From the The Burnham Institute Cancer Research
Center, La Jolla, California 92037
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ABSTRACT |
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NG2 is a transmembrane chondroitin sulfate
proteoglycan that is expressed by immature progenitor cells in several
developmental lineages and by some types of malignant cells. In
vitro studies have suggested that NG2 participates in growth
factor activation of the platelet-derived growth factor- Due to their structural complexity, proteoglycans are highly
interactive macromolecules that participate in a broad range of
cell-cell and cell-matrix interactions, including regulation of cell
adhesion, proliferation, motility, and differentiation (Refs. 1-3; for
review, see Ref. 1). Proteoglycans are important mediators of growth
factor binding. Several examples illustrate how proteoglycans can
function both as extracellular reservoirs for growth factors and as
facilitators of growth factor binding to signal transducing receptors
on the cell surface (4-6).
Most research has focused on the interaction of growth factors with the
glycosaminoglycan (GAG)1
chains of proteoglycans. bFGF (FGF-2) binds tightly to the heparan sulfate chains of extracellular proteoglycans, as well as to free heparin (7, 8). Since the bound growth factor is resistant to
degradation by extracellular proteases, the complex serves as a
reservoir of matrix-bound FGF. Active FGF is released by proteolysis of
the proteoglycan core protein or by partial degradation of the heparan
sulfate chains, processes that occur during tissue development and
remodeling (9, 10).
bFGF also binds to cell-surface heparan sulfate proteoglycans such as
syndecan, which present the bound FGF to its signaling receptor, a
receptor tyrosine kinase whose activation leads to induction of a
variety of cellular processes, including proliferation. Binding of bFGF
has also been demonstrated to the core protein of the chondroitin
sulfate proteoglycan phosphacan (11). TGF- Results from our laboratory have suggested that the integral membrane
chondroitin sulfate proteoglycan NG2 is another example of a
proteoglycan that can influence growth factor activity. NG2 is found on
the surface of several different types of immature progenitor cells,
including oligodendrocyte progenitors, chondroblasts, and smooth muscle
cells (13-17). Some types of neoplasms, such as melanomas,
glioblastomas, osteosarcomas, chondrosarcomas, and lymphomas also
express NG2 (18-21). NG2 thus appears to be a developmental marker
that is expressed at high levels on mitotic cells but is down-regulated
during terminal differentiation. Developmental studies in the central
nervous system have shown that there is a close relationship between
the expression of NG2 and PDGF- In order to investigate the possibility that NG2 is involved in binding
and presentation of PDGF-AA, we have studied the ability of NG2 to bind
to a variety of different growth factors. Our laboratory has produced
and purified several recombinant forms of NG2, including the entire
270-kDa extracellular domain (both with and without GAG chains) and
several well defined fragments of this ectodomain. Use of these species
has allowed us to determine both the binding specificity and affinity
of NG2 for growth factors. Here we report that the NG2 core protein
binds with high affinity to PDGF-AA and bFGF (FGF-2). In addition we
show that the large ectodomain of NG2 appears to contain at least two
binding sites for each of these growth factors. These findings provide
a molecular basis for understanding the role of NG2 in PDGF- Antibodies
Goat polyclonal antibodies against human bFGF, EGF, PDGF-AA,
PDGF-BB, TGF- Purified Proteins
Expression and structural features of the recombinant NG2
fragments have been previously described (23). Four different NG2
fragments were used in our experiments: NG2/EC (extracellular) without
GAG chains, comprising the whole extracellular domain (residues
1-2223); NG2/EC with GAG chains; D2 (domain 2, residues 632-1450) and
D3 (domain 3, residues 1587-2218). The D2 preparation used for our
binding studies also contains GAG chains, while the D3 preparation does
not. Recombinant human bFGF, EGF, PDGF-AA (long isoform), PDGF-BB,
TGF- Reagents
Research grade CM5 SensorChips (carboxymethylated dextran
matrix), amine-coupling kit
(N-ethyl-N'-(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and HBS buffer (10 mM Hepes with 0.15 M NaCl,
3.4 mM EDTA, and 0.005% surfactant P20 at pH 7.4) were all
obtained from Pharmacia Biosensor AB (Uppsala, Sweden). Turbo TMB
(3,3',5,5'-tetramethylbenzidine) was purchased from Pierce.
Surface Plasmon Resonance (SPR)
A BIAcore 2000 surface plasmon resonance-based biosensor
(Pharmacia Biosensor AB) was used to measure kinetic parameters for the
interaction between soluble NG2 fragments (analytes) and immobilized growth factors (ligands). Growth factors were immobilized to the sensor
chip surface by the amine-coupling method (24) according the
manufacturer's suggestion. Each ligand was immobilized at a
concentration of approximately 1500 resonance units (1.5 ng/mm2). The reverse design, immobilizing NG2 and injection
of growth factors as soluble analytes, could not be utilized due to
high nonspecific binding of the basic growth factor proteins to the unmodified chip surface.
For all kinetic measurements, we used a flow path involving all four
flow cells of the BIAcore 2000. Simultaneous measurements were obtained
from one flow cell containing the growth factor-coated sensor chip and
a second flow cell containing an underivatized chip. Parallel
measurements of specific and background binding were thus obtained. The
other two flow cells were not monitored. Solutions of NG2 fragments
were injected into the flow cells using the KINJECT command specifying
a 40-µl analyte volume and a 90-s dissociation time. Each assay cycle
was performed with a constant flow of HBS at 20 µl/min. Between
cycles, the immobilized ligands were regenerated by injecting 20 µl
of 1 M NaCl and activating the EXTRACLEAN command. For each
immobilized growth factor a complete set of sensorgrams was recorded at
three to five different analyte concentrations in the range between 100 and 3,500 nM. This set of sensorgrams was analyzed using
the BIAevaluation version 3.0 software. To prepare the data for
analysis, baselines were adjusted to zero for all curves, and injection
start times were aligned. Background sensorgrams were then subtracted
from the experimental sensorgrams to yield curves representing specific
binding. The association and dissociation phases of the sensorgrams
were fit simultaneously, assuming a simple bimolecular reaction model: A + B Solid-Phase Binding Assays
Growth Factor Binding to Immobilized NG2--
Enyzme-linked
immunosorbent assays were used to evaluate the binding of growth
factors to various NG2 fragments coated into microtiter wells. 96-well
microtiter plates (Greiner, Nuertingen, Germany) were coated overnight
at 4 °C with proteins at 3 µg/ml in 100 µl of coating buffer (50 mM sodium carbonate, pH 9.6). After washing twice with PBST
(10 mM phosphate buffer, pH 7.4, containing 2.7 mM KCl, 137 mM NaCl and 0.05% Tween 20),
residual protein binding sites in the wells were saturated by
incubating for 1 h at room temperature with 200 µl of blocking
solution (PBS, 1% BSA). Growth factor samples diluted in PBS, 1% BSA
were then incubated in the wells either at 4 °C overnight or for
2 h at room temperature. Both incubation protocols yielded similar
results. After four washes with PBST, 100 µl of the growth
factor-specific detection antibody (goat polyclonal antibody/2 µg/ml
in PBS, 1% BSA) was allowed to incubate in the wells for 1.5 h at
room temperature. After another round of washing, 100 µl of
HRP-rabbit anti-goat IgG was added and incubated for 1 h at room
temperature. Wells were then washed and bound HRP was detected by
addition of 100 µl of TMB as a peroxidase substrate.
The reaction was terminated after 10 min by addition of 50 µl of 0.5 M H2SO4. The absorbance of the
yellow reaction product was then measured at 450 nm on a Titertek
microtiter plate reader.
NG2 Binding to Immobilized Growth Factors--
Wells were coated
with 2 µg/ml growth factor. Binding of NG2 fragments to these
immobilized growth factors was then determined as described above, with
the following changes: polyclonal antibodies against NG2/EC or NG2/D3
were used as primary detection antibodies, and HRP-goat anti-rabbit IgG
was used as the secondary antibody.
Binding data from the solid-phase immunoassays were analyzed using
PRISM software (GraphPad, San Diego, CA). Dissociation constants were
determined by nonlinear regression analysis.
ELISA Assays
Binding of Soluble Growth Factors to Immobilized NG2
Fragments--
A solid-phase assay was used to examine the binding
potential of soluble growth factors to plastic-immobilized NG2
fragments representing the entire extracellular domain and the
subdomains D2 and D3. PDGF-AA and bFGF bound strongly to each of these
NG2 species in a concentration-dependent manner (Fig.
1, A and B). In
each case the levels of binding to BSA-coated wells were quite low.
Nonlinear regression analysis yielded apparent dissociation constants
(KD) between 5 and 15 nM for bFGF and
KD values between 10 and 20 nM for
PDGF-AA (Table I). bFGF has a somewhat
higher affinity for NG2/EC with GAG chains than for NG2/EC without GAG
chains, whereas PDGF-AA binds slightly better to the species without
GAG chains.
To further address the specificity of the interaction between bFGF or
PDGF-AA and NG2, we asked whether the binding of these growth factors
to immobilized NG2 could be blocked by increasing concentrations of
soluble NG2. Fig. 2 shows that soluble D2
and D3 inhibited the binding of either bFGF or PDGF-AA to the
respective NG2 subdomains in a concentration-dependent
fashion. A 300-fold molar excess of soluble D2 or D3 almost completely
blocked the respective interaction with bFGF or PDGF-AA.
Neither EGF nor VEGF exhibited binding to any of the NG2 fragments that
was significantly above the low background levels observed with
BSA-coated wells, whereas PDGF-BB and TGF- Binding of NG2 to Immobilized Growth Factors--
As an additional
assessment of the specificity of growth factor binding to NG2, we
examined the interactions in assays in which the roles of the binding
partners were reversed: i.e. the growth factors were
immobilized in plastic wells, and the coated wells were incubated with
soluble NG2. Fig. 3 shows that when bFGF
or PDGF-AA-coated wells were incubated with NG2/EC without GAGs or with
NG2/D3 (inset of Fig. 3) at two different concentrations, each of these NG2 fragments bound effectively to both growth factors. Little background binding of NG2 species was observed to BSA-coated wells.
This reversed assay also provided an opportunity to re-examine the
interaction of NG2 with PDGF-BB and TGF- SPR Measurements of NG2/Growth Factor Interaction
Initial experiments indicated that injecting bFGF or PDGF-AA over
a blank sensor chip yielded a high nonspecific binding response, probably due to electrostatic interaction of the cationic growth factors with the negatively charged carboxymethyl dextran layer on the
chip surface. In contrast, injection of NG2 fragments over a blank chip
surface resulted in a low nonspecific interaction (10-30 resonance
units). We therefore restricted our studies to analysis of soluble NG2
fragments injected over growth factor-coated chips. As shown in Figs.
4 and 5,
sensorgrams obtained from injection of NG2 fragments onto surfaces
coated with bFGF or PDGF-AA had good signal to noise ratios and
exhibited concentration-dependent increases in both the
rate and extent of binding. Bound NG2 could be eluted by injection of 1 M NaCl, suggesting that ionic interactions play an
important part in the association of NG2 with these growth factors.
Curve fitting of the sensorgrams yielded on and off rates and apparent
dissociation constants as presented in Table
II. These values represent the mean of
triplicate measurements made on the same sensor chip. The
KD values obtained from SPR measurements are in good
agreement with those obtained from solid-phase assays (Table I).
receptor.
In this study the ability of recombinant NG2 core protein to interact
with several different growth factors (epidermal growth factor (EGF),
basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor
(VEGF)165 and transforming growth factor (TGF)-
1) was
investigated using two different assay systems: enzyme-linked
immunosorbent assay-type solid-phase binding and an optical biosensor
(BIAcore) system. High-affinity binding of bFGF and PDGF-AA to the core
protein of NG2 could be demonstrated with both types of assays. Using both the BIAcore software analysis program and nonlinear regression analysis of the solid phase binding data, KD values
in the low nanomolar range were obtained for binding of each of these growth factors to NG2. The results further indicate that NG2 contains at least two binding sites for each of these two growth factors. PDGF-BB, TGF-
1, VEGF, and EGF exhibited little or no binding to NG2
in either type of assay. These data suggest that NG2 can have an
important role in organizing and presenting some types of mitogenic
growth factors at the cell surface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is another growth factor
that can bind to proteoglycan core proteins. For example, betaglycan
(also called the type III TGF-
1 receptor) is a transmembrane
proteoglycan with multiple binding sites for TGF-
1 (12). Upon
binding to betaglycan, TGF-
1 is presented to types I and II
receptors, which are serine/threonine kinases that activate
intracellular signaling cascades.
receptor on oligodendrocyte
progenitors (15). Moreover, antibodies against NG2 are capable of
blocking the mitogenic effects of PDGF-AA on both oligodendrocyte
progenitors and vascular smooth muscle cells, suggesting that NG2 is
involved in some way in the operation of the PDGF-AA/PDGF-
receptor
pathway (16, 17). Recently, direct comparisons of aortic smooth muscle
cells from wild type and NG2 null mice have shown that the NG2-negative
cells fail to respond to PDGF-AA in both proliferation and migration
assays and that this lack of activity stems from failure of the
PDGF-
receptor to be activated in the presence of PDGF-AA (22).
receptor activation and suggest that similar effects may be found for
FGF receptors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, and VEGF165 were purchased from R&D
Systems (Minneapolis, MN). Rabbit polyclonal antibodies against rat NG2 were affinity-purified from crude antisera using a matrix of
recombinant NG2 fragments NG2/EC or NG2/D3 coupled to Sepharose CL-4B.
Rabbit anti-goat IgG conjugated to horseradish peroxidase (HRP) was
obtained from Calbiochem. Peroxidase-labeled goat anti-rabbit IgG was
purchased from Bio-Rad.
1, and VEGF (long isoform) were purchased from R&D Systems.
AB. The analysis software corrects for the systematic upward
drift of the base line that occurred during some measurements. Both an
association rate constant ka
(M
1 s
1) and a dissociation rate
constant kd (s
1) were obtained for the
entire data set (global fit), and the dissociation constant
(KD = kd/ka) was derived from the two deduced rate constants. For the determination of ka only the middle portion of the association
curve was used for fitting. For determination of kd
only the initial portion of the curve encompassing the fast
dissociation phase was used for fitting. All of our kinetic data were
fit most adequately by assuming a simple bimolecular model for
interaction between soluble analyte and immobilized ligand, equivalent
to the Langmuir isotherm for adsorption to a surface. The goodness of
fit was assessed by inspecting the statistical value
2
and the residuals (observed-calculated). The
2 values
were low (<5) and the residuals randomly distributed about zero. Fits
were not improved by using a mass transport model or a two-state model
(conformational change), except in the case of the interaction between
bFGF and NG2/EC with GAG chains, which was best fit by the mass
transport model.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Binding of bFGF and PDGF-AA to
plastic-immobilized NG2 fragments. Increasing concentrations of
bFGF (A) or PDGF-AA (B) were added to microtiter
wells coated with NG2 fragments or BSA at a concentration of 3 µg/ml.
Bound bFGF and PDGF-AA were detected with growth factor-specific
polyclonal antibodies. The binding assay was performed as
described under "Experimental Procedures." Curves in NG2-coated
wells represent the best fit determined by nonlinear regression
analysis.
Solid-phase binding data
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Fig. 2.
Inhibition of binding of bFGF or PDGF-AA to
immobilized NG2D2 or NG2D3 by soluble NG2 fragments. Microtiter
wells coated with NG2/D2 (A) or NG2/D3 (B) were
incubated with 15 nM FGF ( ) or 15 nM PDGF-AA
(
) in the presence of increasing concentrations of NG2/D2
(A) or NG2/D3 (B), respectively. Bound growth
factor was detected with growth factor-specific polyclonal antibodies.
The OD at 450 nm obtained with bound growth factors in the absence of
soluble NG2 fragments was set at 100%. Each point represents the
mean ± S.D. of duplicate values from three separate
experiments.
1 bound strongly to both
NG2-coated wells and BSA-coated wells (data not shown). Although the
binding of these two growth factors to NG2-coated wells often appeared
to be slightly higher than that to BSA-coated wells, the extremely high
background makes it difficult to draw definitive conclusions from these results.
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Fig. 3.
Binding of NG2 fragments to
plastic-immobilized growth factors. NG2/EC minus GAGs was diluted
at 20 (black columns) and 100 (gray columns)
ng/ml and added to microtiter wells coated with bFGF, PDGF-AA, PDGF-BB,
TGF- 1, or BSA at a concentration of 2 µg/ml. The inset
shows binding of D3 at a concentration of 40 (black columns)
or 200 (gray columns) ng/ml to bFGF, PDGF-AA, and BSA-coated
wells (2 µg/ml). Bound NG2 fragments were detected with
NG2/EC-specific or NG2/D3-specific polyclonal antibodies. The data
represent the mean ± S.D. of duplicate values from four separate
experiments.
1. As shown in Fig. 3,
NG2/EC exhibited no significant binding to plastic-immobilized PDGF-BB
or TGF-
1.
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Fig. 4.
Analysis of bFGF binding to NG2 fragments by
surface plasmon resonance. Sensorgrams are shown for different
concentrations of NG2/D2, NG2/D3, NG2/EC minus GAGs and NG2/EC plus
GAGs injected on immobilized bFGF. The analysis was performed at a flow
rate of 20 µl/min. Regeneration of bFGF was done with 1 M
NaCl. The apparent equilibrium dissociation constants estimated from
these data are summarized in Table II.
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Fig. 5.
Analysis of PDGF-AA and PDGF-BB binding to
NG2 fragments by surface plasmon resonance. Sensorgrams are shown
for different concentrations of NG2/D2, NG2/D3, NG2/EC minus GAGs, and
NG2/EC plus GAGs injected on immobilized PDGF-AA. The analysis and
regeneration was performed as described in the legend to Fig. 4, and
calculated dissociation constants are listed in Table II. The
inset in the NG2/EC panel shows analysis of PDGF-BB
interaction with NG2 by surface plasmon resonance. Sensorgrams were
obtained from the injection of three different concentration of NG2/EC
minus GAGs on immobilized PDGF-BB.
BIAcore binding data
BIAcore analysis was also used to examine the interaction of soluble
NG2 species with sensor chips coated with EGF, VEGF, PDGF-BB, and
TGF-1. In agreement with the results of the ELISA-type binding
assays, none of the four NG2 fragments exhibited significant affinity
for this set of growth factors. The inset of the NG2/EC
sensorgram in Fig. 5 shows an example of sensorgrams obtained for
injection of different concentrations of NG2/EC without GAGs over a
PDGF-BB-coated chip. The blank chip surface responses are subtracted.
Injection of the other NG2 fragments over immobilized PDGF-BB also
failed to yield positive signals (sensorgrams not shown).
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DISCUSSION |
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Several pieces of information obtained previously in our
laboratory have suggested that NG2 plays an important role in cellular responsiveness to PDGF-AA. After noting the close co-localization of
NG2 and PDGF- receptor on oligodendrocyte progenitor cells during
neural development (15), we found that antibodies against NG2 could
block PDGF-induced proliferation of oligodendrocyte progenitors
in vitro (16). Extension of this work to rat aortic smooth
muscle cells showed that these antibodies blocked both proliferation
and migration in response to PDGF-AA (17). Responses to PDGF-BB were
unaffected by the antibodies, specifically implicating NG2 in
PDGF-AA-mediated activation of the
-receptor. Recent work with
aortic smooth muscle cells from the NG2 knockout mouse has allowed us
to confirm these findings independent of the use of antibodies. While
smooth muscle cells from both wild type and NG2 knockout mice are able
to proliferate and migrate in equivalent fashion in response to
PDGF-BB, only the wild type cells are able to respond to PDGF-AA
(22).
More detailed analysis of growth factor-activated signaling pathways in
the wild type and knockout cells showed that while PDGF-AA did not
activate the MAP kinase pathway in knockout cells, the pathway was
still fully operative in response to other effectors such as PDGF-BB
and PMA. This suggested that the absence of NG2 probably affected a
mechanism upstream of the MAP kinase pathway. In support of this idea
we found that in NG2 null cells PDGF-AA was unable to induce
autophosphorylation, and thus activation, of the PDGF- receptor. We
hypothesized that NG2 could affect
-receptor activation in one of
two ways: 1) by participating in growth factor binding and/or
presentation to the signaling receptor or 2) by interacting with the
-receptor in such a way as to facilitate either growth factor
binding or growth factor-mediated changes in receptor structure (such
as dimerization).
While the findings presented in this report do not address the
possibilities suggested by the second model, they nevertheless demonstrate a clear role for NG2 in the binding of two growth factors,
PDGF-AA and bFGF, as suggested in model 1 above. The specificity of
these interactions is emphasized by the failure of NG2 to bind several
other growth factors, including PDGF-BB, TGF-1, EGF, and VEGF. The
absence of interaction between NG2 and PDGF-BB is consistent with the
observed lack of effect of NG2 on cellular responses to this growth factor.
The pattern and specificity of growth factor binding to NG2 was confirmed using two separate experimental systems, a solid-phase ELISA-type assay and an optical biosensor assay. In addition to demonstrating that NG2 binds to a limited spectrum of growth factors, the two types of assays also yielded essentially identical values for the binding affinity of PDGF-AA and bFGF to NG2. In some cases the apparent dissociation constants obtained from surface plasmon resonance measurements are approximately 2-4-fold higher than the corresponding values obtained from solid-phase assays. Presumably these differences are due to differences in the nature of the two assays. SPR measures the interactions directly in real time, while the solid-phase assays are lengthy and require the use of antibodies for indirect detection of the interaction. Also, differences between the electrostatic environments of the plastic wells and dextran-coated sensor chips may cause changes in the detailed conformation of adsorbed proteins, resulting in slight differences in ligand binding properties on the two sets of surfaces. Still, the two sets of values are in good agreement, and both sets reflect the same types of trends in the binding data. For example, in both sets of data bFGF displays a higher affinity for NG2/EC with GAG chains than for the same fragment without GAG chains. PDGF-AA, on the other hand, appears to bind slightly better in both assays to NG2 without GAG chains.
These latter observations may be indicative of subtle differences in
the mechanism of binding of the two growth factors to NG2. It is known
that negatively charged heparan sulfate chains are critical for the
binding of bFGF to heparan sulfate proteoglycans, so interaction of
bFGF with chondroitin sulfate chains may also contribute to the binding
of bFGF to NG2. This interaction may be responsible for the very large
plasmon resonance response of bFGF-coated chips exposed to soluble NG2
containing chondroitin sulfate chains (Fig. 4). While the binding
affinities of NG2/EC+ and NG2/EC differ by a factor of two in these
experiments (Table II), the capacity of NG2/EC+ for binding bFGF
appears to be 5-6-fold greater than that of NG2/EC
. A similar,
although much smaller trend can be seen in the ELISA-type binding
assays (Fig. 1A). Whether electrostatic interactions between
bFGF and the negatively charged chondroitin sulfate chains of NG2 are
somehow magnified in the plasmon resonance experiments, and whether the
large bFGF binding capacity of NG2 with chondroitin sulfate chains is
preserved in a biological environment, are questions that remain to be resolved.
This large contribution of chondroitin sulfate is not seen in the case of PDGF-AA, even though both bFGF and PDGF-AA are basic proteins with isoelectric points between 8.5 and 9.5 (25-29). In fact, it is apparent that the major burden of binding both of these growth factors is borne by the NG2 core protein, independent of the presence of chondroitin sulfate chains. This therefore represents a major difference between the modes of action of NG2 and heparan sulfate proteoglycans and is reminiscent of the ability of the phosphacan core protein to interact with bFGF (11).
Even though their interaction with GAG chains may not be critical for binding of bFGF and PDGF-AA to NG2, the cationic nature of both growth factors is likely to be important for their interaction with the NG2 core protein. This is suggested by the sensitivity of both interactions to high ionic strength. While hydrophobic moieties are often thought to provide the bulk of free energy for binding between proteins and to help slow dissociation of the complex, hydrophilic interactions between polar residues are more likely to determine the specificity of protein-protein interactions and to increase the rate of association (30, 31). The core protein of NG2 has a net negative charge at physiologic pH (pI 5-6) and contains numerous clusters of acidic residues (32). In view of these multiple clusters of negative charges, it may be hypothesized that ligands that bind to NG2 would have properly positioned clusters of basic amino acids. The human PDGF-A chain contains two highly basic amino acid sequences, Arg-Lys-Lys-Pro-Lys (amino acids 73-77) and Lys-Lys-Arg-Lys-Arg-Lys-Arg (amino acids 114-120) (33). A somewhat less imposing sequence (Lys-Asp-Pro-Lys-Arg) is found in bFGF (28). Although PDGF-BB and VEGF are also highly basic proteins, having isoelectric points above 9 (34, 35), they fail to bind to NG2, underscoring the specificity of the interaction of NG2 with PDGF-AA and bFGF.
The above hypothesis might also predict the existence of multiple sites for interaction of NG2 with other molecules, depending on the number and positioning of charged clusters. Experimentally, we found that bFGF and PDGF-AA each could bind effectively to two distinct subdomains of NG2, the central domain 2 and the juxtamembrane domain 3, suggesting at least two separate sites of interaction for these growth factors. Consistent with this observation is the fact that domains 2 and 3 both have numerous clusters of acidic residues, including pairs and triplets of aspartic and glutamic acid (32). Multiple binding sites for PDGF isoforms have also been found in the large basement membrane proteoglycan perlecan (36), indicating that this phenomenon is not unique to NG2. The fact that our growth factor binding data can be fit most adequately by the use of a simple Langmuir adsorption isotherm suggests that the multiple binding sites on NG2 are noninteractive (i.e. not conformationally linked) and that they have similar binding affinities for the growth factors. This similarity in binding affinities can be observed in our comparisons of bFGF or PDGF-AA binding to the D2 and D3 fragments of NG2, each of which contains at least one binding site.
The high proportion of arginine and lysine residues in PDGF-AA and bFGF makes both proteins sensitive targets for proteolysis by plasmin and other proteases that cleave at basic amino acids. Thus the association of these growth factors with proteoglycans can be an important mechanism for protecting them from degradation (37-39). Information concerning such a role for NG2 remains to be obtained. It is of interest to note in this regard that, although NG2 is an integrated membrane protein, large soluble forms of the ectodomain are shed from the surface after proteolytic processing of the core protein (40). Therefore NG2 could be important not only for sequestering growth factors at the cell surface, but also in protecting them from degradation in the extracellular matrix and in body fluids. These possibilities will be addressed experimentally in future investigations.
Our current findings suggest that in addition to the previously
observed effects of NG2 on PDGF-AA-mediated activation of the PDGF-
receptor, we might also expect to find similar effects of NG2 on
bFGF-mediated receptor activation. PDGF-AA and bFGF are ubiquitous
growth factors that have profound effects on proliferation, differentiation, and survival of cells from many different tissues and
developmental lineages. NG2 is also widespread in a variety of
developing tissues (13-17), thus providing a number of potential choices for study. One extremely interesting system involving NG2 and
both of the relevant growth factors is that of developing oligodendrocyte progenitors. In the absence of bFGF and PDGF-AA, these
progenitors differentiate through a series of intermediates into mature
oligodendrocytes (41, 42). In contrast, the combination of bFGF and
PDGF-AA, produced by neurons and astrocytes, delays this
differentiation and promotes proliferation and expansion of the pool of
progenitor cells (43-47). NG2 is present on the undifferentiated
progenitors during the period in which they are sensitive to PDGF-AA
and bFGF and therefore is in place to potentate the effects of the
growth factors (13, 15, and 16). NG2 is then down-regulated after
progenitors progress past the pre-oligodendrocyte stage, at which time
they have become insensitive to the effects of these two growth
factors. This would appear to offer an excellent system for studying
the functional importance of NG2 in events mediated by bFGF and
PDGF-AA.
In summary, the current work shows that PDGF-AA and bFGF have the
ability to bind directly and specifically to the NG2 proteoglycan. NG2
may thus be important for regulating the extracellular localization and
levels of these two growth factors and possibly for presentation of
these ligands to their respective signaling receptors. Further work
will be required to ascertain the degree to which NG2 functions as a
co-receptor for these growth factors and whether its involvement in
receptor activation includes interaction with the receptor to induce
structural transitions (such as dimerization) that facilitate signal transduction.
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ACKNOWLEDGEMENT |
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We thank Dr. Christian R. Lombardo for helpful advice and assistance with the BIAcore 2000 system and the Biaevaluation software.
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FOOTNOTES |
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* The work was supported by National Institutes of Health Grants RO1 NS21990 (to W. B. S.), F32 CA72220 (to M. A. B.), F32 HL09541 (to K. A. G.), and T32 CA09579 (to L. G.).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. Tel.: 619-455-6480 (ext. 3220); Fax: 619-646-3197; E-mail: goretzki{at}ljcrf.edu.
¶ Current address: Selective Genetics Inc., 11035 Roselle St., San Diego, CA 92121.
Current address: Mitokor, 11494 Sorrento Valley Rd., San
Diego, CA 92121.
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ABBREVIATIONS |
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The abbreviations used are:
GAG, glycosaminoglycan;
bFGF, basic fibroblast growth factor;
BSA, bovine
serum albumin;
EGF, epidermal growth factor;
PDGF, platelet-derived
growth factor;
VEGF, vascular endothelial growth factor;
SPR, surface
plasmon resonance;
TGF-1, transforming growth factor-
1;
HRP, horseradish peroxidase;
PBS, phosphate-buffered saline;
ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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