Journal of Cell Science 105, 1085-1093 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
1085
Heparan sulfate proteoglycan and FGF receptor target basic FGF to
different intracellular destinations
Jane Reiland and Alan C. Rapraeger*
Department of Pathology and Laboratory Medicine, 1300 University Avenue, School of Medicine, University of Wisconsin,
Madison, WI 53706, USA
*Author for correspondence
SUMMARY
Basic FGF is a prototype of a family of heparin binding growth factors that regulate a variety of cellular
responses including cell growth, morphogenesis and
differentiation. At least two families of receptors bind
bFGF and could mediate its response: (1) tyrosine
kinase-containing FGF receptors, designated FGFR-1 to
FGFR-4, and (2) heparan sulfate proteoglycans that
bind bFGF through their heparan sulfate chains. Both
are known to undergo internalization and thus bFGF
bound to the different receptors may be internalized via
more than one pathway. It is not known whether the
intracellular fate of bFGF differs depending upon which
receptor binds it at the cell surface. To investigate the
respective roles of these receptors in the intracellular
targeting of bFGF, we utilized NMuMG cells that bind
and internalize bFGF through their heparan sulfate
proteoglycans, but do not express detectable levels of
FGFRs nor respond to bFGF. Basic FGF conjugated to
saporin (bFGF-saporin) was used as a probe to study
targeting of bFGF by the different receptors. Saporin is
a cytotoxin that has no effect on cells if added exogenously. However, it kills cells if it gains access to the
cytoplasm. The NMuMG cells internalize bFGF-saporin
but are not killed. Transfecting these cells with FGFR1 results in bFGF-responsive cells, which bind and internalize bFGF through FGFR-1, and are killed. Removing
the heparan sulfate from these cells eliminates killing by
bFGF-saporin. Therefore, endocytosis of bFGF-saporin
by these receptors can lead to two fates: (i) bFGFsaporin internalized by heparan sulfate proteoglycan,
which is not targeted to the cytoplasm, and (ii) a bFGFsaporin internalized by the bFGF-saporin bound to a
complex of heparan sulfate proteoglycan and FGFR-1
from which the saporin can gain access to the cytoplasm.
INTRODUCTION
Cells also bind bFGF through the heparan sulfate chains
of cell surface proteoglycans, such as syndecan-1, a well
characterized heparan sulfate proteoglycan, which has been
shown to bind bFGF with a dissociation constant in the
nmolar range (Kiefer et al., 1991). Syndecan-1 is a member
of a family of related proteoglycans (syndecans 1 - 4) that
are defined by a highly conserved cytoplasmic domain
(reviewed by Bernfield et al., 1992). Basic FGF also binds
to other cell surface heparan sulfate proteoglycans, including a lipid linked proteoglycan (Brunner et al., 1991).
Heparan sulfate proteoglycans are obligate partners in binding of FGFs to their FGFRs; bFGF, aFGF and K-FGF do
not bind to FGFRs unless heparan sulfate, or its analog
heparin, is present (Rapraeger et al., 1991; Yayon et al.,
1991; Olwin and Rapraeger, 1992; Ornitz et al., 1992; Kan
et al., 1993), and heparan sulfate proteoglycans are required
for FGF induction of fibroblast mitogenesis and negative
regulation of myoblast differentiation (Rapraeger et al.,
1991; Olwin and Rapraeger, 1992). These results suggest
that a complex of bFGF, FGFR and heparan sulfate pro-
Basic fibroblast growth factor (bFGF) is a prototype of the
heparin binding growth factor family, which is composed
of aFGF (FGF-1), bFGF (FGF-2), int-2 (FGF-3), K-FGF
(FGF-4), FGF-5, FGF-6 and KGF (FGF-7) (Burgess and
Maciag, 1989; Finch et al., 1989; Marics et al., 1989). Basic
FGF promotes the proliferation and differentiation of a wide
range of cells of mesenchymal and neuroectodermal origin
and is an important regulator of angiogenesis and wound
healing (Burgess and Maciag, 1989). Basic FGF binds to a
family of tyrosine kinase-containing FGF receptors
(FGFRs) that includes at least four members, designated
FGFR-1 through FGFR-4 (Lee et al., 1989; Dionne et al.,
1990; Houssaint et al., 1990; Partanen et al., 1991;
Pasquale, 1990; Reid et al., 1990; Keegan et al., 1991; Stark
et al., 1991). In addition, splicing variants have been found
for these receptors that have altered affinities for members
of the FGF family (Johnson et al., 1991; Miki et al., 1992;
Werner et al., 1992).
Key words: basic fibroblast growth factor, heparin sulfate
proteoglycan, saporin, endocytosis, receptors, fibroblast growth
factor
1086 J. Reiland and A. C. Rapraeger
teoglycan is required for bFGF binding and biological
response.
Intracellular targeting of bFGF after binding and endocytosis by surface receptors is not well defined but may
play a role in bFGF-mediated responses. Unlike some other
growth factors that are rapidly degraded once internalized,
bFGF is long-lived (Moscatelli, 1988) and may play a regulatory role after internalization. A fraction of the exogenously added bFGF escapes degradation in the endosomal
pathways and is targeted to the nucleus (Bouche et al.,
1987; Amalric et al., 1991). Targeting of the internalized
bFGF could therefore play a role in the divergent responses
mediated by this growth factor.
It is not known if the intracellular fate of bFGF is determined by the receptor that binds bFGF at the cell surface.
The multiple bFGF receptors have different rates of internalization and intracellular fates. The FGFRs are rapidly
down-regulated in response to binding bFGF (Burgess and
Maciag, 1989). In contrast, the proteoglycans are constitutively internalized by several pathways, one being a fast
pathway in which the proteoglycans are completely
degraded with a half-life of 30 minutes and a second pathway in which the cells accumulate heparan sulfate fragments cleaved from the internalized proteoglycans (Yanagishita and Hascall, 1984). Thus, bFGF could have altered
fates depending which proteoglycan bound bFGF.
In this study, we used bFGF conjugated to saporin
(bFGF-saporin) as a facile means of determining if binding
of bFGF by different cell surface receptors results in bFGF
targeting to different sites. Saporin is a ribosomal inactivating protein that is cytotoxic once it enters the cytoplasm.
However, saporin lacks cell binding capabilities. Binding
and uptake of saporin require that it be conjugated to
another protein, in this case bFGF, for which cell surface
receptors are available. Saporin conjugated to bFGF (bFGFsaporin) is cytotoxic to cells that are bFGF responsive
(Lappi et al., 1989, 1991; Beattie et al., 1990). We find that
Swiss 3T3 cells that express heparan sulfate proteoglycan
and FGFR are killed by exposure to bFGF-saporin. However, NMuMG cells, which express little or no FGFRs and
have abundant proteoglycan, internalize bFGF-saporin but
are resistant to its cytotoxicity. Transfection of these cells
with one particular member of the FGFR family, FGFR-1,
results in the transfected cells being killed by bFGF-saporin.
In addition, blocking synthesis of heparan sulfate chains in
cells expressing both FGFRs and proteoglycans conferred
resistance to bFGF-saporin. This suggests that the intracellular fate is dependent upon the receptor that internalizes
the bFGF. Basic FGF-saporin endocytosed on heparan sulfate proteoglycan enters a pathway in which it cannot exert
its cytotoxic effect, possibly leading to the lysosome where
it would be degraded. Alternatively, FGFR-1, perhaps in a
complex with heparan sulfate, targets bFGF-saporin to a
different destination. This latter destination ultimately
allows the saporin to gain access to the cytoplasm.
MATERIALS AND METHODS
Cell culture
The NMuMG (normal murine mammary gland) epithelial cells,
the resulting transfected cell lines, and the Swiss 3T3 cells were
routinely maintained in 10% fetal bovine serum (FBS; Tissue Culture Biologics, Tulane, CA) in Dulbecco’s modified Eagle’s
medium (DME; Gibco, Grand Island, NY), with 4.5 g/l glucose,
penicillin/streptomycin (P/S), and 10 µg/ml bovine pancreatic
insulin (Sigma, St. Louis, MO). Cells were not allowed to exceed
70% confluence before passage.
Transfection
NMuMG cells were transfected using Lipofectin Reagent (Gibco
BRL, Gaithersburg, MD) with the FGFR-1 gene in a Bluescript
vector containing the maloney virus promoter (Mo/mFR1/SV), a
gift from Dr David Ornitz (Harvard Medical School, Boston, MA),
and PKO-neo plasmid that confers neomycin resistance (Ornitz et
al., 1992). Lipofectin-DNA solution was prepared according to the
manufacturer’s directions. Briefly, 19 µg Mo/mFR1/SV and 1 µg
PKO-neo in 50 µl water were incubated with 50 µl of 1 mg/ml
Lipofectin Reagent 15 min, then 3 ml DME with P/S was added.
NMuMG cells at 50% confluence were washed twice with
DME. The Lipofectin/FGFR-1 DNA solution was incubated with
the cells for 5.5 h at 37°C in a 5% CO 2 incubator. Then 3 ml 20%
FBS/DME with P/S was added. After 2 d cells were transferred
to selection medium: 10% FBS/DME with P/S and 2 mg/ml
geneticin (Gibco, Grand Island, NY). Two weeks later, clones
were selected, grown to sufficient density and screened for FGFR
binding sites. Several clones were subcloned in 96-well plates and
resulting clones were again screened for FGFR binding sites.
NMuMG cells transfected with the PKO-neo plasmid alone
were prepared as above with the exception that the cells were
transfected with a total of 50 µg PKO-neo DNA alone. Clones
were selected for resistance to geneticin.
mRNA analysis
Poly-adenylated RNA was prepared from cells essentially as
described by Bradley et al. (1988). Briefly, confluent monolayers
of cells were rinsed, suspended by treatment with trypsin, washed
three times with PBS containing 25 µM aurin tricarboxylic acid,
pelleted and frozen for storage. The pellets were resuspended in
lysis buffer (0.2 M NaCl, O.2 M Tris-HCl, pH 7.5, with 0.15 mM
MgCl2, 2% SDS, 200 µg/ml Proteinase K and 20 µM aurin tricarboxylic acid) at 1×107 cells/ml and homogenized by passage
through a 18 gauge needle 4 times and then through a 22 gauge
needle an additional 4 times. The cell lysate was incubated for 2
h at 45°C with intermittent mixing, adjusted to 0.5 M NaCl and
incubated with oligo(dT)-cellulose for 60 min at room temperature. The oligo(dT)-cellulose was pelleted and washed four times
with 0.5 M NaCl, 0.1 M Tris-HCl, pH 7.5, and then eluted with
5 washes of 0.1 M Tris-HCl, pH 7.5.
For northern blot analysis, RNA samples (3 µg) were electrophoresed in a formaldehyde-agarose gel and transferred to
Nytran membrane (Schleicher and Schuell, Keene, NH) by capillary action. The RNA on the filter was hybridized with FGFR-1
cDNA probe labeled with 32P using Prime-a-gene labeling system
(Promega, Madison, WI: Feinberg and Vogelstein, 1984). Hybridization was carried out at 42°C for 16 h in 50% formamide, 2.5×
Denhardt’s, 0.1% SDS, 100 µg/ml sssDNA (sonicated salmon
sperm DNA) and 5× SSPE. The filter was washed twice in 6×
SSPE at room temperature and then 1× SSPE at 65°C.
Iodination of bFGF and bFGF-saporin
Human recombinant bFGF, a gift from Dr Brad Olwin (University of Wisconsin, Madison, Wisconsin), was iodinated by the
chloramine T method to a specific activity of approximately 200
µCi/fmole (Burrus and Olwin, 1989). Specific activity was determined with a mitogenic assay on Swiss 3T3 cells in comparison
to unlabeled bFGF. Basic FGF-saporin, a gift from Dr Andrew
Baird (The Whittier Institute for Diabetes and Endocrinology, La
Differential targeting of basic FGF 1087
Jolla, CA), was iodinated by the same procedure with the exception that the iodination reaction was terminated with a saturated
tyrosine solution instead of dithiothreitol. Specific activity of the
bFGF-saporin was determined with a cytotoxic assay on Swiss
3T3 cells in comparison to unlabeled bFGF-saporin.
bFGF binding and crosslinking assay
For bFGF binding, cells were plated in 24-well plates at approximately 150,000 cells/well and incubated with 50 pM 125I-bFGF
for 2 h at 4°C in DME with 20 mM Hepes (pH 7.4) and 0.1%
bovine serum albumin (BSA). The labeling solution was removed
and the cells were washed 3 times with 150 mM NaCl, 10 mM
Tris-HCl, pH 7.4. Basic FGF binding to heparan sulfate sites was
determined as the bFGF removed during 3 washes of 2 M NaCl,
0.1% BSA, 10 mM Tris-HCl, pH 7.4 (Moscatelli, 1987). Subsequently, the FGFR sites were determined as the bFGF removed
during an additional 3 washes with 2 M NaCl, 0.1% BSA, 10 mM
sodium acetate, pH 4. Nonspecific binding in the pH 4 washes
was assessed by competition with 5 nM unlabeled bFGF to equivalent wells during binding.
For crosslinking, cells were plated in 6-well plates at 500,000
cells/well and incubated with 500 pM 125I-bFGF 4°C for 2.5 h.
The labeling solution was replaced with 0.1 mg/ml disuccinimidyl
suberate, DSS (Pierce Chemical Company, Rockford, IL), in PBS,
pH 7.7 and incubated 30 min at 4°C. Cells were washed as
described for the binding assay to reduce background and then
twice with PBS. Cells were then extracted in 200 µl sample buffer
and cell extract equivalent to 200,000 cells was separated by 7.5%
SDS-PAGE. The gel was dried and exposed to Kodak X-Omat
AR film (Eastman Kodak Co., Rochester, NY).
Mitogenic assay
Cells at 50-80% confluence in 24-well plates were washed twice
with DME and incubated 24 h with DME containing 1 mg/ml
BSA and P/S. The medium was removed and replaced with DME
containing 1 mg/ml BSA, 10 µg/ml insulin and P/S with 0-270
pM bFGF and incubated for 18 h. The insulin does not stimulate
growth in the cells, however it potentiates the mitogenic response
to bFGF. DNA synthesis was measured by adding 1 µCi/ml
[3H]thymidine (NEN Research Products, Boston, MA) and incubating at 37°C for 4-6 h. The cells were then incubated at room
temperature for 10 min with 5% trichloroacetic acid, washed and
solubilized in 0.1% NaOH. Incorporated [3H]thymidine was
counted in a LS 5800 liquid scintillation counter (Beckman Instruments, Inc., Irvine, CA).
bFGF-saporin internalization
Approximately 500,000 cells in 60 mm dishes were incubated with
5 ml 100-200 pM 125I-FGF in 10% FBS/DME and P/S for 8 h at
37°C. Cells were washed as in the binding assay to remove surface bound 125I-bFGF-saporin, then washed once with TES (160
mM NaCl, 5 mM EDTA and 20 mM Tris-HCl, pH 7.6). Residual surface bound 125I-bFGF was removed with 0.1% trypsin in
TES. The cells were pelleted and counted in a LKB Gamma
counter (Stockholm, Sweden). Incubation of cells at 4°C followed
by treatment with trypsin contain less than 10% of the c.p.m.
measured in cells incubated at 37°C.
bFGF-saporin cytotoxicity assay
Cells were plated in 10% FBS/DME at 5000 cells/well in 24-well
plates and incubated overnight. Basic FGF-saporin or unconjugated bFGF and saporin together were then added to the final concentration indicated in the figures. Cells were incubated an additional 24 h and DNA synthesis was measured as for mitogenic
assays. In addition cells cultured in bFGF-saporin do not increase
in cell number and after 3-5 days the cells round up and detach
from the dish.
Chlorate treatment and bFGF-saporin cytotoxicity
Cells were plated in 24-well plates at 5% confluence in sulfatefree DME with 10% FBS, 30 mM chlorate, P/S, 10 µg/ml insulin
and 100 pM EGF (Collaborative Research Inc. Lexington, MA)
with the proper adjustments in salt concentration (Baeuerle and
Huttner, 1986; Rapraeger et al., 1991). After 1-2 days pretreatment, the medium was removed and replaced with chlorate
medium or chlorate medium with 10 mM sulfate added. Basic
FGF-saporin or bFGF and non-conjugated saporin were added to
a final concentration of 100 pM each. After a 24 h incubation
DNA synthesis was measured as for mitogenesis.
RESULTS
Swiss 3T3 cells are susceptible to bFGF-saporin
whereas NMuMG cells are resistant
Both proteoglycans and FGFRs bind bFGF at the cell surface and are capable of internalizing the ligand. In order to
determine if the intracellular fate of bFGF depends upon the
receptor by which it is internalized, the ability of the cell to
target FGF into the cytoplasm was studied using bFGFsaporin. These studies utilized two cell types: Swiss 3T3
fibroblasts, which express FGFRs and FGF-binding heparan
sulfate proteoglycan and which respond to bFGF, and
NMuMG epithelial cells, which express heparan sulfate proteoglycans, but have little or no expression of FGFRs and
show no mitogenic response to the growth factor.
Swiss 3T3 cells were incubated with various concentrations of bFGF-saporin for 24 hours and DNA synthesis was
measured as a convenient assay for saporin-mediated cytotoxicity. Cells were incubated in serum-containing medium
to promote full growth potential. Therefore, addition of
bFGF to these cultures had no stimulatory or inhibitory
effect on cell growth (data not shown). However, bFGFsaporin conjugate at 10-100 pM inhibits growth by 40-80%
as measured by inhibition of DNA synthesis (Fig. 1). Cells
left in bFGF-saporin for 3-5 days round up and detach from
the dish. At equivalent concentrations, nonconjugated
saporin has no effect. Basic FGF-saporin was cytotoxic in
the same concentration range as bFGF exerts its mitogenic
effects, suggesting that bFGF-saporin-mediated cytotoxicity and bFGF-mediated mitogenesis employ the same receptors (data not shown).
NMuMG cells, which do not respond mitogenically to
bFGF, are refractory to bFGF-saporin. At concentrations
where 80% of the Swiss 3T3 cells are killed (100 pM),
NMuMG cells are unaffected by the bFGF-saporin (Fig. 1).
One explanation for this difference may be differences in
the receptors mediating bFGF binding to these cell types.
Therefore, binding of bFGF to NMuMG cells was compared to that of Swiss 3T3 cells. Binding occurs to two categories of receptors, heparan sulfate proteoglycans and
FGFRs. Basic FGF bound to heparan sulfate proteoglycan
is released by a 2 M salt wash at neutral pH, whereas bFGF
bound to the FGFRs is resistant to this wash and requires
a subsequent acid wash (Moscatelli, 1987). Basic FGF
binds heparan sulfate proteoglycan on both NMuMG cells
and Swiss 3T3 cells (Fig. 2A). It also binds to FGFRs on
the Swiss 3T3 cells; however, these sites are few or absent
1088 J. Reiland and A. C. Rapraeger
Fig. 1. Cytotoxicity of bFGF-saporin on Swiss 3T3 cells and
NMuMG cells. Swiss 3T3 cells or NMuMG cells were treated for
24 h with the indicated concentrations of bFGF-saporin or
unconjugated saporin, then DNA synthesis was measured with a 4
h pulse of [3H]thymidine. Results are expressed as the amount of
[3H]thymidine incorporation compared to cells incubated in the
absence of bFGF-saporin or saporin.
on the NMuMG cells. Therefore, a major difference
between these two cell types is the presence of the FGFRs.
Heparan sulfate proteoglycans are constitutively endocytosed by cells, providing a means of bFGF uptake (Yanagishita and Hascall, 1984). NMuMG cells are thus equipped
to internalize bFGF or bFGF-saporin. The Swiss 3T3 cells
also internalize bFGF or bFGF-saporin via this mechanism
as well as via endocytosis of bFGF bound to FGFRs. The
Swiss 3T3 cells express several members of the FGFR
family. One specific member of the FGFR family, FGFR1, is present on Swiss 3T3 cells as shown by Northern analysis of poly(A)+ RNA (Fig. 2B). These cells express an abundant message of 4.4 kb. In contrast, the NMuMG cells do
not express any detectable FGFR-1 mRNA (Fig. 2B).
Expression of FGFR-1 by NMuMG cells
transfected with receptor cDNA
The potential role of FGFR-1 in bFGF targeting was
explored by expressing this receptor in the NMuMG cells.
Cells were co-transfected with the plasmid Mo/mFR1/SV
containing the FGFR-1 cDNA and the PKO-neo plasmid
that confers neomycin resistance. Of approximately 20
clones generated, two clones expressing receptor (R1-1 and
R1-2) and two clones expressing neomycin resistance alone
(Neo-10 and Neo-5) were used in these studies. FGFR-1
expression was confirmed in three ways. First, the cells
were examined for expression of FGFR-1 mRNA (Fig. 3).
Poly(A)+ RNA was isolated from the cells and screened by
northern analysis. Clone R1-1 expresses a 4.4 kDa mRNA
that hybridizes with radiolabeled FGFR-1 probe; the mRNA
is the same size as that of the Swiss 3T3 cells. FGFR-1
mRNA is not detectable in clones Neo-10 and Neo-5.
Secondly, expression of the FGFR-1 protein at the cell
surface was examined by binding of iodinated bFGF. Clones
R1-2 and R1-1 bound bFGF to FGFRs (Fig. 4A). The
Fig. 2. Basic FGF binding and expression of FGFR-1 mRNA in
Swiss 3T3 cells and NMuMG cells. (A) Cells were incubated with
100 pM 125I-bFGF for 2 h at 4°C, washed three times with 2 M
salt, pH 7.4, to determine binding to heparan sulfate proteoglycan
(open bar), then washed three times with 2 M salt, pH 4, to assess
bFGF binding to FGFRs (shaded bar). (B) Northern blot analysis
of mRNA from Swiss 3T3 cells (1) or NMuMG cells (2) probed
for FGFR-1 expression.
amount of binding observed was similar to that of Swiss
3T3 cells. Binding to heparan sulfate proteoglycan is not
significantly changed by the transfection (data not shown).
The Neo-10 and Neo-5 clones retained the negative bFGF
binding characteristics of the parental NMuMG cell line.
Thirdly, the size of the cell surface receptor was examined. Iodinated bFGF was cross-linked to the surface of the
transfected cells using DSS, a protein-protein cross-linker,
and the size of the resulting complexes was analyzed on
SDS-PAGE. A complex with apparent molecular mass of
170 kDa was seen in cells transfected with FGFR-1, which
corresponds to a similar complex found in Swiss 3T3 cells
(Fig. 4B). The Swiss 3T3 cells also express another band
of approximately 150 kDa, which may be another FGFR or
4.4 kb
Fig. 3. FGFR-1 mRNA expression in NMuMG transfectants.
Northern blot analysis of FGFR-1 expression demonstrates that
R1-1 cells (1) express FGFR-1 mRNA whereas two neomycin
resistant clones Neo-10 (2) and Neo-5 (3) lack detectable FGFR-1
mRNA expression.
Differential targeting of basic FGF 1089
A
B
Fig. 5. Growth response of transfectants to bFGF. Quiescent cells
were incubated 18 h with 0-270 pM bFGF in DME with 10 µg/ml
insulin and 1 mg/ml BSA. DNA synthesis was measured by the
incorporation of [3H]thymidine in a 4 h pulse.
Fig. 4. Basic FGF binding and crosslinking to FGFRs on
NMuMG transfectants. (A) For binding, cells were incubated with
50 pM 125I-bFGF for 2 h at 4°C, washed with 2 M salt, pH 7.4, to
remove binding to heparan sulfate proteoglycan. Basic FGF bound
to FGFRs was eluted with three washes with 2 M salt, pH 4, and
quantified. Non-specific binding, determined as binding in the
presence of 100-fold excess unlabeled bFGF, was subtracted.
(B) For crosslinking, cells were incubated with 500 pM 125I-bFGF
for 2.5 h at 4°C. The labeling medium was removed and replaced
with 0.1 mg/ml DSS for 30 min. Cells were washed and extracted
in SDS-PAGE sample buffer and electrophoresed on a 7.5%
polyacrylamide gel: lane 1, Swiss 3T3 cells; lane 2, R1-2; lane 3,
R1-1; lane 4, Neo-10; lane 5, Neo-5; lane 6, NMuMG cells.
alternatively spliced form of FGFR-1 (Fig. 4B). No complex was seen in the Neo-10 and Neo-5 clones. Heparan
sulfate binding of bFGF is not detected as DSS does not
cross-link the bFGF to the heparan sulfate chains. Taken
together, these results demonstrate that FGFR-1 is
expressed and binds bFGF in the transfected cells.
Mitogenic activity of bFGF on transfected cells
To determine if the cell surface FGFR-1 is functional in
the transfected cells, the growth response of the cells to
bFGF was measured. Cells were serum starved and then
stimulated with various concentrations of bFGF for an
additional 18 hours before DNA synthesis was measured
by incorporation of [ 3H]thymidine in a 6 hour pulse. The
R1-1 cell line responds to bFGF with half maximal stimulation at approximately 10 pM (Fig. 5). This concentration of bFGF is similar to that required for half-maximal
stimulation of other bFGF-responsive cell lines. The Neo10 cells fail to respond to bFGF at the concentrations tested
(Fig. 5). Thus, FGFR-1 is functional in the FGFR-1 transfected NMuMG cells and capable of mediating bFGFinduced growth. This implies also that parental NMuMG
cells must contain the signal transduction machinery
required for the bFGF response and lack only sufficient
expression of FGFRs.
FGFR-1 confers susceptibility to bFGF-saporin to
NMuMG transfected clones
The NMuMG clones expressing or not expressing FGFR1 were now used for comparison of bFGF-saporin intracellular pathways. However, it was important to ensure that
equivalent amounts of the toxin were internalized and thus
available for targeting by both cell types.
Uptake of bFGF-saporin
Uptake of iodinated bFGF-saporin was measured at 37°C
in the culture medium used for the cytotoxicity experiments.
The integrity of the iodinated compound was monitored by
examining the iodinated complex by SDS-PAGE, which
demonstrated that 90% of the radiolabel was in the bFGFsaporin conjugate (data not shown). The concentration of
iodinated bFGF-saporin was determined by comparison to
unlabeled bFGF-saporin in a cytotoxicity concentration
curve on Swiss 3T3 cells (data not shown).
A range of bFGF-saporin concentrations was used to
measure its accumulation in the Neo-10 and the R1-1 cells
(data not shown). Accumulation, of course, is a function of
uptake and loss from the cells. 125I-bFGF accumulation in
these cells is a direct measure of uptake, however, as no loss
of intracellular iodinated material is seen during chases of
up to 6 hours. This demonstrated that the R1-1 cells takeup more bFGF-saporin than equivalent numbers of the Neo10 cells. This is to be expected as both cell types internalize bFGF on heparan sulfate proteoglycan and the R1-1 cells
also have additional uptake due to the FGFR-1. Uptake data
are shown at two concentrations where the R1-1 and the
Neo-10 transfects accumulate equivalent amounts of bFGFsaporin. The R1-1 cells internalized 0.42 fmoles of 125IbFGF-saporin/500,000 cells over an 8-hour period when
incubated with 100 pM 125I-bFGF-saporin; the Neo-10 cells
1090 J. Reiland and A. C. Rapraeger
Fig. 6. Basic FGF-saporin uptake and
cytotoxicity. (a) For measurement of uptake,
cells were incubated for 8 h at 37°C in 10%
FBS/DME with 125I-bFGF-saporin: Neo-10,
(200 pM) or R1-1 (100 pM). Cells were then
washed to remove receptor bound 125I-bFGFsaporin, treated with trypsin to remove residual
surface bound 125I-bFGF-saporin, pelleted and
counted. Results are given as %125I-bFGFsaporin internalized with the neo 10-4 cells
(0.44 fmoles/500,000 cells) set at 100%. (b) For
cytotoxicity assays, cells were incubated 24 h at
37°C in 10% FBS/DME with bFGF-saporin:
Neo-10 (200 pM) or R1-1 (100 pM). DNA
synthesis was measured by a 4 h pulse-label of [3H]thymidine. Control cells were treated with equivalent concentrations of both
unconjugated bFGF and saporin. Results are expressed as the [3H]thymidine incorporation compared to that by cells incubated with 10%
FBS/DME with no additions.
take up at least this amount (0.44 fmoles/500,000 cells)
when incubated with 200 pM 125I-bFGF-saporin over an 8hour period (Fig. 6a). These relatively long time points were
employed to determine the total amount of bFGF-saporin
the cells would be exposed to during the cytotoxicity assay.
Uptake for shorter (4-hour) periods showed a similar relationship, with 0.31 fmole uptake/500,000 cells by the Neo10 clone and 0.25 fmole uptake/500,000 cells for the R1-1
clone. Evaluation of uptake for periods of time longer than
8 hours was not deemed feasible, as bFGF-saporin began to
be cytotoxic to the R1-1 clone. The 125I-bFGF-saporin is not
degraded to iodinated products that are released to the culture medium because chloroquine, a lysosomal inhibitor, had
no affect on the total cellular accumulation of 125I-bFGFsaporin (data not shown). Also cells chased in the absence
of bFGF following a 3-hour uptake show no loss of label
during ensuing 6-hour chases (data not shown).
Cytotoxicity of bFGF-saporin
Basic FGF-saporin had different cytotoxic effects at the
concentrations where the two clones internalize equivalent
amounts of bFGF-saporin; bFGF-saporin killed the R1-1
cells that express the FGFR-1 and had no effect on the Neo10 clone (Fig. 6b). Non-conjugated bFGF together with free
saporin had no cytotoxic effect on either clone. Because the
Neo-10 cells were resistant to bFGF-saporin despite its
internalization by these cells, the two cell types must target
the bFGF-saporin differently once it is internalized.
Basic FGF-saporin is cytotoxic to FGFR-1 clones at concentrations that are at least 5-fold lower than concentrations
at which cells without FGFR-1 are resistant. Cytotoxicity
is exhibited at concentrations as low as 30 pM on R1-1 and
R1-2 clones (Fig. 7), but the Neo-10 cells and Neo-5 cells
are resistant to at least 150 pM bFGF-saporin. Basic FGF
together with unconjugated saporin at concentrations equivalent to the bFGF-saporin had no effect on any of the clones
(data not shown). An additional three FGFR-1 clones and
three neomycin clones were studied with similar results;
clones that express FGFR-1 at levels within a 2-fold range
of Swiss 3T3 cells are susceptible to bFGF-saporin, while
clones expressing little or no FGFR-1 are resistant (data not
shown). Thus FGFR-1 negative cells are resistant to bFGFsaporin despite internalizing as much if not more bFGFsaporin than is cytotoxic to the cells expressing FGFR-1.
Elimination of functional heparan sulfate
proteoglycan abolishes bFGF-saporin cytotoxicity
Cell surface heparan sulfate is required for bFGF binding to
FGFRs and for bFGF-induced mitogenesis of Swiss 3T3 cells.
To verify that heparan sulfate proteoglycan must be part of
the complex that endocytoses bFGF-saporin, we blocked the
sulfation of newly synthesized heparan sulfate by the addition
of chlorate to the culture medium of growing cells, thus
Fig. 7. Concentration dependence of bFGF-saporin cytotoxicity.
R1-1 (m), R1-2 (v), Neo-10 (n), and Neo-5 (V) cells were
treated with 0-150 pM bFGF-saporin for 24 h. DNA synthesis was
then measured as the amount of [3H]thymidine incorporated
during a 4 h pulse. Results are expressed as the percentage of
[3H]thymidine incorporation compared to cells not treated with
bFGF-saporin. Basic FGF with unconjugated saporin had no
effect on these cells at the equivalent concentrations.
Differential targeting of basic FGF 1091
Fig. 8. Basic FGF-saporin cytotoxicity on chlorate-treated R1-1
cells. R1-1 cells were treated with chlorate medium for 2 days and
then the medium was replaced with medium containing fresh
chlorate or chlorate plus sulfate and either 100 pM bFGF-saporin
(FGF-saporin cells) or 100 pM each unconjugated bFGF and
saporin (control cells). Cells were incubated 24 h and then labeled
4 h with [3H]thymidine. Results are expressed as the percentage of
[3H]thymidine incorporation compared to cells incubated in
chlorate or chlorate plus sulfate medium without additions.
inhibiting bFGF binding to the glycosaminoglycan (Baeuerle
and Huttner, 1986; Rapraeger et al., 1991). Addition of excess
sulfate restores the sulfation of the chains and the bFGF binding characteristics. Treatment for 24 h with 30 mM chlorate
reduces radiolabeled sulfate incorporation into heparan sulfate of NMuMG cells by greater than 90% (data not shown)
and reduces 125I-bFGF binding by 85%. Treatment of the R11 clone with 30 mM chlorate completely blocked the cytotoxic effect of the FGF-saporin conjugate on these cells, as
compared to untreated controls (Fig. 8). Treatment of the cells
with non-conjugated saporin together with bFGF also was not
cytotoxic (Fig. 8). As expected, however, restoring functional
heparan sulfate proteoglycan by the addition of 10 mM sulfate in the continued presence of 30 mM chlorate restores the
cytotoxic effect of FGF-saporin (Fig. 8).
DISCUSSION
Signaling of growth and differentiation by bFGF involves
multiple pathways, and internalization of bFGF may be
involved in one or more of these pathways. We have therefore used bFGF-saporin as a means of detecting differences
in the intracellular targeting of bFGF when bound and internalized by different receptors. NMuMG cells bind bFGF
and bFGF-saporin via cell surface heparan sulfate proteoglycans; however, they express little or no FGFRs and are
not responsive to bFGF. Nevertheless, bFGF and bFGFsaporin are internalized by these cells, but the cells apparently do not target the bFGF-saporin to a compartment from
which it can gain access to cytoplasmic ribosomes. In contrast, NMuMG clones expressing a specific tyrosine kinasecontaining receptor, FGFR-1, bind bFGF via a proteoglycan/FGFR-1 complex, respond to bFGF mitogenically at
concentrations similar to other bFGF-responsive cell lines
and are killed by bFGF-saporin. Thus, endocytosis of the
FGF-saporin conjugate on FGFR/heparan sulfate proteoglycan complexes results in its targeting to a compartment
from which the bFGF-saporin gains access to the cytoplasm, leading to inactivation of the ribosomes.
There are at least four FGFRs, designated FGFR-1 to
FGFR-4, that bind members of the FGF family. NMuMG
cells were transfected with the FGFR-1 as a representative
member of the FGFR family. The transfectants express
FGFR-1, as chemical crosslinking of 125I-bFGF cell surface
proteins reveals a protein-125I-bFGF complex of 170 kDa, an
appropriate size for FGFR-1 cross-linked to bFGF. The binding and crosslinking characteristics are similar to those of
Swiss 3T3 cells, a cell line which expresses endogenous
FGFR-1. The FGFR-1 transfected into the NMuMG cells is
active as these cells now respond mitogenically to bFGF at
concentrations that stimulate other bFGF-responsive cell lines.
Intracellular sorting of bFGF-saporin is dependent upon
the receptor by which the ligand is internalized. We have
shown here that bFGF-saporin is internalized by both HSPG
and FGFR. This supports other work demonstrating that
bFGF is internalized by both types of receptors (Rusnati et
al., 1993; Roghani and Moscatelli, 1992). Basic FGFsaporin internalized after binding heparan sulfate proteoglycan at the cell surface is not targeted to a compartment
from which the saporin has cytoplasmic access. It is possible that it is targeted to lysosomes where it is degraded. The
susceptibility of cells expressing FGFR-1 to bFGF-saporin
cytotoxicity suggests that the FGFR provides sorting information that results in the bFGF-saporin being transported to
a different compartment. This sorting may occur at the cell
surface, perhaps by localization to specific sites for endocytosis. There are several different pathways for endocytosis, including clathrin-coated pits and clathrin-independent
pathways (VanDeuers et al., 1989). The most abundant proteoglycan on NMuMG cells is syndecan-1, a member of a
family of proteoglycans (syndecans 1-4) characterized by
their well-conserved cytoplasmic domains (Bernfield et al.,
1992). The role of this conserved region is not known,
although it contains several conserved tyrosines, which
could mediate endocytosis through coated pits as is the case
with several other receptors such as the mannose 6-phosphate receptor and the LDL receptor (Pearse, 1988; Glickman et al., 1989; Pearse and Robinson, 1990). This domain
could therefore play an important role in the endocytosis of
the bFGF-receptor complex. Alternatively, the sorting may
occur once in the endosomal pathway, perhaps by retrieving the bFGF/heparan sulfate complex from a route leading
to lysosomes and targeting it to another pathway. Such a
retrieval could possibly occur in the endosomes where
FGFR would be captured and localized in discrete vesicles.
Alternatively, the bFGF-saporin may be targeted to a single
site whether bound either to receptor or to proteoglycan
1092 J. Reiland and A. C. Rapraeger
alone, but might gain access to the cytoplasm from this site
only if bound to receptor. Other members of the FGF receptor family, which are highly homologous (Kornbluth et al.,
1988; Pasquale, 1990; Keegan et al., 1991; Partanen et al.,
1991; Stark et al., 1991), may also direct intracellular targeting of FGFs in the same manner.
Intracellular sorting of bFGF out of the endocytic pathway
may be significant in light of the proposed signal transduction pathway in which bFGF is targeted directly to the
nucleus. Basic FGF has been found in the nucleus of a number
of bFGF-responsive cells (Bouche et al., 1987; Baldin et al.,
1990; Renko et al., 1990; Speir et al., 1991; Woodward et al.,
1992). Studies on exogenously added bFGF indicate that
endocytosed bFGF can be targeted to the nucleus despite lacking a nuclear localization sequence (Bouche et al., 1987;
Bugler et al., 1991). Such a pathway would require bFGF to
leave the vesicular endocytic system and traverse the cytoplasm to reach the nucleus. Basic FGF-saporin may be detecting such a pathway by its cytotoxic effect on cells. Several
studies indicate that nuclear targeting of bFGF may signal
physiological responses. Expression of bFGFs containing
nuclear localization sequences is reported to regulate growth
and cellular transformation (Couderc et al., 1991; Quarto et
al., 1991), suggesting that they act by an internal autocrine
mechanism. Also, deleting a putative nuclear localization
sequence of aFGF eliminates nuclear localization of the
growth factor and abolishes its ability to stimulate cell growth
(Imamura et al., 1990; Imamura et al., 1992). However, the
mutant still binds and triggers cell surface receptors as shown
by stimulation of receptor phosphorylation and induction of
c-fos. Nuclear localization of the deletion mutant and acquisition of mitogenic activity can be restored by adding the
nuclear localization sequence of Histone 2B to the mutant
growth factor (Imamura et al., 1990, 1992). Thus, the biological action of FGFs may require intracellular sorting of the
ligand in addition to receptor activation.
Several criteria demonstrate that the sorting of bFGFsaporin faithfully monitors intracellular targeting of bFGF.
First, unconjugated saporin is not cytotoxic to cells. Second,
endocytosis of the bFGF-saporin does not necessarily lead
to cell killing, but is dependent on the receptor that carries
out internalization. Third, the amount of bFGF-saporin necessary for killing is similar to concentrations of bFGF that
promote growth. Thus, bFGF-saporin appears to be using
the same physiological mechanism as bFGF. Taken together
these results indicate that bFGF-saporin is binding to the
bFGF receptors for internalization and is specifically sorted
depending on receptor type.
The targeting of bFGF-saporin requires heparan sulfate as
well as FGFR-1. This is not surprising, as it has been established that bFGF requires this glycosaminoglycan in order to
recognize FGFR (Rapraeger et al., 1991; Yayon et al., 1991;
Ornitz et al., 1992). However, heparan sulfate proteoglycan
may also play an important role in the uptake and sorting of
FGF. A hallmark feature of heparan sulfate proteoglycan
uptake is cleavage of the heparan sulfate chains into discrete
fragments in a prelysosomal compartment (Yanagishita and
Hascall, 1984). Several different pathways are utilized, however. One is a fast pathway in which heparan sulfate of the
proteoglycan is completely degraded with a half-life of 30
min. The heparan sulfate of this fast pathway is derived from
phosphoinositol-linked proteoglycan such as glypican
(Yanagishita, 1992). An additional pathway results in much
degradation of the heparan sulfate fragments in a prelysosomal compartment with a much slower a half-life of ~4 hours.
Recent work by Yanagishita, 1992) has demonstrated that
endocytosis of membrane-spanning proteoglycans, presumably syndecans, are the source of heparan sulfate in the slow
pathway that accumulates heparan sulfate fragments. The
generation of such fragments would allow the
FGFR/FGF/heparan sulfate fragment complex to be divorced
from the core protein of the proteoglycan and be targeted
separately. Thus, intracellular heparan sulfate fragments may
also sustain binding of bFGF to the FGFRs, and protect bFGF
from acid and proteolytic denaturation as the bFGF moves
though the endocytic pathway (Gospodarowicz et al., 1987;
Saksela and Rifkin, 1990). Heparan sulfate is found in the
nucleus in a sequence-specific and cell cycle-specific manner
(Fedarko and Conrad, 1986; Fedarko et al., 1989), suggesting that the heparan sulfate fragments accompany bFGF to
the nucleus.
The authors thank Dr Brad Olwin for providing bFGF, Dr
Andrew Baird for providing bFGF-saporin, and Dr David Ornitz
for providing the Mo/mFR1/SV plasmid. This work was supported
by grants from the National Institutes of Health (HD21881). Jane
Reiland was supported by Developmental Biology Training grant
(HD017118) and is currently supported by American Heart Association Grant/Wisconsin Affiliate, Inc.
REFERENCES
Amalric, F., Baldin, V, Bosc, B. I., Bugler, B., Couderc, B., Guyader, M.,
Patry, V., Prats, H., Roman, A. M. and Bouche, G. (1991). Nuclear
translocation of basic fibroblast growth factor. Ann. N Y Acad Sci. 638,
127-138.
Baeuerle, P. A. and Huttner, W. B. (1986). Chlorate—A potent inhibitor
of protein sulfation in intact cells. Biochem. Biophys. Res. Commun. 141,
870-877.
Baldin, V., Roman, A. M., Bosc-Bierne, I., Amalric, F. and Bouche, G.
(1990). Translocation of bFGF to the nucleus is G1 phase specific in
bovine aortic endothelial cells. EMBO J. 9, 1511-1517.
Beattie, G. M., Lappi, D. A., Baird, A. and Hayek, A. (1990). Selective
elimination of fibroblasts from pancreatic islet monolayers by basic
fibroblast growth factor-saporin mitotoxin. Diabetes 39, 1002-1005.
Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M. T., Spring, J., Gallo,
R. L. and Lose, E. J. (1992). Biology of the syndecans. Annu. Rev. Cell
Biol. 8, 365-394.
Bouche, G., Gas, N., Prats, H., Baldin, V., Tauber, J. P., Teissie, J. and
Alamric, F. (1987). Basic fibroblast growth factor enters the nucleolus
and stimulates the transcription of ribosomal genes in ABAE cells
undergoing G0 to G1 transition. Proc. Nat. Acad. Sci. USA 84, 6770-6774.
Bradley, J. E., Bishop, G. A., St. John, T. and Frelinger, J. A. (1988). A
simple, rapid method for the purification of poly A+ RNA. Biotechniques
6, 114-116.
Brunner, G., Gabrilove, J., Rifkin, D. B. and Wilson, E. L. (1991).
Phospholipase C release of basic fibroblast growth factor from human
bone marrow cultures as a biologically active complex with a
phosphatidylinositol-anchored heparan sulfate proteoglycan. J. Cell Biol.
114, 1275-1283.
Bugler, B., Amalric, F. and Prats, H. (1991). Alternative initiation of
translation determines cytoplasmic or nuclear localization of basic
fibroblast growth factors. Mol. Cell. Biol. 11, 573-577.
Burgess, W. H. and Maciag, T. (1989). The heparin-binding (fibroblast)
growth factor family of proteins. Annu. Rev. Biochem. 58, 575-606.
Burrus, L. W. and Olwin, B. B. (1989). Isolation of a receptor for acidic
and basic fibroblast growth factor from embryonic chick. J. Biol. Chem.
264, 18647-18653.
Differential targeting of basic FGF 1093
Couderc, B., Prats, H., Bayard, F. and Amalric, F. (1991). Potential
oncogenic effects of basic fibroblast growth factor requires cooperation
between CUG and AUG-initiated forms. Cell Regul. 2, 709-718.
Dionne, C. A., Crumley, G., Bellot, F., Kaplow, J. M., Searfoss, G., Ruta,
M., Burgess, W. H., Jaye, M. and Schlessinger, J. (1990). Cloning and
expression of two distinct high-affinity receptors cross-reacting with
acidic and basic fibroblast growth factors. EMBO J. 9, 2685-2692.
Feinberg, A. P. and Vogelstein, B. (1984). A technique for radiolabelling
DNA restriction endonuclease fragments to high specific activity. Anal.
Biochem. 137, 266-267.
Fedarko, N. S. and Conrad, H. E. (1986). A unique heparan sulfate in the
nuclei of hepatocytes: structural changes with the growth state of the cells.
J. Cell Biol. 102, 587-599.
Fedarko, N. S., Ishihara, M. and Conrad, H. E. (1989). Control of cell
division in hepatoma cells by exogenous heparan sulfate proteoglycan. J.
Cell. Physiol. 139, 287-294.
Finch, P. W., Rubin, J. S., Miki, T., Ron, D. and Aaronson, S. A. (1989).
Human KGF is FGF-related with properties of a paracrine effector of
epithelial cell growth. Science 245, 752-755.
Glickman, J. N., Conibear, E. and Pearse, B. M. F. (1989). Specificity of
binding of clatherin adapters to signals on the mannose-6phosphate/insulin-like growth factor II receptor. EMBO. J. 8, 1041-1047.
Gospodarowicz, D., Ferrara, N., Schweigerer, L. and Neufeld, G.
(1987). Structural charcterization and biological functions of fibroblast
growth factor. Endocrinol. Rev. 8, 95-113.
Houssaint, E., Blanquet, P. R., Champion, A. P., Gesnel, M. C.,
Torriglia, A., Courtois, Y. and Breathnach, R. (1990). Related
fibroblast growth factor receptor genes exist in the human genome. Proc.
Nat. Acad. Sci. USA 87, 8180-8184.
Imamura, T., Engleka, K., Zhan, X., Tokita, Y., Forough, R., Roeder,
D., Jackson, A., Maier, J. A. M. Hla,T. and Maciag, T. (1990).
Recovery of mitogenic activity of a growth factor mutant with a nuclear
translocation sequence. Science 249, 1567-1570.
Imamura, T., Tokita, Y. and Mitsui, Y. (1992). Identification of a heparinbinding growth factor-1 nuclear translocation sequence by deletion
mutation analysis. J. Biol. Chem. 267, 5676-5679.
Johnson, D. E., Lu, J., Chen, H., Werner, S. and Williams, L. T. (1991).
The human fibroblast growth factor receptor genes: a common structural
arrangement underlies the mechanisms for generating receptor forms that
differ in their third domain. Mol. Cell. Biol. 11, 4627-4634.
Kan, M., Wang, F., Xu, J., Crabb, W. C., Hou, J. and McKeehan, W. L.
(1993). An essential heparin-binding domain in the fibroblast growth
factor receptor kinase. Science 259, 1918-1921.
Keegan, K., Johnson, D. E., Williams, L. T. and Hayman, M. J. (1991).
Isolation of an additional member of the fibroblast growth factor receptor
family, FGFR-3. Proc. Nat. Acad. Sci. USA 88, 1095-1099.
Kiefer, M. C., Baird, A., Nguyen, T., George, N. C., Mason, O. B., Boley,
L. J., Valenzuela, P. and Barr, P. J. (1991). Molecular cloning of a
human basic fibroblast growth factor receptor cDNA and expression of a
biologically active extracellular domain in a baculovirus system. Growth
Factors 5, 115-127.
Kornbluth, S., Paulson, K. E. and Hanafusa, H. (1988). Novel tyrosine
kinase identified by phosphotyrosine antibody screening of cDNA
libraries. Mol. Cell. Biol. 8, 5541-5544.
Lappi, D. A., Maher, P. A., Martineau, D. and Baird, A. (1991). The
basic fibroblast growth factor-saporin mitotoxin acts through the basic
fibroblast growth factor receptor. J Cell Physiol. 147, 17-26.
Lappi, D. A., Martineau, D. and Baird, A. (1989). Biological and
chemical characterization of basic FGF-saporin mitotoxin. Biochem.
Biophys. Res. Commun. 160, 917-923.
Lee, P. L., Johnson, D. E., Cousens,L. S., Fried, V. A. and Williams, L.
T. (1989). Purification and complementary DNA cloning of a receptor for
basic fibroblast growth factor. Science 245, 57-60.
Marics, I., Adelaide, J., Rayboud, F., Mattei, M.-G., Coulier, F.,
Planche, J., Lapeyriere, O. D. and Birnbaum, D. (1989).
Characterization of the HST-related FGF-6 gene, a new member of the
fibroblast growth factor gene family. Oncogene 4, 335-340.
Miki, T., Bottaro, D. P., Fleming, T. P., Smith, C. L., Burgess, W. H.,
Chan, A. M. and Aaronson, S. A. (1992). Determination of ligandbinding specificity by alternative splicing: two distinct growth factor
receptors encoded by a single gene. Proc. Nat. Acad. Sci. USA 89, 246-250.
Moscatelli, D. (1987). High and low affinity binding sites for basic
fibroblast growth factor on cultured cells: absence of a role for low
affinity binding in the stimulation of plasminogen activator production by
bovine capillary endothelial cells. J. Cell. Physiol. 131, 123-30.
Moscatelli, D. (1988). Metabolism of receptor-bound and matrix bound
basic fibroblast growth factor by bovine capillary endothelial cells. J. Cell
Biol. 107, 753-760.
Olwin, B. B. and Rapraeger, A. (1992). Repression of Myogenic
differentiation by aFGF, bFGF, and K-FGF is dependent on cellular
heparan sulfate. J. Cell Biol. 118, 631-639.
Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E. and
Leder, P. (1992). Heparin is required for cell-free binding of basic
fibroblast growth factor to a soluble receptor and for mitogenesis in whole
cells. Mol. Cell. Biol. 12, 240-247.
Partanen, J., Makela, T. P., Eerola, E., Korhonen, J., Hirvonen, H.,
Claesson-Welsh, L. and Alitolo, K. (1991). FGFR-4, a novel acidic
fibroblast growth factor receptor with a distinct expression pattern.
EMBO. J. 10, 1347-1354.
Pasquale, E. B. (1990). A distinctive family of embryonic protein tyrosine
kinase receptors. Proc. Nat. Acad. Sci. USA 87, 5812-5816.
Pearse, B. M. F. (1988). Receptors compete for adaptors found in plasma
membrane coated pits. EMBO J. 7, 3331-3336.
Pearse, B. M. F. and Robinson, M. S. (1990). Clathrin, adaptors, and
sorting. Ann. Rev. Cell Biol. 6, 151-171.
Quarto, N., Finger, F. P. and Rifkin, D. (1991). The NH2-terminal
extension of high molecular weight bFGF is a nuclear targeting signal. J.
Cell. Physiol. 147, 311-318.
Rapraeger, A. C., Krufka, A. and Olwin, B. B. (1991). Requirement of
heparan sulfate for bFGF-mediated fibroblast growth and myoblast
differentiation. Science 252, 1705-1708.
Reid, H. H., Wilks, A. F. and Bernard, O. (1990). Two forms of the basic
fibroblast growth factor receptor-like mRNA are expressed in the
developing mouse brain. Proc. Nat. Acad. Sci. USA 87, 1596-1600.
Renko, M., Quarto, N., Morimoto, T. and Rifkin, D. B. (1990). Nuclear
and cytoplasmic localization of different basic fibroblast growth factor
species. J. Cell. Physiol. 144, 108-114.
Roghani, M. and Moscatelli, D. (1992). Basic fibroblast growth factor is
internalized through both receptor-mediated and heparan sulfatemediated mechanisms. J. Biol. Chem. 267, 22156-22162.
Rusnati, M., Urbinati, C. and Presta, M. (1993). Internalization of basic
fibroblast growth factor (bFGF) in cultured endothelial cells: role of the
low affinity heparin-like bFGF receptors. J. Cell. Physiol. 154, 152-161.
Saksela, O. and Rifkin, D. B. (1990). Release of basic fibroblast growth
factor-heparan sulfate complexes from endothelial cells by plasminogen
activator-mediated proteolytic activity. J. Cell Biol. 110, 767-775.
Speir, E., Sasse, J., Shrivastav, S. and Casscells, W. (1991). Culturedinduced increase in acidic and basic fibroblast growth factor activities and
their association with the nuclei of vascular endothelial and smooth
muscle cells. J. Cell. Physiol. 147, 362-373.
Stark, K. L., McMahon, J. A. and McMahon, A. P. (1991). FGFR-4, a
new member of the fibroblast growth factor receptor family, expressed in
the definitive endoderm and skeletal muscle lineages of the mouse.
Development 113, 641-651.
VanDeuers, B., Petersen, O. W., Olsnes, S. and Sandvig, K. (1989). The
ways of endocytosis. Int. Rev. Cytol. 117, 131-176.
Werner, S., Duan, D.-S. R., de Vries, C., Peters, K. G., Johnson, D. E.
and Williams, L. T. (1992). Differential splicing in the extracellular
region of fibroblast growth factor receptor 1 generates receptor variants
with different ligand-binding specificities. Mol. Cell. Biol. 12, 82-88.
Woodward, W. R., Nishi, R., Meshul, C. K., Williams, T. E., Coulombe,
M. and Eckenstein, F. P. (1992). Nuclear and cytoplasmic localization
of basic fibroblast growth factor in astrocytes and CA2 hippocampal
neurons. J. Neurosci. 12, 142-152.
Yanagishita, M. (1992). Glycosylphosphatidylinositol-anchored and core
protein-intercalated heparan sulfate proteoglycans in rat ovarian
granulosa cells have distinct secretory, endocytic, and intracellular
degradative pathways. J. Biol. Chem. 267, 9505-9511.
Yanagishita, M. and Hascall V. C. (1984). Metabolism of proteoglycans in
rat ovarian granulosa cell culture: multiple intracellular degradative
pathways and the effect of chloroquine. J. Biol. Chem. 259, 10270-10283.
Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P. and Ornitz, D. M.
(1991). Cell surface, heparin-like molecules are required for binding of
basic fibroblast growth factor to its high affinity receptor. Cell 64, 841848.
(Received 5 March 1993 - Accepted 27 April 1993)