, have been isolated. In addition, an alternative splicing mechanism gives rise to multiple isoforms of FGFR1-FGFR3 (for review, see (6) and (7) ). An important feature of the relationship between FGFRs and their ligands is a high degree of cross-reactivity. Thus, all receptors that have been examined interact with more than one type of FGF, and likewise, a given FGF can bind more than one type of FGFR. For example, bFGF binds with high affinity to FGFR1(8, 9) , FGFR2 form IIIc(10) , and FGFR4(11) , whereas acidic FGF (aFGF) binds all four receptors(8, 9, 10, 11, 12, 13) . KGF is unique among FGFs since it interacts only with one FGFR type. This receptor is an isoform of FGFR2 known as the KGFR or FGFR2 form IIIb(10, 14) . Basic FGF can also bind this isoform albeit with 10-15-fold lower affinity than KGF (14, 15, 16) .
Besides binding to FGFRs, FGFs interact with heparin-like molecules. These molecules are heparan sulfate proteoglycans that exist on the surface of many cell types and in the extracellular matrix(17, 18, 19) . Heparan sulfate (HS) and heparin protect bFGF from heat inactivation and proteolitic degradation(20) . In addition, HS modulate the biological activities of FGFs. In cells expressing HS, depending on the cell or FGF receptor type, heparin or HS can either stimulate or inhibit the biological activities of FGFs(5, 16, 21, 22) . In cells deficient in HS, the mitogenic activity of aFGF and bFGF is absolutely dependent on the addition of heparin(13, 23, 24, 25) . In this type of cells, heparin also facilitate the binding of aFGF and bFGF to their receptors(13, 16, 25, 26) , whereas the binding of KGF to KGFR is inhibited(16) . The mechanism by which HS or heparin exert these multiple effects is not clear. To date, four different mechanisms were proposed(27, 28, 29, 30) .
Although much is known concerning the multiple forms of FGFRs and their ligand binding properties, less is known about the structural domains that determine the receptor binding characteristics of FGFs. Because bFGF and KGF exhibit distinct receptor binding specificity, we reasoned that the generation of chimeric molecules between the two growth factors will help to identify determinants that confer receptor binding specificity. In this study, such chimeric molecules were generated, and their biological properties were characterized.
Figure 1: Construction of bFGF/KGF chimeric molecules. Panel A, schematic representation of bFGF and KGF chimeric molecules. Arrowheads indicate the position of the recombination sites. Panel B, illustration of the approach used to generate the chimeric molecules. The chimeric molecules were generated in two steps by polymerase chain reaction as described under ``Experimental Procedures.''
Purification of the recombinant proteins was performed by applying the bacterial supernatant to a heparin-Sepharose column. The column was washed with phosphate buffer (50 mM, pH 7.2, containing 0.2 mM NaCl) until the absorbance fell to near base line and then was subjected to a linear step gradient of increasing NaCl concentration. Aliquots from the fractions were analyzed by SDS-PAGE (37) and immunoblotting and tested for mitogenic activity. The recombinant BK chimeras were eluted at 0.45 M NaCl, and the KB chimeras were eluted at 0.85 M NaCl. Selected fractions were concentrated 10-20-fold with a Centricon-10 microconcentrator (Amicon). The concentrated fractions were diluted in 50 mM phosphate buffer, pH 6.8, and subjected to fast protein liquid chromatography using a gradient of increasing NaCl concentrations.
Immunoprecipitation, when indicated in the text, was done using 200 ng of purified bFGF or KGF. The proteins were incubated for 2-12 h with either a polyclonal antibody directed against KGF or a monoclonal antibody directed against bFGF (Type I antibody) in IP buffer(40) . The complex was recovered by protein A-Sepharose and then separated by electrophoresis and blotted onto nitrocellulose filter paper. Immunodetection was done with a polyclonal antibody directed against either bFGF or KGF, and development was done using enhanced chemiluminescence reagents.
DNA
synthesis was measured by [H]thymidine
incorporation assay. NIH/3T3 or Balb/MK cells, plated in fibronectin (1
µg/cm
) coated 96-well microtiter plates, were
serum-starved for 48 h, treated with growth factor, and processed as
described elsewhere(42) .
The receptor recognition properties of the chimeras were assessed by examining their ability to compete for the binding of radiolabeled KGF to KGFR and for the binding of bFGF to FGFR1 and FGFR4. The binding experiments were performed in the rat myoblast cell line, L6E9, that lacks detectable high affinity binding sites for FGFs and was engineered to overexpress functional FGFR1, FGFR4, and KGFR(11, 43) . Some of the experiments were also carried out in NIH/3T3 cells overexpressing the KGFR. The results are shown in Fig. 2and Fig. 3. The two KB chimeras, which contain the amino-terminal part of KGF and carboxyl-terminal part of bFGF (KB1 and KB2), did not compete with bFGF for binding either to L6/R1 or to L6/R4 cells (Fig. 2, A and B). In contrast, both KB chimeras competed with KGF for binding to the L6/KGFR cells (Fig. 2C). KB2 competed for bound KGF about 6-fold more efficiently than KB1 but about 20-fold less efficiently than KGF. Thus, the two KB chimera displayed the receptor binding specificity of KGF.
Figure 2:
Receptor binding specificities of the KB
chimeras. L6/R1 (panel A) or L6/R4 cells (panel B)
were bound to I-bFGF. L6/KGFR cells (panel C)
were bound to
I-KGF. Binding was competed by increasing
concentrations of the above indicated ligands. Binding assays were
performed as described
previously(16) .
Figure 3:
Receptor binding specificities of the BK
chimeras. L6/R1 (panel A) or L6/R4 (panel B) were
bound to I-bFGF, and
I-KGF was bound to
NIH/KGFR cells (panel C) in the presence of increasing
concentrations of unlabeled KGF, bFGF, BK1, and BK2. The assay was
performed in the presence of 3 µg/ml of
heparin.
In contrast to the results
with the KB chimeras, the reciprocal chimeras, BK1 and BK2, competed
for the binding of I-bFGF to both L6/R1 and L6/R4 cells (Fig. 3, A and B). The efficiency of
displacement for both BK chimeras was about 20-fold lower than that
obtained with unlabeled bFGF. These findings indicate that with respect
to the interaction with FGFR1 and FGFR4, the BK chimera exhibited
bFGF-like receptor binding specificity.
In order to complete the
assessment of the binding profile of the BK chimeras, it was necessary
to examine their interaction with the KGFR. Due to the strong
inhibitory effect of heparin on the binding of KGF to L6/KGFR cells (16) and the observed dependence of the BK chimeras on heparin
for receptor binding, it was not possible to perform the assay with I-KGF on L6/KGFR cells. Therefore, competition was
carried out using NIH/KGFR cells. In these cells, heparin inhibits the
binding of KGF to its receptor as well, but in heparin concentrations
higher than 3 µg/ml(16) . Alternatively, the assay was
carried out in L6/KGFR cells but on bound
I-aFGF. Unlike
KGF, the binding of aFGF to the KGFR is not inhibited by
heparin(16) . As shown in Fig. 3C, the binding
of KGF to NIH/KGFR cells was inhibited by both BK chimeras, but BK1 was
slightly more efficient competitor than BK2. Half-maximal displacement
was obtained at about 80 and 250 ng/ml of BK1 and BK2, respectively. It
should be noted that a comparison of the efficiency of competition with
that of bFGF in this experiment is complicated by the fact that heparin
inhibits the binding of bFGF to the KGFR (compare Fig. 2C and Fig. 3C). Similar results were obtained when
the competition assay was performed in L6/KGFR cells using
I-aFGF (data not shown).
To further confirm the
receptor binding specificities of the chimeric molecules, we examined
the pattern of cross-linking of I-bFGF to L6/R1 and
I-KGF to NIH/KGFR cells in the absence or presence of
unlabeled chimeric molecules. As shown in Fig. 4, a band
corresponding to
I-bFGF cross-linked to FGFR1 (Fig. 4A) and of
I-KGF to KGFR (Fig. 4B) was observed in the absence of competitors.
Excess of bFGF, BK1 and BK2 efficiently competed for the binding of
radiolabeled bFGF to FGFR1 and of KGF to KGFR (Fig. 4, A and B). The KB chimeras, similar to KGF, could only
compete for the binding of
I-KGF to the KGFR but not for
the binding of
I-bFGF to FGFR1 (compare Fig. 4, A and B, lanes 6-10).
Figure 4:
Covalent affinity cross-linking of I-bFGF to L6/R1 cells (panel A) and
I-KGF to NIH/KGFR cells (panel B). Specifically
labeled proteins are competed in each case by 200-fold excess (2
µg/ml) of the corresponding unlabeled ligands. The binding assay
was performed in the presence (lanes 1-5 in both panels)
or the absence (lanes 6-10 in both panels) of 3
µg/ml heparin.
We next
examined whether the receptor binding properties of the chimeras
correlated with the known specificities of bFGF and KGF in eliciting a
mitogenic response. To this end, we performed
[H]thymidine incorporation assay in NIH/3T3 and
Balb/MK cells. Basic FGF is highly mitogenic to NIH/3T3 cells and, to a
lesser extent, to Balb/MK cells, while KGF induces a mitogenic response
only in Balb/MK cells(5, 42) . As shown in Fig. 5, the two BK chimera induced an efficient and identical
level of DNA synthesis in NIH/3T3 cells (Fig. 5A). The
two BK chimera were also mitogenic to Balb/MK cells, but in these cells
BK1 induced a higher level of DNA synthesis than BK2 or even bFGF (Fig. 5B). To exclude the possibility that the lower
biological activity of BK2 in Balb/MK cells was due to instability, we
incubated equal amounts of BK1 and BK2 at 37 °C in Dulbecco's
modified Eagle's medium in the presence and absence of Balb/MK
cells for 16 h. The medium was than removed, clarified by
centrifugation, and tested for mitogenic activity on NIH/3T3 cells.
Both chimeras exhibited identical activity, indicating that BK2 is not
more labile than BK1. The lack of instability problem and the fact that
both chimera exhibited an identical activity in NIH/3T3 cells, suggests
that the additional 20 residues of KGF (residues 91-110), which
are present only in BK1, confer on this molecule a better ability to
bind (see Fig. 3C) and to activate the KGFR.
Figure 5:
Growth of NIH/3T3 and Balb/MK cells in
response to the chimeric molecules. Increasing concentrations of the
indicated growth factors were added to serum-starved confluent cultures
of NIH/3T3 (A and C) or Balb/MK cells (B and D). 16 h later, [H]thymidine was added
for 6 h, and incorporation was measured as described
previously(21, 42) . Heparin (10 µg/ml) was added
during the assay along with the BK chimeras in both cell types. For
bFGF heparin was added only in the assay with NIH/3T3 cells, since
heparin inhibits the mitogenic activity of bFGF in Balb/MK cells ( (22) and Fig. 7C). Maximal counts/min were
151,000 for bFGF (panels A and C) and 138,000 for KGF (panels B and D). The results are representative of
at least four different experiments.
Figure 7:
The effect of heparin on the biological
activities of the chimeric and native molecules. The biological
activity of the above indicated ligands was determined utilizing
[H]thymidine incorporation assay as described in
the legend to Fig. 5. The assay was performed in the absence or
presence of 10 µg/ml heparin. The above indicated concentrations of
growth factors give rise to half-maximal stimulation under the optimal
conditions for mitogenic activity.
The KB chimeras, similar to KGF, induced DNA synthesis only in Balb/MK cells (Fig. 5, C and D). The KB2 chimera, which contains residues 31-110 of KGF, was significantly more potent than KB1 that has residues 31-90 of this growth factor (Fig. 5D). Taken together, the results of the mitogenic assay correlated well with the receptor recognition properties of the chimeric molecules.
Figure 6:
Neutralization of the biological activity
of the BK chimera by a monoclonal antibody against bFGF. Panel
A, inhibition of the binding of I-bFGF (
) to
L6/R1 and
I-KGF (
) to L6/KGFR cells by type I
antibody; panel B, lack of cross-reactivity between KGF and
type-I antibody. Purified KGF or bFGF were first immunoprecipitated (IP) by either a type I antibody or a monoclonal antibody
against KGF. The complex was recovered using protein A-Sepharose,
separated on 12% SDS-PAGE, and immunoblotted with polyclonal antiserum
against KGF or bFGF. Panel C, neutralization of the biological
activity of the BK chimeras by type-I antibody. The indicated growth
factors were added in the absence (closed bars) or in the
presence of 5 µg/ml of Type I antibody (shaded bars) or
nonspecific IgG (open bars). The mitogenic assay was carried
out as described in the legend to Fig. 5.
Basic FGF and KGF display different affinities for heparin. Dissociation of bFGF from heparin-Sepharose or cell-associated heparan sulfates occurs at 1.5-2 M NaCl, while KGF displacement from these glycosaminoglycans occurs at 0.5 M salt(17, 21) . Apparently, the BK chimeras displayed KGF-like affinity for heparin, whereas the affinity of the KB chimeras for this glycosaminoglycan was about 1.6-fold higher than that of KGF (eluted at 0.85 M NaCl). The results shown in Fig. 7demonstrate the effect of heparin on the biological activities of the parental and chimeric molecules. Similar to KGF, the mitogenic activity of KB2 was inhibited by about 10-fold in the presence of heparin (Fig. 7A). The mitogenic activity of bFGF was enhanced by heparin in NIH/3T3 cells but inhibited in Balb/MK cells (Fig. 7, B and C), in agreement with previous report(22) . In contrast to the situation with bFGF, heparin enhanced the biological activity of BK1 in both cell types (Fig. 7, B and C). Similar results were obtained with the other two chimeras (BK2 and KB1), and similar modulation of heparin was observed on receptor binding (data not shown).
In the present study, we generated chimeric molecules between
bFGF and KGF in order to find out how their unique receptor binding
characteristics will be segregated. The BK chimeras containing the
amino-terminal part of bFGF (BK1,
bFGF:KGF
; BK2,
bFGF
:KGF
) interacted
similarly with FGFR1 and FGFR4 and exhibited an identical mitogenic
activity in fibroblasts that responds to bFGF but not to KGF. Since
only bFGF but not KGF can bind and activate FGFR1 and FGFR4, these
findings strongly suggest that the first 54 residues of bFGF confer
bFGF-like receptor recognition. The BK chimera, like bFGF, interacted
with the KGFR. The affinity of BK1 to this receptor was only slightly
higher than that of BK2. However, BK1 induced a significantly higher
level of mitogenic response in keratinocytes that naturally express the
KGFR and at high concentrations reached a maximal response that is
similar to that obtained with KGF. In these cells, BK1 (at
concentrations
10 ng/ml) also induced higher level of DNA synthesis
than bFGF. These results point to the importance of residues
91-110 of KGF for the activation of the KGF receptor since these
residues are only present in BK1 but not in BK2.
The reciprocal chimeras, KB1 and KB2, displayed KGF-like receptor recognition and did not recognize FGFR1 and FGFR4. Therefore, residues 59-110 of KGF contain a determinant that is involved in conferring KGF-like receptor recognition (residues 31-58 are not required for receptor binding as we have previously shown(21) ). The finding that KB2 (KGF 31-110; bFGF 75-155) has higher affinity for KGFR and is a much more potent mitogen than KB1 (KGF 31-90; bFGF 55-155) points again to the importance of residues 91-110 of KGF for KGF receptor recognition and activation. Apparently, the corresponding 20-residue segment of bFGF (amino-acids 55-74) does not play a similar role in the interaction with FGFR1 and FGFR4, as both BK chimeras similarly interacted with these two receptors.
Taken
together, our results indicate that the amino-terminal part of bFGF and
KGF is involved in determining their receptor binding specificities.
Additionally, a segment from the middle part of KGF also has an
important contribution for KGF receptor recognition. The amino-terminal
part of other FGFs is likely to play a similar role, since a chimera
between aFGF and KGF
(aFGF:KGF
) displays an aFGF
receptor recognition pattern (data not shown). The high degree of
amino-acid sequence divergence within the amino-terminal part of FGFs
further supports a role for this region in determining their distinct
receptor binding characteristics.
In addition to the differential receptor recognition, the chimeric molecules exhibited distinct behavior with respect to the modulation of their activities by heparin. The biological activities of the KB chimeras were inhibited, whereas those of the BK chimeras were enhanced by heparin. Because heparin inhibits the biological activities of KGF (16, 21) and in general potentiates the activities of bFGF(3) , it seems that the heparin dependence of the chimeric molecules resembled that of the parent molecule that displayed similar receptor binding specificities. An exception from this general behavior was the effect of heparin on the interaction of bFGF and the BK chimeras with the KGFR. While heparin inhibited the KGFR-mediated biological activities of bFGF, it enhanced those of the BK chimeras. The reason for this is not clear, but it might be the consequence of the significantly lower affinity of the BK chimeras for heparin compared with bFGF (as judged from the salt concentration required for elution from heparin-Sepharose).
The receptor binding site of bFGF
was recently mapped by site-directed mutagenesis(30) . It was
proposed that bFGF contains two separated binding sites. A primary site
that is responsible for high affinity interaction of bFGF with its
receptor and is comprised of six discontinuous residues that are
localized on the same face in the three-dimensional
structure(44, 45) . The other binding site plays only
a secondary role in receptor binding since its affinity to FGFR1 is
extremely low and is located within a loop structure between the ninth
and tenth -strands in the carboxyl-terminal part of
bFGF(45, 46) . Out of the six residues comprising the
proposed primary binding site, four are identical in bFGF and KGF. The
residues that differed (Tyr-24 and Arg-44 in bFGF, Phe-71 and Gln-91 in
KGF), are located within the region that is suggested by the present
study to be important for receptor binding specificity. Although not
yet proven, we believe that these two different residues are unlikely
to be responsible for the distinct receptor binding characteristics of
KGF and bFGF because they represent a conservative change(30) .
Moreover, the proposed primary binding site of bFGF (except for a
conservative change in the carboxyl-terminal tail) aligns with
identical residues in aFGF, yet the two growth factors display distinct
receptor binding specificities(13, 15) . Therefore,
other residues within the amino-terminal part of FGFs are likely to be
involved in the determination of their receptor recognition
characteristics. Distinct determinants conferring receptor recognition
and high affinity binding were reported for ligands of the
integrins(46) .
The proposed secondary binding site of FGFs is suggested to play a role in receptor dimerization(30) . Because the amino acid sequence of this site shows a high degree of diversity among FGFs, it was also suggested that it might provide their distinct receptor binding characteristics or tissue specificity(30, 47) . The ability of the BK chimera (in which the secondary binding site of bFGF was replaced with that of KGF) to induce a biological activity in fibroblasts that do not bind or respond mitogenically to KGF, strongly suggest that, albeit the diversity, the secondary binding site can be exchanged among FGFs without affecting their receptor binding and target cell specificity. If this site, indeed, plays a role in receptor dimerization, we predict that our chimeric molecules will induce the formation of FGFR1 and KGFR heterodimers. Studies are currently under way to address this question.