(Received for publication, November 22, 1995; and in revised form, December 22, 1995)
From the
High affinity fibroblast growth factor (FGF) receptors contain a cluster of acidic amino acids in their extracellular domains that is reminiscent of the calcium binding domains of some cell adhesion molecules. Based on this observation, we used a calcium blotting technique to show that FGFR-1 binds calcium and that calcium binding is not observed in a mutagenized form of the receptor that lacks the acidic box region. The acidic box also binds other divalent cations, including copper. This latter interaction appears unique since the binding of copper to FGFR-1 mediates the binding of the receptor to immobilized heparin. While this observation may help explain the angiogenic properties of copper, divalent cation binding to FGF receptors may also mediate the interaction between FGF receptors, cell adhesion molecules and other proteoglycan components of the extracellular matrix.
Basic (FGF-2) ()and acidic (FGF-1) fibroblast growth
factors are the prototypes for a family of multifunctional growth
factors which have been identified in a wide variety of tissues (for
reviews, see Baird and Bohlen(1990), Burgess and Maciag(1989),
Wagner(1991), Fernig and Gallagher(1994)). The nine known members of
this growth factor family all share some sequence homology and
associate with heparan sulfate proteoglycans on the cell surface and in
the extracellular matrix. FGF-1 and FGF-2 are mitogenic for a variety
of different cell types, but predominantly for those of mesenchymal or
neuroectodermal origin. FGFs can also modulate a number of other
cellular functions such as differentiation, chemotaxis, and protease
synthesis and secretion.
FGFs interact with two classes of FGF receptors; high affinity receptors which bind FGFs with picomolar affinity and are thought to mediate the cellular responses to FGF and low affinity heparan sulfate containing proteoglycans which bind FGFs with nanomolar affinity. The family of high affinity FGF receptors contains four major members (for reviews, see Givol and Yayon(1992), Johnson and Williams(1993), Partanen et al.(1993), and Fernig and Gallagher(1994)), each of which exists in multiple isoforms generated by alternate splicing of their mRNAs. The four different FGF receptor genes encode proteins that are closely related and share a number of characteristic structural features which distinguish them from other tyrosine kinase receptors (Hanks et al., 1988). One of these structural features is the presence of a cluster of acidic amino acids in the extracellular domain of the receptor between the first and second Ig-like loops. This acidic box is found in all FGF receptor isoforms except a variant of the keratin growth factor receptor, an isoform of FGFR-2. Nevertheless, the role of this characteristic sequence with regard to receptor function is, at present, unknown.
Initially, the acidic box was postulated to play a role in ligand binding to the receptor (Lee et al., 1989). Since, FGF-2, as its original name would indicate, is quite basic, this idea made sense. Furthermore, the acidic box is either very short (Partanen et al., 1991) or absent (Miki et al., 1991) from forms of the FGF receptor to which FGF-2 binds poorly (Partanen et al., 1991; Miki et al., 1991). However, more recent studies have suggested that the ligand binding domains of the FGF receptors reside in the second and third Ig domains and do not include the acidic box (Zimmer et al., 1993; Chellaiah et al., 1994). In addition, in two separate studies, deletion of the acidic box from FGFR-1 had no effect on ligand binding (Byers et al., 1992; Hou et al., 1992).
Our approach to
determining the role of the acidic box in FGF receptor function was to
search for similar sequences in other proteins and to compare the role
of those acidic boxes to the one in FGF receptors. Specifically, an
acidic box similar to that found in the FGF receptors is found in some
cell adhesion molecules such as uvomorulin (Kemler et al.,
1989). In this instance, the acidic box binds calcium (Ringwald et
al., 1987), and the calcium binding is critical to the activity of
this Ca-dependent cell adhesion molecule (Ozawa et al., 1990). In the experiments described below, we tested
the ability of FGFR-1 to bind calcium, and, after obtaining a positive
result, proceeded to explore the role of divalent cation binding in
FGFR-1 function.
If the acidic box region of FGFR-1 binds calcium, then it
should be possible to detect calcium binding to either intact FGF
receptors or their extracellular domains. We used a radioactive
Ca blotting technique (Maruyama et al.,
1984), in combination with the recombinant extracellular domain of
FGFR-1 expressed in either insect cells (Kiefer et al., 1991)
or bacteria to determine whether FGFR-1 could bind calcium. As shown in Fig. 1, the extracellular domain of FGFR-1 shows strong labeling
with
Ca
by this technique. Bovine serum
albumin, a protein of approximately the same size, is unlabeled.
Similar results were obtained with bacterial (unglycosylated) FGFR-1 (Fig. 2), indicating that the carbohydrate groups are not
playing a role in calcium binding. However, the binding of
Ca
is competed completely by either 5
mM Ca
or Mn
(Fig. 1). Ca
binding to FGFR-1 is also
blocked by a peptide corresponding to the acidic box region (FGFR-1
125-133) (Fig. 1). These data indicate that FGFR-1 can
bind calcium, and perhaps other divalent cations, and that this binding
may be mediated by the acidic box.
Figure 1:
Ca
blotting of the extracellular domain of FGFR-1 produced in Sf9
cells (lane 1) and bovine serum albumin (lane 2),
alone (A) or in the presence of 5 mM CaCl
(B), 5 mM MnCl
(C), 100
µg/ml acidic box peptide (D), or 5 mM CuCl
(E). F shows the Amido Black staining of one of
the panels. Similar results were obtained in three independent
experiments of identical design.
Figure 2:
Ca
blotting
of the extracellular domain of FGFR-1 produced in bacteria. Wild type (wt) receptor and the acidic box deletion mutant (del) were examined for Ca
binding (A), and then the same blot was stained with Amido Black to
confirm equal protein loading in both lanes (B). The lower
band indicated with an arrowhead in B is
maltose-binding protein, which proved difficult to separate completely
from the mutant receptor. Similar results were obtained in three
independent experiments of identical design. Molecular weights (
10
) are indicated at right.
To explore further the role of
the acidic box in calcium binding, this region was deleted from FGFR-1
using PCR-based mutagenesis. The mutant was expressed in bacteria and
purified, and the calcium binding activity of the mutant protein was
compared with that of the wild type protein. As shown in Fig. 2,
when equal amounts of the wild type and mutant proteins are subjected
to Ca
blotting, only the wild type
protein is labeled. These data provide strong support for the
hypothesis that the acidic box mediates divalent cation binding by
FGFR-1.
To characterize further the calcium binding of FGFR-1, we
used a rapid ultrafiltration method (Fuchs, 1972) to estimate the
calcium-FGFR-1 binding constants. These studies demonstrated that both
the glycosylated and unglycosylated forms of recombinant FGFR-1
extracellular domain bind one calcium molecule with a similar affinity (K
20 µM). In contrast, when the
mutant receptor was tested in the same assay, no specific binding of
calcium was detected.
Several experiments were carried out to
determine a role for calcium binding in FGF receptor function. Neither
the addition nor the removal of Ca affected either
ligand binding, as determined by
I-FGF-2 binding to whole
cells, or signal transduction, as monitored by changes in protein
tyrosine phosphorylation (data not shown). Because of the role copper
plays in regulating angiogenesis (for review, see Gullino(1986)), we
tested the possibility that Cu
, rather than
Ca
, was the true divalent cation which binds to
FGFRs. FGFs have been clearly implicated (for reviews, see Baird and
Bohlen(1990), Burgess and Maciag(1989), Wagner (1991), and Fernig and
Gallagher(1994)) in the control of angiogenesis, further supporting the
notion that the physiological divalent cation affecting FGFR-1 could be
Cu
. Accordingly, we first tested whether
Cu
could block
Ca
binding in the Ca
blotting assay. As shown in Fig. 1, Cu
effectively competes with
Ca
in this assay, suggesting that the
acidic box can bind copper as well as calcium. To explore further the
interaction of Cu
with FGFR-1, we evaluated the
effect of increasing amounts of Cu
on Ca
binding in the rapid ultrafiltration binding assay.
Cu
efficiently reduces the binding of Ca
to both glycosylated and nonglycosylated FGFR-1 in a similar
manner (Fig. 3), providing additional evidence for an
interaction of Cu
with the acidic box region of
FGFR-1. In yet other experiments, we tested the ability of the wild
type and mutant receptors to bind specifically to chelating Sepharose
charged with Cu
. Only the wild type receptor was
found to bind in a specific manner to Cu
-charged
chelating Sepharose (not shown), further supporting the idea that the
acidic box region of FGFR-1 binds Cu
.
Figure 3:
The effect of Cu on
Ca
binding by the extracellular domain of FGFR-1 (ECD) produced in bacteria (unglycosylated)
(
-
) or Sf9 cells (glycosylated)
(
-
). Increasing amounts of Cu
were included in the rapid ultrafiltration assay for
Ca
binding along with a fixed amount (10
µM) of Ca
. Each point was tested in
duplicate. The results shown are from a single experiment. Similar
results were obtained in three independent experiments of identical
design.
Since the
effects of copper on angiogenesis are heparin-dependent, we suspected
that divalent cations could play a role in modulating the interactions
of FGF receptors with components of the extracellular matrix. In order
to test this idea, we examined FGFR-1 binding to heparin-Sepharose in
the absence or presence of divalent cations. As shown in Fig. 4,
only low levels of either glycosylated or nonglycosylated FGFR-1 bind
to heparin-Sepharose when evaluated in PBS. The binding of glycosylated
receptor to heparin-Sepharose is completely eliminated if the reaction
is performed in 0.5 M NaCl (Fig. 4A), in
agreement with previous results (Kan et al., 1993), whereas
the binding of nonglycosylated receptor to heparin-Sepharose is not
affected by salt (Fig. 4B). The presence of 1 or 5
mM Ca in the binding buffer has no effect on
the interaction of either glycosylated or nonglycosylated receptor with
heparin-Sepharose. In contrast, the addition of 1 or 5 mM Cu
to the binding buffer significantly increases
the level of FGFR-1 binding to heparin-Sepharose in both PBS and 0.5 M NaCl (Fig. 4). The mutant receptor which lacks the
acidic box shows a slightly increased level of basal heparin binding in
the absence of divalent cations (Fig. 4C). However,
unlike the wild type receptor, Cu
has little or no
effect on the interaction of the mutant receptor with heparin-Sepharose (Fig. 4C). These observations provide additional
evidence that the acidic box binds copper and suggest that this
interaction plays an important role in mediating the binding of the
receptor to extracellular matrix.
Figure 4:
Heparin-Sepharose binding of the wild type (A and B) extracellular domain of FGFR-1 produced in
Sf9 cells (A) or bacteria (B) and the acidic box
deletion mutant produced in bacteria (C). Heparin-Sepharose
binding was carried out in PBS or 0.5 M NaCl in the absence of
divalent cations (lanes 2, 3, 8, and 9) or in the presence of 1 mM CaCl (+Ca
) or 1 mM CuCl
(+Cu
) as described under
``Materials and Methods.'' In all cases, lane 1 is
the receptor prior to the addition of heparin-Sepharose; lanes
2, 4, 6, 8, 10, and 12 show the receptor remaining in an aliquot (25%) of the supernatant
following precipitation with heparin-Sepharose, and lanes 3, 5, 7, 9, 11, and 13 show
the total amount of receptor that binds to heparin-Sepharose. Similar
results were obtained in five independent experiments of identical
design.
The results presented here provide the first indication of a
specific role for the acidic box in FGF receptor function. The acidic
box, similar to homologous regions in other calcium-binding proteins,
binds divalent cations. The binding of a specific cation,
Cu, to this region modulates the interaction between
the FGF receptor and heparin. It may also affect the interaction of FGF
receptors with other proteins, particularly those in the extracellular
matrix.
Our results are consistent with our failure to identify a
role for the FGF receptor acidic box in either ligand binding or signal
transduction. Since receptor activation and consequent signal
transduction is thought to require receptor dimerization (Ueno et
al., 1992), it is unlikely that the acidic box plays a role in
receptor-receptor interactions. Instead, there is now considerable
evidence for an interaction between FGF receptors, cell adhesion
molecules (Williams et al., 1994), and the extracellular
matrix (Hanneken et al., 1995). Although some matrix binding
is mediated by heparan sulfate proteoglycans, there is also evidence
for heparin-independent interactions with extracellular matrix
(Hanneken et al., 1995). All of these interactions could be
modulated by divalent cations, particularly Cu.
Although our data suggest a role for copper in FGF receptor-matrix
interactions, they do not preclude the possibility that calcium could
also mediate a subset of receptor interactions, distinct from those
that are copper-dependent. Thus, interactions with heparin could
involve copper whereas interactions with other extracellular matrix
components might require Ca or even another, as yet
unidentified, divalent cation. Further studies will be needed to sort
out the physiological relevance of these different interactions.
Two
types of calcium binding sites in proteins are known. The better known
calcium binding site is the EF hand (Kretsinger, 1980), whereas the
sequence in the FGF receptors resembles the calcium binding sequence
found in -lactalbumin (Stuart et al., 1986). In both
cases, these sites form loops which coordinate around the divalent
cation. Thus, it is likely that the acidic box also forms a loop in the
presence of Ca
or other divalent cations. Such a loop
could stabilize the receptor in a conformation conducive to interaction
with extracellular matrix components or other proteins. Given the
calculated affinity of the acidic box for Ca
, the
interaction of the receptor with this divalent cation is likely to play
a regulatory role in FGF receptor function. Thus, local changes in
Ca
concentration could trigger a conformational
change (e.g. loop formation) in the receptor. This change then
could be detected by other proteins at, or near, the cell surface.
Divalent cations could also directly mediate protein-protein
interactions. These potential functions of calcium binding domains are
consistent with the proposed structural role for the acidic box
(Chaudhuri et al., 1993).
There is some recent evidence
demonstrating that cell adhesion molecules such as N-cadherin, N-CAM,
and L1 can interact with FGF receptors and, in so doing, activate the
FGF signaling pathway (Williams et al., 1994) that leads to
neurite outgrowth. It is interesting to note that an antibody to the
acidic box region was shown to block FGF receptor activation by these
cell adhesion molecules. Furthermore, a reduction in extracellular
Ca inhibits neurite outgrowth induced by L1 and
N-CAM, even though these cell adhesion molecules mediate Ca
independent adhesion. Thus, it is possible that these
interactions of cell adhesion molecules with the FGF receptor are
regulated by divalent cation binding to the acidic box. Since several
domains of the FGF receptor are implicated in the interaction between
FGF receptors and cell adhesion molecules, divalent cation binding to
the acidic box may simply stabilize a tertiary conformation that is
conducive to the interaction between FGF receptors and cell adhesion
molecules.
The data presented here on the interaction of FGFR-1 with
divalent cations may help to resolve a controversy in the literature
regarding the ability of FGFR-1 to interact directly with heparin
(Kiefer et al., 1991; Ornitz et al., 1992; Kan et
al., 1993; Fernig and Gallagher, 1994). Thus, these differences in
experimental results could be due to the absence or presence of low
levels of Cu in the preparations of recombinant
receptor used in the various studies.
Finally, the results presented above may serve to consolidate a large body of work that has described the angiogenic activities of copper. A number of studies (for review see Gullino(1986)) have shown how copper can promote neovascularization and how heparin can acquire angiogenic properties when bound to copper. Although the mechanism whereby this divalent cation and glycosaminoglycans induce vascular growth remains unclear (McAuslan and Reilly, 1980; Raju et al., 1984; Terrell and Swain, 1991), perhaps it is through their synergistic ability to interact with FGF receptors. If so, changes in the ionic milieu may play a critical role in regulating the cellular response to FGF, equally important as that of matrix.
Taken together, all of these results suggest that the acidic box region of FGF receptors plays an important role in their interaction with cell adhesion molecules, extracellular matrix, and heparin.