(Received for publication, December 11, 1996)
From the Laboratory of Cellular and Molecular Biology, Hepatocyte growth factor/scatter factor (HGF/SF)
is a heparin-binding polypeptide that stimulates cell proliferation,
motility, and morphogenesis by activation of its receptor, the c-Met
tyrosine kinase. HGF/SF consists of a series of structural units,
including an amino-terminal segment with a hairpin loop, four kringle
domains, and a serine protease-like region. In this study, we
demonstrate that the amino-terminal (N) domain retains the
heparin-binding properties of full-length HGF/SF. In contrast to a
previous hypothesis, selected basic amino acid residues in the hairpin
loop are not critical for heparin binding, although alanine
substitution at a subset of these sites markedly reduced the biological
activity of the HGF/SF isoform, HGF/NK1. Covalent cross-linking
experiments performed with wild-type and heparan sulfate
glycosaminoglycan (HSGAG)-deficient Chinese hamster ovary (CHO) cells
revealed that Met-HGF/NK1 binding was strongly dependent on HSGAG.
Addition of heparin to HSGAG-deficient CHO cells not only restored
ligand binding, but also increased ligand-dependent Met
tyrosine phosphorylation and c-fos expression. Moreover,
our results showed that heparin stimulated ligand oligomerization
through an interaction with the N domain. These findings establish the
importance of the N domain for heparin-ligand and ligand-ligand
interactions, and demonstrate a crucial role for HSGAG in receptor
binding and signal transduction.
During the past dozen years, several mitogens have been identified
that possess the distinctive property of binding to heparin or closely
related heparan sulfate glycosaminoglycan
(HSGAG)1 (1, 2). Heparin-based affinity
chromatography has been a convenient tool for purification of these
factors, and several studies have shown that this binding phenomenon
has functional relevance as well (2-10). HSGAG-dependent
stabilization (3) and/or localization of the growth factor on the cell
surface may promote interaction with less abundant, higher affinity
tyrosine kinase receptors involved in signal transduction (2, 4, 5).
Coupling of heparin or HSGAG either to the growth factor or its
tyrosine kinase receptor may induce conformational changes in these
molecules that augment signaling (3, 6). Several laboratories have
reported that heparin binding can provide a framework for ligand
oligomerization, which may enhance signaling by stimulating
dimerization of the tyrosine kinase receptor (5, 8-10).
Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding
polypeptide that can function as a mitogen, motogen, or morphogen on a
broad spectrum of cellular targets (11-13). HGF/SF is related to
plasminogen and macrophage-stimulating protein, sharing with these
molecules approximately 40-45% amino acid sequence identity and
several structural motifs (14, 15). HGF/SF is synthesized as an
inactive monomer which undergoes internal proteolysis to yield a
biologically active, disulfide-linked heterodimer (16-18). The heavy
chain of the HGF/SF dimer (~60 kDa) is derived from the amino
terminus of the precursor and contains an amino-terminal segment
followed by four kringle domains. A kringle consists of ~80 amino
acids and has a characteristic folding pattern defined by three
internal disulfide bonds and additional conserved residues (19). Two
alternative transcripts have been identified that encode truncated
variants of HGF/SF, terminating after either the first or second
kringle domain (20-23). These smaller isoforms, designated HGF/NK1 and
HGF/NK2, respectively, behave as antagonists or partial agonists,
depending on the molecule and assay conditions (20, 22-25). All of
these HGF/SF isoforms bind with high affinity to the tyrosine kinase
transmembrane HGF/SF receptor, the Met proto-oncogene product (20, 22,
23, 26, 27).
As with other heparin-binding growth factors, the higher capacity,
lower affinity cell surface binding sites for HGF/SF were attributed to
heparan sulfate proteoglycan (27-30). Based on deletion analysis,
kringle 2 (K2) and the hairpin loop in the amino-terminal (N) domain
were implicated in heparin binding (31, 32), although recent work
raised doubt about the importance of K2 in this regard (23). The
potential significance of HGF/SF-heparin interactions has been inferred
from a variety of observations. HGF/SF-Met covalent cross-linking was
diminished following treatment of primary hepatocytes with
heparitinase, indicating that HSGAG might be required for binding (33).
Administration of heparin to cells altered the biological response to
HGF/SF, although the impact on DNA synthesis was either positive or
negative depending on the assay system (34-37). Recent evidence
indicated that heparin stimulated dimerization of HGF/SF and HGF/NK1 in
a cell-free setting, raising the possibility that a similar effect on
the cell surface could facilitate Met dimerization and activation (25,
37) as suggested for FGF-1 and FGF-2 (5, 9, 10).
In the present study, we have investigated the hypothesis that heparin
binding of HGF/SF isoforms is dependent on K2 and specific basic amino
acid residues in the hairpin loop of the N domain. We also provide new
evidence that HSGAG is required for the binding of HGF/SF variants to
Met and subsequent signal transduction. Finally, we demonstrate that
heparin stimulates the oligomerization of HGF/SF isoforms in both
cell-free and cell culture models, and that the N domain has an
important role in this process.
BALB/MK mouse epidermal keratinocytes were
maintained as described previously (38, 39). Madin-Darby canine kidney
cells (MDCK), kindly provided by Dr. R. Furlong (ICRF, Cambridge
University Medical School, Cambridge, United Kingdom) were cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum (Biofluids). Chinese hamster
ovary (CHO) cell lines, both wild-type (WT) and mutant pgsA-745
(hereafter designated CHO-745), which is defective in glycosaminoglycan
chain initiation (40), were grown in Ham's F-12 medium (Biofluids) supplemented with 5% fetal calf serum (Biofluids).
Recombinant human HGF/SF was produced using a
baculovirus expression system and purified as described previously
(23). Recombinant human HGF/NK1 and HGF/NK2, as well as the individual
N and K1 domains, were expressed in bacteria as
described.2 In brief, the DNAs encoding the
various HGF/SF derivatives were generated as
NdeI-BamHI fragments using the polymerase chain
reaction (41). These DNA fragments were then cloned into expression
vector pET11a, the Escherichia coli expression system
developed by Studier (42). Site-directed mutants, designated 2A/NK1,
3A/NK1, and 5A/NK1, with alanine substitution in the
NH2-terminal domain of HGF/NK1 were prepared using the
technique of Higuchi et al. (43). The following positively
charged residues were replaced with alanine: 2A/NK1, Arg-76 and Lys-78;
3A/NK1, Lys-91, Arg-93, and Lys-94; 5A/NK1, combined set of five. The
mutant proteins were expressed in E. coli, refolded, and
purified as described for wild type HGF/NK1.2
HGF/SF and its derivatives
(each at 1 mg/ml) were individually dissolved in phosphate-buffered
saline (PBS), and 0.1 ml was applied to heparin-Sepharose CL-6B resin
(0.3 ml bed volume, Pharmacia Biotech Inc.) equilibrated in PBS.
Protein was eluted with a stepwise gradient of increasing NaCl
concentration (1.5 ml/step, as indicated) and detected by
immunoblotting.
For detection of HGF/SF derivatives eluted
from heparin-Sepharose, 10 µl of each fraction was resolved by
SDS-PAGE in a 4-20% polyacrylamide mini-gel (Novex) under reducing
conditions. Immunoblotting was performed as described previously (44),
with the following modifications: proteins were transferred to
Immobilon-P (polyvinylidene fluoride) filters (Millipore), and probed
with a mixture of GammaBind (Pharmacia Biotech Inc.)-purified rabbit
polyclonal antisera raised against either full-length human HGF/SF (36)
or a synthetic peptide corresponding to HGF/SF residues 158-172
(numbering according to Ref. 36).
Met was detected by sequential immunoprecipitation and immunoblotting
of protein isolated from equivalent amounts of whole cell lysates of
serum-starved BALB/MK, CHO-WT, and CHO-745 cells prepared essentially
as described previously (26). Samples were incubated with anti-mouse
Met polyclonal antiserum (1 µg/ml; Santa Cruz Biotechnology Inc.,
catalog no. sc-162) for 2 h at 4 °C. After immunoprecipitation,
SDS-PAGE and transfer to Immobilon filters, blotting was performed with
anti-mouse Met polyclonal antiserum (0.5 µg/ml; Santa Cruz, catalog
no. sc-162).
DNA synthesis by BALB/MK cells was
measured by [3H]thymidine incorporation as described
(45), except that IGF-I (200 ng/ml; Pepro Tech Inc., catalog no.
100-11) was included in the assay medium. The scatter assay was
performed with a subclone of MDCK cells, according to published methods
(46). After 20 h, the cells were fixed for 20 min with 2.5%
formaldehyde, and for an additional 20 min with
methanol/formaldehyde/phosphate-buffered saline (7/2/1) prior to
staining with Giemsa (pH 6.8).
Recombinant human HGF/NK1 was radiolabeled with
[125I]NaI (Amersham) by the chloramine-T method as
described (26, 44). Reactions performed with 10 µg of protein and 1 mCi of [125I]NaI yielded tracer with a specific activity
of ~65 µCi/µg.
Cross-linking was performed as described (44), with the following
modifications. Confluent serum-starved monolayers were incubated for 45 min at room temperature in HEPES binding buffer (44) containing 0.9 nM of 125I-HGF/NK1 (6.6 × 106
cpm) in the presence or absence of heparin (Fisher, catalog no. H-19),
unlabeled HGF/SF isoforms or IGF-I at the indicated concentrations. Cells were washed twice with HEPES-buffered saline (10 mM
HEPES, 150 mM NaCl, pH 7.4) and then incubated for 16 min
at room temperature with 0.1 mM disuccinimidyl suberate
(Pierce) dissolved in dimethyl sulfoxide (Me2SO). The
cross-linking reaction was terminated with quench buffer (200 mM glycine, 10 mM Tris-HCl, 2 mM
EDTA, pH 7.4). Cell lysates, clarified by centrifugation (14,000 × g, 15 min), were boiled in sample buffer containing 2%
Confluent monolayers (10-cm dishes)
of CHO-WT and CHO-745 cells were serum-starved overnight, exposed to
100 ng/ml HGF/NK1 and/or heparin at either 0.3 or 3 µg/ml for 5 min
at 37 °C, lysed in anti-phosphotyrosine RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 2.5 mM
EDTA, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium vanadate, 1% Nonidet P-40, plus protease
inhibitors, pH 7.5), and immunoprecipitated with anti-phosphotyrosine
monoclonal antibody 4G10 bound to agarose beads (50 µl of
beads/lysate; Upstate Biotechnology, Inc.). Immunoprecipitated proteins
were resolved by 7.5% SDS-PAGE and immunoblotted for mouse Met as
described above.
Serum-starved confluent CHO-WT and CHO-745 monolayers (10 cm dishes) were treated with HGF/NK1 (100 ng/ml) and/or heparin (0.3 µg/ml) for 15 or 60 min, or with acidic FGF (10 ng/ml) kindly provided by W. Burgess (Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross) (47) and heparin (0.3 µg/ml) for 60 min
at 37 °C. RNA was prepared with RNAzol (Tel-Test, Inc.) as described
(48). 20 µg of total RNA was electrophoresed in 1% agarose
formaldehyde gels, transferred to nylon membranes, and hybridized to a
32P-labeled c-fos cDNA probe kindly provided
by T. Curran (49) or 32P-labeled 18 S ribosomal RNA
cDNA probe (ATCC, catalog no. 77242).
Experiments were performed according to a protocol
similar to one described in Ref. 25. 1 µg HGF/NK1, N, or K1 domain
was incubated in 20 µl of PBS for 20 min at room temperature with or
without cross-linking reagent bis(sulfosuccinimidyl) suberate (1 mM); Pierce) in the presence or absence of heparin (1 µg). Next, 5 µl of 1 M Tris-HCl, pH 6.8, was added,
followed by 6 µl of 5 × SDS-sample buffer containing 10%
To analyze the structural features of HGF/SF responsible
for its interaction with heparin, we determined the NaCl concentration required to elute various HGF/SF isoforms and derivatives from heparin-Sepharose resin. Immunoblotting of fractions from each step in
the salt gradient revealed that HGF/SF and the naturally occurring
variants, HGF/NK1 and HGF/NK2, all were released from the resin with
0.8 and 1.0 M NaCl (Fig. 1). To further
localize the heparin-binding site of HGF/SF, we produced the N and K1
domains separately in a bacterial expression system, and purified them to homogeneity. Analysis of their circular dichroism spectra suggested that the recombinant domains attained their native
conformation.2 When subjected to heparin-Sepharose
chromatography, the K1 domain eluted at
In their investigation of the sites required
for heparin binding, Mizuno et al. (32) observed that
deletion of the hairpin loop region (amino acid residues 70-96) in the
N domain resulted in loss of heparin binding. After comparing the
heparin-binding properties and hairpin loop sequences of HGF/SF and the
structurally related molecules, plasminogen (14) and
macrophage-stimulating protein (15), they noted a correlation between
the net charge of the hairpin region and avidity for heparin.
Specifically, the net charge of this region in HGF/SF was +6 and the
protein eluted from heparin-Sepharose with ~1.0 M
NaCl, while the corresponding values of these parameters for
plasminogen were
The effect of alanine substitution on HGF/SF biological activity was
evaluated by testing the 2A, 3A, and 5A/NK1 derivatives in a
[3H]thymidine incorporation bioassay using BALB/MK cells
(Fig. 3A). 5A/NK1 was approximately 30-fold
less potent than wild-type HGF/NK1 in this assay. This decrease in
activity could not be attributed to the small reduction in heparin
affinity associated with this molecule, because 2A/NK1, which exhibited
a comparable decline in affinity to heparin-Sepharose, was as active as
wild type HGF/NK1. Moreover, the derivative that most closely retained
the heparin-binding characteristics of HGF/SF, 3A/NK1, was markedly
less potent than HGF/NK1 in the mitogenic bioassay. A similar pattern
of relative potency was observed with these reagents in the standard
scatter assay using MDCK cells (Fig. 3B). At a concentration
where wild type HGF/NK1 and 2A/NK1 elicited a marked scatter response,
3A/NK1 and 5A/NK1 stimulated only modest cellular retraction and fewer signs of the spiky morphology characteristic of the scatter phenomenon (46). Preliminary experiments suggest that the diminished activity of
3A/NK1 and 5A/NK1 may result from reduced stability of these derivatives during overnight incubation at
37 °C.3 Nonetheless, regardless of the
loss in biological activity, alanine substitution of the
HGF/SF-specific basic residues in the hairpin loop had little impact on
heparin binding.
In
addition to characterizing the structural elements responsible for
heparin binding of HGF/SF isoforms, we also explored the relevance of
the heparin interaction for ligand binding to Met. To address this
issue, we performed a series of experiments with wild type and
glycosaminoglycan-deficient CHO cells (CHO-745). The latter are lacking
in enzymatic activity required for synthesis of heparan sulfate (40),
and have been used extensively to analyze the role of HSGAG in
FGF-receptor binding (7, 9, 50). Using an antiserum raised against a
carboxyl-terminal peptide sequence of murine Met, our initial studies
established that a 145-kDa protein band corresponding to the Met
Covalent cross-linking experiments performed with
125I-HGF/NK1 and BALB/MK or CHO cells revealed a high
molecular mass band with an estimated mass of 165 kDa in lysates of
BALB/MK and wild type CHO cells (Fig. 5A). However, no
band was observed in lysates from the glycosaminoglycan-deficient
CHO-745 cells. The ligand specificity of this binding interaction was
verified by competition with an excess of unlabeled HGF/NK1 but not
IGF-I (Fig. 5A). Immunoprecipitation of the cross-linked
complex with mouse Met peptide antiserum in the absence but not
presence of peptide confirmed that Met was in the complex (Fig.
5B). To demonstrate that the lack of HGF/NK1-Met cross-linking in CHO-745 cells was due to a deficiency in HSGAG, the
experiment also was performed in the presence of varying concentrations of added soluble heparin. Consistent with earlier observations (33),
when heparin was added to cells such as BALB/MK and wild type CHO,
which have an ample supply of HSGAG on their surfaces, cross-linking of
ligand to Met was diminished (Fig. 5A). In contrast, heparin
at 0.3 µg/ml (and to a lesser extent at 3 µg/ml) facilitated cross-linking of 125I-HGF/NK1 to Met in the
glycosaminoglycan-deficient CHO-745 cells. These results established
that the shortage of endogenous HSGAG in CHO-745 cells was specifically
responsible for the paucity of their HGF/NK1-Met binding.
To explore the functional
significance of HGF/NK1 binding to Met observed in response to
exogenous heparin, we measured tyrosine phosphorylation of Met
following treatment of CHO cells with HGF/NK1 in the presence or
absence of heparin. As shown in Fig. 6, incubation of
wild type CHO cells with HGF/NK1 for 5 min resulted in a prominent Met
protein band in the anti-phosphotyrosine immunoprecipitate. When
heparin also was present in the HGF/NK1-containing medium, there was
little or no additional stimulation. In contrast, while HGF/NK1 and
heparin each by itself caused only a slight increase in the amount of
Met immunoprecipitated from CHO-745 cells by phosphotyrosine antibody,
together they stimulated a large increase in the amount of
immunoprecipitated Met protein. This result strongly suggested that the
addition of heparin to glycosaminoglycan-deficient CHO cells not only
enhanced HGF/NK1 binding to Met, but also triggered tyrosine kinase
activity.
Further evidence of signaling was obtained by analysis of
c-fos expression. c-fos induction is a sensitive
measure of rapid cellular responses to mitogenic stimulation (51),
which served as a convenient indicator of HGF/NK1 signaling. When
wild-type CHO cells were incubated with HGF/NK1 at 100 ng/ml, an
increase in c-fos transcript was observed after 15 min and
was more pronounced at 60 min (Fig. 7). Heparin alone
did not stimulate c-fos expression, and had only a modest
positive effect when tested in combination with HGF/NK1 on CHO-WT
cells. In contrast, HGF/NK1 alone stimulated only a slight increase in
c-fos expression by CHO-745 cells after either 15 or 60 min.
However, when heparin (3 µg/ml) was included in the incubation
medium, c-fos induction in CHO-745 was much stronger after
60 min, comparable to that observed with the wild-type cells. Heparin
itself had no effect on c-fos expression in this assay.
These findings indicated that the heparin-dependent
interaction of HGF/NK1 with Met in CHO-745 cells was capable of
activating the signal transduction process.
Because of earlier reports that heparin enhanced
the activity of FGFs (8-10) and HGF/SF (37) by promoting ligand
oligomerization, we investigated the possibility that the positive
impact of heparin or cell surface heparan sulfate proteoglycan on
Met-ligand binding and signaling might involve oligomerization of
HGF/SF isoforms. We first examined the effect of heparin on HGF/NK1
oligomerization in a cell-free setting. In the absence of heparin,
there was little evidence of cross-linked HGF/NK1 dimer as detected by
Coomassie Blue staining after SDS-PAGE. However, when heparin was
present during the incubation with cross-linking agent, subsequent
analysis revealed that a substantial portion of HGF/NK1 protein
migrated as a dimer (Fig. 8, left panel).
Weak bands corresponding to higher order oligomers occasionally were
also seen. When similar experiments were performed with separately
prepared recombinant N and K1 domains, the ability to oligomerize
correlated unequivocally with the N domain. As shown in the
middle panel of Fig. 8, a small amount of putative N domain
dimer was evident in the absence of heparin, and in the presence of
heparin a set of bands were observed which represent a series of
oligomers. Frequently the series included bands corresponding in size
to hexamers and even octamers. In contrast, there was no evidence of
oligomerization of the K1 domain either in the absence or presence of
heparin (right panel, Fig. 8).
Ligand oligomerization also was observed when incubations were
performed with cells in culture. Cross-linked 125I-HGF/NK1
dimer was readily detected when tracer was added to BALB/MK or CHO-WT
cells, but was far less conspicuous with the glycosaminoglycan-deficient CHO-745 cells (Fig. 9). The
addition of 25 nM unlabeled HGF/NK1 increased the amount of
dimer in lysates of all three cell types, and even trimer and tetramer
were seen with BALB/MK and CHO-WT cells. Exogenous heparin only
promoted oligomer formation in CHO-745 lysates, while it inhibited
oligomerization in cultures of cells having normal levels of
proteoglycan. These findings suggested that a moderate amount of
heparin or endogenous heparan sulfate proteoglycan could facilitate
oligomerization on the cell surface, but that excessive heparin would
block this process.
Several reports suggest that the interaction of heparin-binding
growth factors with heparan sulfate proteoglycan has a major impact on
their biological effects (2-5, 7-10, 25, 34-37). In practical terms,
the clinical utility of such growth factors may be strongly influenced
by their binding to the proteoglycan on blood vessel walls and in
extracellular matrix, which could hamper delivery of factor to cellular
targets. The present study was undertaken to identify the
heparin-binding site(s) of HGF/SF and investigate the role of heparin
in growth factor binding and signal transduction. Mapping of the
primary heparin-binding site of HGF/SF to the N domain is a first step
in designing mutants with altered heparin affinity. Such derivatives
would be useful in testing the importance of the proteoglycan
interaction for growth factor activity and might have greater efficacy
in therapeutic applications.
Previous analyses of HGF/SF deletion mutants had implicated the N
domain (31) and more specifically the hairpin loop (32) in heparin
interactions. However, in deletion studies loss of function could
result not only from removal of the targeted domain but also from
improper folding of the residual protein that could reduce its
stability or alter tertiary structure. For instance, the K2 deletion
mutant also reportedly bound poorly to heparin (32), suggesting a
critical role for K2 in this interaction that was inconsistent with our
results. By showing that the N domain retained the binding properties
of full-length HGF/SF, we directly demonstrate that the heparin-binding
site of HGF/SF resides in this domain.
Because the heparin-binding motifs of many proteins involve basic amino
acid residues clustered in a linear sequence (52, 53), the proposed
role of two such sites in the hairpin region of the N domain was
tested. The failure of alanine substitution at these positions to have
much impact on the interaction with heparin-Sepharose suggested that
other residues must be responsible. Two clusters of basic residues near
the amino terminus of the N domain, RKRR (residues 33-36) and KKSAK
(residues 43-47), probably can be excluded from consideration because
corresponding sequences were absent from a truncated form of rat
HGF/SF, which bound tightly to heparin-Sepharose (14, 54). Two other
segments, KIKTKK (residues 58-63) and KK (residues 109-110), are
worthy of investigation, as well as other nearby residues that may
contribute to heparin binding (52, 53, 55).
While our evidence that HSGAG is important for Met-ligand binding
complements other recent work, it would appear to be in conflict with
earlier observations made in a cell-free setting. When Mark et
al. (56) detected high affinity binding of HGF/SF isoforms to a
fusion protein containing the extracellular domain of Met and the heavy
chain Fc portion of immunoglobulin G, no heparin or HSGAG had been
added to the assay. These ostensibly contradictory observations could
be reconciled in several ways. First, HSGAG may have inadvertently
co-purified with the Met-IgG fusion protein, and consequently been
present in the cell-free assay. Such co-purification is plausible given
the evidence that the extracellular domain of another heparin-binding
growth factor, tyrosine kinase receptor can itself bind heparin (6).
Second, it is possible that nonspecific adsorption of HGF/SF isoforms in the solid phase binding assay used by Mark et al. (56)
resulted in high local concentrations of ligand, obviating one of the
potential mechanisms of HSGAG action in promoting high affinity
receptor binding (5). Third, heparin-dependent protection
of ligand from proteolytic degradation may not have been important in a cell-free binding assay, where proteases presumably are scarce. Finally, if the HSGAG requirement for Met-ligand binding involves receptor dimerization as has been suggested both for FGFs (5, 8-10)
and HGF/SF isoforms (25, 37), this probably would have been unnecessary
in the cell-free assay because immunoglobulin fusion proteins typically
already exist as dimers.
The present findings indicated that heparin or endogenous HSGAG was
required both for binding and activation of receptor signaling. The
potent stimulation of tyrosine phosphorylation and c-fos
expression in CHO-745 cells elicited by the combination of HGF/NK1 and
exogenous heparin established the biological relevance of
heparin-dependent binding reconstituted in these cells.
Occasionally weak responses to HGF/NK1 alone were observed in CHO-745
cells, presumably due to a low level of receptor binding that was below
the sensitivity of detection by cross-linking analysis. Such binding
might arise either from small amounts of endogenous HSGAG on CHO-745
cells or occur independent of HSGAG. Similarly, heparin itself at times appeared to elicit a minor tyrosine phosphorylation signal. While not a
consistent finding, this may reflect a capacity of the
glycosaminoglycan to promote tyrosine phosphorylation, as has been
reported in cells expressing the tyrosine kinase receptor FGFR-4
(57).
Our results emphasized the importance of heparin not only for Met
binding but also ligand oligomerization. As recently reported for
HGF/NK1 (25), cross-linking experiments performed in a cell-free setting suggested that HGF/NK1 and especially the N domain itself have
an intrinsic tendency to oligomerize. This predisposition is markedly
enhanced by the addition of heparin. For the N domain, a series of
oligomers ranging up to octamers formed in the presence of heparin.
When 125I-labeled HGF/NK1 (Fig. 9) or N domain3
was incubated with cells and cross-linking agent, oligomers again were
observed if endogenous heparan sulfate proteoglycan or added heparin
was present. Because the latter experiments were performed with whole
cells and ng/ml concentrations of tracer, the potential physiological
relevance of ligand oligomerization was reinforced. Of note, the effect
of exogenous heparin on ligand oligomerization varied with cell type:
while heparin increased dimer formation on glycosaminoglycan-deficient
CHO cells, it reduced oligomerization on BALB/MK and wild type CHO
cells. In the latter instances, HGF/SF ligands probably were dispersed
among the abundant heparin molecules such that ligand-ligand
interactions were less likely to occur. Thus, the impact of heparin on
oligomerization is dependent on the content of endogenous HSGAG. If
ligand oligomerization is important for signal transduction, the effect
of added heparin on the biological activity of HGF/SF ligands will vary
with the quantity and perhaps composition of proteoglycan on the cell
surface.
We thank Dr. J. D. Esko for providing us with
wild type and glycosaminoglycan-deficient CHO cells and Dr. D. P. Bottaro for critical reading of the manuscript.
Protein Expression Laboratory,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Cell Culture
-mercaptoethanol and resolved by 6% SDS-PAGE. Alternatively, Met
protein was immunoprecipitated from cell lysates as described above, in
the absence or presence of 10 µg/ml of competing peptide (Santa Cruz,
catalog no. sc-162 P). Following SDS-PAGE, radiolabeled cross-linked
complexes were detected by autoradiography of dried gels exposed to
Kodak X-Omat AR film at
70 °C.
-mercaptoethanol. After boiling for 3 min, samples were resolved by
SDS-PAGE in a 4-20% mini-gel (Novex). Protein was detected by
staining with colloidal Coomassie Blue (Novex).
N Domain Retains Heparin-binding Properties of Full-length
HGF/SF
0.2 M NaCl, while
the N domain eluted at the same concentration as HGF/SF (Fig. 1). These
data suggest that the determinants of HGF/SF heparin binding are
confined to the N domain.
Fig. 1.
Heparin-Sepharose chromatography of HGF/SF
isoforms and derivatives. Various HGF/SF-related polypeptides were
applied in parallel to columns containing 0.3 ml of heparin-Sepharose resin equilibrated in PBS. After the resins were washed twice with 1.5 ml of PBS, protein was eluted with a stepwise gradient of increasing
NaCl molar concentrations. Aliquots of starting material (lane
1), eluted fractions (lanes 2-10), and
heparin-Sepharose beads following chromatography (lane 11)
were processed for SDS-PAGE and immunoblotting as described under
"Experimental Procedures." Molecular size markers are indicated at
the right.
[View Larger Version of this Image (36K GIF file)]
2.5 and ~ 0.1 M NaCl, and for
macrophage-stimulating protein were +2 and ~ 0.4 M
NaCl. This led to the suggestion that clusters of basic amino acid
residues in the HGF/SF hairpin loop mediated the interaction with
heparin. We tested this hypothesis directly by substituting alanine for
these residues in a series of site-directed mutants. As illustrated in
Fig. 2, HGF/NK1 derivatives were generated that had 2, 3, or 5 alanine substitutions, as follows: R76A and K78A (2A/NK1),
K91A, R93A, and K94A (3A/NK1), or a combined set (5A/NK1). When these
reagents were chromatographed on heparin-Sepharose, they all bound
tightly to the resin and eluted either with 0.6-0.8 M
(2A/NK1 and 5A/NK1) or 0.8-1.0 M (3A/NK1) NaCl (Fig. 1).
Although replacement of R76 and K78 resulted in slightly earlier
elution of protein, our findings indicated that the five basic residues did not have a critical role in heparin binding of HGF/SF isoforms.
Fig. 2.
Amino acid sequences of hairpin loop region
from wild type human HGF/NK1 and site-directed mutants. Positively
charged residues are indicated by + in the wild type (WT)
sequence, and sites of alanine substitutions are designated by
shaded circles labeled A in the mutant sequences
(2A, 3A, and 5A). Residues in the wild
type sequence are numbered according to Rubin et al. (36).
[View Larger Version of this Image (17K GIF file)]
Fig. 3.
Biological activity of HGF/NK1 and three
site-directed mutants. A, stimulation of DNA synthesis in
BALB/MK cells by HGF/NK1 (closed square), 2A/NK1 (open
square), 3A/NK1 (closed circle), and 5A/NK1 (open
circle). Mean values of triplicate measurements of
[3H]thymidine incorporation from one of three independent
experiments are expressed as counts/min. Standard deviations were
approximately 10%. B, effect of HGF/NK1, 2A/NK1, 3A/NK1,
and 5A/NK1 in scatter assay with MDCK cells. Concentration of each
growth factor is 12.5 nM. Original magnification, ×100.
These pictures are representative fields from multiple wells and
reflect consistent differences in serial dilutions of the
samples.
[View Larger Version of this Image (27K GIF file)]
-subunit in BALB/MK cells was detected in CHO cells (Fig.
4). Moreover, the wild type and
glycosaminoglycan-deficient CHO strains expressed similar levels of
hamster Met protein.
Fig. 4.
Immunodetection of Met in BALB/MK, wild type
and glycosaminoglycan-deficient CHO cells. Lysates from BALB/MK,
CHO-WT, and CHO-745 cells were immunoprecipitated and immunoblotted
with an antiserum raised against the carboxyl-terminal sequence of murine Met. The arrow indicates a 145-kDa protein band
corresponding to the Met -subunit in murine and hamster cells.
[View Larger Version of this Image (43K GIF file)]
Fig. 5.
Covalent affinity cross-linking of
125I-HGF/NK1 to Met in BALB/MK, CHO-WT, and CHO-745 cells.
A, cross-linking of 125I-HGF/NK1 in the absence
or presence of 25 nM unlabeled HGF/NK1, heparin (0.3 or 3.0 µg/ml), or 25 nM IGF-I. Following incubation with tracer
and the indicated reagents, cells were treated with disuccinimidyl
suberate and subsequently lysed. Proteins were resolved by 6% SDS-PAGE
and gels dried for autoradiography. B, Met specificity of
125I-HGF/NK1 cross-linking. After cross-linking
125I-HGF/NK1 to three cell lines as described in
A, 10% of each lysate was removed and the remainder divided
in half for immunoprecipitation with Met antiserum in the absence
(Met) or presence (+Comp) of competing peptide
(10 µg/ml). For CHO-745, cross-linking was performed in the presence
of heparin at 0.3 µg/ml. In both A and B, the arrow indicates a 165-kDa band corresponding to
125I-HGF/NK1-Met complex with an apparent stoichiometry of
1:1. Positions of molecular mass markers are at right.
[View Larger Version of this Image (65K GIF file)]
Fig. 6.
Tyrosine phosphorylation of Met in response
to HGF/NK1 and/or heparin in CHO-WT and CHO-745 cells.
Serum-starved cells were treated with 100 ng/ml HGF/NK1, 3 µg/ml
heparin, both, or neither reagent for 5 min at 37 °C. Whole cell
lysates were immunoprecipitated with phosphotyrosine antibody bound to
agarose beads, resolved by 8% SDS-PAGE, blotted, and probed with Met
antiserum. The band migrating at 145 kDa corresponds to the -subunit
of Met; the positions of molecular mass markers are at the
right.
[View Larger Version of this Image (46K GIF file)]
Fig. 7.
HGF/NK1 and heparin-mediated activation of
c-fos expression in CHO-WT and CHO-745 cells. Cultures
of CHO-WT and CHO-745 cells were stimulated with HGF/NK1 (100 ng/ml),
acidic FGF (aFGF; 10 ng/ml) and/or heparin (0.3 µg/ml),
for 0, 15 or 60 min. Total RNA samples (20 µg) were fractionated by
electrophoresis in a formaldehyde agarose gel, transferred to nylon
membrane, hybridized with a 32P-labeled c-fos or
18S-rRNA cDNA probe, and subjected to autoradiography.
[View Larger Version of this Image (28K GIF file)]
Fig. 8.
Oligomerization of HGF/NK1, N, and K1 in a
cell-free setting. Cross-linking reactions were performed as
described under "Experimental Procedures." Protein was incubated
alone or with cross-linking agent bis(sulfosuccinimidyl)suberate in the
absence or presence of heparin. Subsequently, protein was resolved by SDS-PAGE and detected by staining with colloidal Coomassie Blue. In
addition to oligomers of HGF/NK1 and the N domain, a doublet was
observed when cross-linker was added either to HGF/NK1 or the K1
domain. This probably resulted from intramolecular cross-linking of
compact, non-reduced protein, as earlier work indicated that HGF/NK1
migrated more rapidly in SDS-PAGE under non-reducing conditions (23).
The positions of molecular mass markers are at the
right.
[View Larger Version of this Image (28K GIF file)]
Fig. 9.
Oligomerization of 125I-HGF/NK1
in cell culture. Covalent cross-linking of HGF/NK1 to itself was
analyzed by incubating tracer and cross-linking agent with BALB/MK,
CHO-WT, or CHO-745 cells in the presence or absence of various
combinations of unlabeled HGF/NK1 (500 ng/ml) and heparin (0.3 or 3 µg/ml). After the reaction was terminated, cell lysates were resolved
by SDS-PAGE and labeled proteins detected by autoradiography of dried
gel. Position of HGF/NK1 dimer (a), trimer (b),
and tetramer (c) is indicated by label at left;
location of molecular mass markers is shown at right.
[View Larger Version of this Image (50K GIF file)]
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: NCI/DBS/LCMB,
Bldg. 37, Rm. 1E24, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255. Tel.: 301-496-4265; Fax: 301-496-8479; E-mail: rubinj{at}dc37a.nci.nih.gov.
1
The abbreviations used are: HSGAG, heparan
sulfate glycosaminoglycan; HGF/SF, hepatocyte growth factor/scatter
factor; HGF/NK1, truncated HGF/SF isoform containing amino-terminal (N)
domain and kringle 1; HGF/NK2, truncated HGF/SF isoform containing N domain and kringles 1 and 2; K2, second kringle domain in HGF/SF; CHO-WT, wild type Chinese hamster ovary (CHO) cell line; MDCK, Madin-Darby canine kidney; 2A/NK1, 3A/NK1, and 5A/NK1, alanine substitution mutants in HGF/NK1 with 2, 3, or 5 substitutions as
described in the text; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; IGF, insulin-like growth
factor.
2
S. J. Stahl, P. T. Wingfield, J. D. Kaufman, L. K. Pannell, V. Cioce, H. Sakata, W. G. Taylor, J. S. Rubin, and D. P. Bottaro, submitted for publication.
3
H. Sakata, W. G. Taylor, J. M. Rosenberg, and J. S. Rubin, unpublished observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.