A Receptor-like Protein-tyrosine Phosphatase PTP
/RPTP
Binds a Heparin-binding Growth Factor Midkine
INVOLVEMENT OF ARGININE 78 OF MIDKINE IN THE HIGH AFFINITY
BINDING TO PTP
*
Nobuaki
Maeda
,
Keiko
Ichihara-Tanaka§,
Terutoshi
Kimura¶,
Kenji
Kadomatsu§,
Takashi
Muramatsu§, and
Masaharu
Noda
From the
Division of Molecular Neurobiology, National
Institute for Basic Biology, and Department of Molecular Biomechanics,
Graduate University for Advanced Studies, Okazaki 444-8585, the
§ Department of Biochemistry, Nagoya University School
of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466-8550, and the
¶ Peptide Institute Inc., 4-1-2, Ina,
Minoh, Osaka 562-0015, Japan
 |
ABSTRACT |
Midkine is a 13-kDa heparin-binding growth factor
with 45% sequence identity to pleiotrophin. Pleiotrophin has been
demonstrated to bind to protein-tyrosine phosphatase
(PTP
) with
high affinity. In this study, we examined the binding of midkine to
PTP
by solid-phase binding assay. Midkine and pleiotrophin binding
to PTP
were equally inhibited by soluble pleiotrophin and also by
some specific glycosaminoglycans. For both bindings, Scatchard analysis
revealed low (3.0 nM) and high (0.58 nM)
affinity binding sites. These results suggested that PTP
is a common
receptor for midkine and pleiotrophin. Midkine is structurally divided
into the N- and C-terminal halves, and the latter exhibited full
activity for PTP
binding and neuronal migration induction. The
C-terminal half contains two heparin-binding sites consisting of
clusters of basic amino acids, Clusters I and II. A mutation at
Arg78 in Cluster I resulted in loss of the high affinity
binding and reduced neuronal migration-inducing activity, while
mutations at Lys83 and Lys84 in Cluster II
showed almost no effect on either activity. Chondroitinase ABC-treated
PTP
exhibited similar low affinity binding both to the native
midkine and midkine mutants at Arg78. These results
suggested that Arg78 in midkine plays an essential role in
high affinity binding to PTP
by interacting with the chondroitin
sulfate portion of this receptor.
 |
INTRODUCTION |
PTP
/RPTP
1 is a
receptor-like protein-tyrosine phosphatase, which is abundantly
expressed in the central nervous system as a chondroitin sulfate
proteoglycan (1-4). PTP
is composed of an N-terminal carbonic
anhydrase-like domain, a fibronectin type III domain, a serine,
glycine-rich domain that is thought to be chondroitin sulfate
attachment region, a transmembrane segment, and two tyrosine
phosphatase domains (1, 2). There are three splice variants of this
molecule: (a) the full-length PTP
(PTP
-A); (b) the short form of PTP
, in which most of the serine,
glycine-rich region is deleted (PTP
-B); and (c) the
secreted form (PTP
-S), which corresponds to the extracellular region
of PTP
-A and is also known as 6B4 proteoglycan/phosphacan (3, 5).
All these splice variants are expressed as chondroitin sulfate
proteoglycans in the brain (6), suggesting that chondroitin sulfate
plays an essential role in receptor function.
Several proteins such as contactin, tenascin, L1, NCAM, and TAG1 have
been reported to bind PTP
(7-9). Contactin is thought to be a
neuronal receptor of PTP
expressed on glial cells (7). Recently, we
found that PTP
binds with pleiotrophin/heparin-binding growth-associated molecule (10), in that a chondroitin sulfate portion
of PTP
constitutes a part of the pleiotrophin binding site and
regulates the affinity of PTP
-pleiotrophin binding (10). We further
demonstrated that pleiotrophin-induced neurite outgrowth and neuronal
migration were suppressed by chondroitin sulfate, polyclonal antibodies
against the extracellular domain of PTP
, and sodium vanadate, a
protein-tyrosine phosphatase inhibitor. These findings suggested that
PTP
expressed on neurons is a signal transducing receptor for
pleiotrophin (10, 11).
Pleiotrophin has 45% sequence identity to midkine, forming a new
family of heparin-binding growth factors. These molecules share many
biological activities (12, 13); both proteins promote neurite outgrowth
(14-16), enhance plasminogen activator activity in aortic endothelial
cells (17), and oncogenically transform NIH3T3 cells (18, 19). These
findings suggest that they use a common or highly related receptors.
Midkine and pleiotrophin are structurally composed of two domains (the
N- and C-terminal halves), each of which is tightly held through three
or two disulfide bridges, respectively (20). The C-terminal half of
midkine binds strongly to heparin and exhibits neurite
outgrowth-promoting and plasminogen activator-enhancing activities (21,
22). On the other hand, the N-terminal half of midkine, which shows
relatively weak heparin binding activity, does not promote neurite
outgrowth or enhance plasminogen activator activity (21, 22). NMR
spectroscopy revealed two clusters of basic amino acids in the
C-terminal half of midkine, Clusters I and II, both of which interact
with heparin oligosaccharides (23). Experiments using various midkine
mutants indicated that Cluster II plays an essential role in its
plasminogen activator-enhancing effect (22).
In this study, we examined the PTP
-midkine interaction using various
midkine mutants. Native PTP
exhibited high affinity binding to
midkine, and the binding properties were essentially the same as those
of pleiotrophin. Moreover, PTP
-midkine binding was inhibited by the
presence of pleiotrophin. These observations suggested that midkine and
pleiotrophin share a common binding site on PTP
. PTP
bound to the
C-terminal half of midkine, but not to the N-terminal half. A mutation
R78Q in Cluster I reduced the binding affinity, while mutations K83Q,
K84Q, and K83Q/K84Q in Cluster II did not affect binding. Furthermore,
in these midkine mutants, the strength of binding affinities and the
neuronal migration-inducing activities were highly correlated. These
findings suggested that basic amino acids in Cluster I of midkine and
pleiotrophin are crucial for high affinity binding to PTP
to
transduce signals in neurons.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Chondroitin sulfate A from whale cartilage,
chondroitin sulfate B from pig skin, chondroitin sulfates C and D from
shark cartilage, chondroitin sulfate E from squid cartilage, heparan
sulfate from bovine kidney, keratan sulfate from bovine cornea, and
chondroitinase ABC were purchased from Seikagaku Corp. Heparin was
obtained from Sigma. 125I-Bolton-Hunter reagent was
purchased from DuPont NEN. Chroma Spin columns were obtained from
CLONTECH. Maxisorp immunoplates were purchased from
Nunc. Dulbecco's modified Eagle's medium, F-12 medium, and B-27
supplement were purchased from Life Technologies, Inc.
TranswellsTM were obtained from Corning Coster Corp. Micro
BCA kit was from Pierce. PTP
-S was purified as reported elsewhere
(24). The N- and C-terminal half domains of human midkine (1-59 and
60-121, respectively) were synthesized as described previously (25). Mouse midkine mutants, R78Q, K83Q, K84Q, K83Q/K84Q and R78Q/K83Q/K84Q were prepared by site-directed mutagenesis (21, 22). Mutations are
indicated by the amino acid residues (in one-letter code) in the
wild-type and the mutant, preceding and following the numbers of the
altered residues, respectively.
125I Labeling of PTP
-S--
PTP
-S was purified
from rat brain and labeled as described previously (10, 24). Briefly,
dried 125I-Bolton-Hunter reagent (100 µCi) was
solubilized with samples (10 µg of protein in 100 µl of 100 mM sodium phosphate buffer, pH 8.0), followed by incubation
for 3 h on ice and then mixed with 30 µl of 1 M
glycine, pH 7.5. After a 2-h incubation at 4 °C, free
125I-Bolton-Hunter reagent was removed by passing through a
Chroma Spin 30 column equilibrated with 0.05% Triton X-100, 0.5 mg/ml BSA, 0.15 M NaCl, 10 mM sodium phosphate, pH
7.2. The specific radioactivity of the sample thus prepared was
3.3 × 106 cpm/µg.
Binding Assay--
Wells of Nunc Maxisorp Immunoplates were
coated with 35 µl of 1~5 µg/ml midkine or pleiotrophin in 5 mM Tris-HCl, pH 8.0, at 4 °C overnight. The wells were
washed three times with phosphate-buffered saline and then blocked with
1% BSA/phosphate-buffered saline for 1 h at room temperature.
125I-PTP
-S diluted in 0.5% BSA, 2 mM
CaCl2, 2 mM MgCl2, 0.1% CHAPS, 0.15 M NaCl, 10 mM sodium phosphate, pH 7.2, was added to the coated wells. When inhibition experiments were
performed, inhibitors (pleiotrophin or glycosaminoglycans) were
premixed with 125I-PTP
-S before addition to the wells.
The plates were incubated for 5 h at room temperature and then the
wells were washed three times with 1 mM CaCl2,
1 mM MgCl2, 0.15 M NaCl, 10 mM Tris-HCl, pH 7.2. The bound materials were released by
adding 200 µl of 0.1 M NaOH, 0.2% SDS to the wells. The
plates were shaken for 15 min at room temperature and then the eluted
radioactivity was measured using a
counter.
125I-Labeled PTP
-S was digested with chondroitinase ABC
as described previously (10). Briefly, 125I-PTP
-S was
diluted with 100 µl of 0.5% BSA, 2 mM MgCl2,
2 mM CaCl2, 0.15 M NaCl, 10 mM sodium acetate, 10 mM Tris-HCl, pH 7.5, to a
final concentration of 2 µg/ml. Aliquots (5 milliunits) of protease-free chondroitinase ABC was added to the samples, and the
solutions were incubated for 30 min at 30 °C for use in binding assays.
Other Methods--
Boyden chamber cell migration assays were
performed using cortical neurons from embryonic day-17 Sprague-Dawley
rats as described previously (11). Protein concentration was determined
using a Micro BCA kit using BSA as a standard.
 |
RESULTS |
Binding of PTP
-S to Midkine--
Fig.
1 shows the binding profile of
125I-labeled PTP
-S to human midkine-coated ELISA plates.
Scatchard analyses of the binding of PTP
-S to midkine showed low
(Kd = 3.0 nM) and high (Kd = 0.58 nM) affinity binding sites
(Fig. 1B), which were similar to those of
pleiotrophin-PTP
binding (10). As shown in Fig. 1, the C-terminal
half of midkine exhibited exactly the same binding properties to
PTP
-S as native midkine. On the other hand, the N-terminal half of
midkine showed no binding activity to PTP
-S (Fig. 1). Soluble
pleiotrophin premixed with PTP
-S inhibited the binding of PTP
-S
to pleiotrophin-coated ELISA plates (Fig.
2). In a similar
dose-dependent manner, soluble pleiotrophin also inhibited
the binding of PTP
-S to midkine on the plates (Fig. 2), suggesting
that pleiotrophin and midkine bind to the same binding site on PTP
-S
with a similar affinity. However, fairly high concentrations of
pleiotrophin were required for inhibition (IC50 = ~600
nM) compared with the Kd values of
midkine- or pleiotrophin-PTP
-S binding obtained by solid-phase
binding assay. These observations suggested that substrate-bound forms of midkine and pleiotrophin exhibit orders of stronger affinity to
PTP
-S than the soluble forms.

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Fig. 1.
Midkine binds to PTP
through the C-terminal half. A, wells of ELISA
plates were coated with wild-type human midkine ( ), the C-terminal
half ( ), or the N-terminal half of midkine ( ), and the binding of
125I-PTP -S was measured by solid-phase binding assay.
B, 125I-PTP -S binding to midkine ( ),
C-terminal half ( ), or N-terminal half of midkine ( ) was analyzed
using Scatchard plots. Midkine exhibited high (Kd = 0.58 nM) and low (Kd = 3 nM)
affinity binding sites. The C-terminal half of midkine also exhibited
high (Kd = 0.55 nM) and low
(Kd = 2.4 nM) affinity binding sites,
but the N-terminal half of midkine showed no binding.
|
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Fig. 2.
Pleiotrophin inhibits the binding of midkine
to PTP -S. Binding of
125I-PTP -S to midkine ( ) or pleiotrophin ( ) on
ELISA plates was measured by solid-phase binding assay in the presence
of various concentrations of soluble pleiotrophin. Soluble pleiotrophin
inhibited midkine-PTP -S binding as well as pleiotrophin-PTP -S
binding.
|
|
Midkine has two clusters of basic amino acids (Clusters I and II)
located at the surface on one side of the C-terminal half domain, which
are considered to be heparin binding sites (23). Cluster I contains
Lys76, Arg78, and Lys99, and
Cluster II contains Lys83, Lys84, and
Arg86; amino acids were numbered according to mouse
midkine. Among these, Lys76, Arg78,
Lys83, and Lys99 are conserved in midkine and
pleiotrophin of all species examined to date. On the other hand,
Lys84 is conserved only in midkine of various species but
is changed to Arg in pleiotrophin, and Arg86 is changed to
Leu in pleiotrophin of various species and midkine of some species
(23).
Five mouse midkine mutants were prepared, in which some of the basic
amino acids in the Cluster I and/or II were changed to glutamine: R78Q,
K83Q, K84Q, K83Q/K84Q, and R78Q/K83Q/K84Q (21, 22). As shown in Fig.
3, K83Q, K84Q, and K83Q/K84Q exhibited essentially the same binding activities to PTP
-S as the native midkine, suggesting that Cluster II is not essential for midkine-PTP
binding. In contrast, R78Q and R78Q/K83Q/K84Q exhibited only low affinity binding to PTP
-S, suggesting that Cluster I plays an important role in the high affinity binding between PTP
and midkine (Fig. 3 and Table I).

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Fig. 3.
Loss of high affinity binding of midkine to
PTP by a mutation of Arg78 of
midkine. A, the binding of 125I-PTP -S to
midkine mutants R78Q ( ) or R78Q/K83Q/K84Q ( ) was measured by
solid-phase binding assay and analyzed using Scatchard plots.
B, the binding of 125I-PTP -S to K83Q ( ),
K84Q ( ), or K83Q/K84Q ( ) was measured by solid-phase binding
assay and analyzed using Scatchard plots. Dotted lines
correspond to the binding of wild-type mouse midkine. Mutation at
Arg78 resulted in the loss of high affinity binding. On the
other hand, mutations at Lys83 and Lys84 showed
no effect on PTP -binding. The Kd values for the
each midkine mutants are summarized in Table I.
|
|
Effects of Chondroitinase ABC Digestion of PTP
-S on the
PTP
-midkine Binding--
Chondroitin sulfate chains of PTP
play
an essential role in its high affinity binding to pleiotrophin (10).
Chondroitinase ABC digestion of PTP
-S reduced its affinity also to
midkine (Fig. 4). In contrast to the
intact PTP
-S showing high (Kd = ~0.5
nM) and low (Kd = ~3 nM)
affinity binding sites, chondroitinase ABC-digested PTP
-S exhibited
only a low affinity binding site (Kd = 8.8 nM) (Fig. 4, A and B). In addition, R78Q (Fig. 4, C and D) and R78Q/K83Q/K84Q (Table
I), which have a mutation at Arg78, showed a single binding
site to intact PTP
-S with a Kd value of 2.8 nM, in a similar affinity range to the chondroitinase ABC-digested PTP
-S (~8 nM). This suggested that
Arg78 is involved in binding to chondroitin sulfate to make
up the high affinity binding site.

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Fig. 4.
Involvement of chondroitin sulfate chains in
the binding of PTP to midkine. Binding of
intact ( ) or chondroitinase ABC-treated ( )
125I-PTP -S to native midkine (A,
B) or R78Q (C, D) was measured by
solid-phase binding assay. Scatchard analysis indicated that
chondroitinase ABC-treated PTP -S contained a single low affinity
binding site for both R78Q (Kd = 8.0 nM)
and native midkine (Kd = 8.8 nM).
|
|
Influence of Glycosaminoglycans on PTP
-midkine
Binding--
Previously, we reported that pleiotrophin-PTP
-S
binding is inhibited strongly by heparin, moderately by heparan sulfate
and chondroitin sulfate C, and very weakly by chondroitin sulfate A
(10). Glycosaminoglycans inhibited midkine-PTP
-S interactions similarly (Fig. 5). Heparin strongly
inhibited binding of PTP
-S to midkine (IC50 = 10 ng/ml),
heparan sulfate showed moderate inhibition (IC50 = 100 ng/ml), and keratan sulfate exerted almost no effect. On the other
hand, various types of chondroitin sulfate exerted diverse influences
on midkine-PTP
-S binding. Chondroitin sulfate D and chondroitin
sulfate E strongly inhibited binding (IC50 = ~70 ng/ml
for both types of chondroitin sulfate). Chondroitin sulfate B and
chondroitin sulfate C showed moderate inhibitory effects
(IC50 = 500 ng/ml and 1000 ng/ml, respectively), but
chondroitin sulfate A exerted almost no effect (IC50 > 100 µg/ml). Similar sensitivities to the various chondroitin sulfates
were observed for pleiotrophin-PTP
binding (data not shown; data
partly shown in Ref. 10).

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Fig. 5.
Influence of various glycosaminoglycans on
the binding of PTP to midkine. Binding of
125I-PTP -S to midkine was measured by a solid-phase
binding assay in the presence of various concentrations of
glycosaminoglycans. The effects of heparin ( ), heparan sulfate
( ), chondroitin sulfate A ( ), chondroitin sulfate B (*),
chondroitin sulfate C ( ), chondroitin sulfate D (+), chondroitin
sulfate E ( ), and keratan sulfate ( ) are shown.
|
|
Cell Migration-inducing Activity of Midkine--
We reported
previously that pleiotrophin induced cell migration of cortical neurons
(11). Midkine also induced neuronal migration in Boyden chamber cell
migration assay with essentially the same dose dependence profile as
that of pleiotrophin (data not shown; see Fig. 3A of Ref.
11). Boyden chamber cell migration assay indicated that the C-terminal
half of midkine exhibited full cell migration-inducing activity but the
N-terminal half was devoid of activity (Fig.
6A). Midkine mutants, K83Q,
K84Q, and K83Q/K84Q, which have amino acid replacements in Cluster II, showed normal levels of activity. In contrast, R78Q and R78Q/K83Q/K84Q exhibited low cell migration-inducing activity (Fig. 6B).
These results suggested that Cluster I is sufficient for the neuronal migration-inducing activity of midkine.

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Fig. 6.
Neuronal migration-inducing activity of
midkine mutants. A, cortical neurons were analyzed by
Boyden chamber cell migration assay using membranes coated with 70 µg/ml human midkine (wild), the N-terminal half
(N-half), or C-terminal half (C-half) of midkine.
The C-terminal half of midkine exhibited normal level of neuronal
migration-inducing activity, whereas the N-terminal half was devoid of
activity. B, cortical neurons were analyzed by Boyden
chamber cell migration assay using membranes coated with 70 µg/ml
mouse midkine (wild-type), or midkine mutants R78Q, K83Q, K84Q,
K83Q/K84Q, or R78Q/K83Q/K84Q. R78Q and R78Q/K83Q/K84Q exhibited reduced
neuronal migration-inducing activity.
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|
Influence of Glycosaminoglycans on Midkine-induced Neuronal
Migration--
Midkine-induced neuronal migration was inhibited
strongly by heparin, moderately by heparan sulfate, but not by keratan
sulfate (Fig. 7). As in the case of
midkine-PTP
-S binding, various types of chondroitin sulfate exerted
diverse effects on midkine-induced neuronal migration. Chondroitin
sulfate A exhibited almost no effect (Fig. 7). On the other hand,
midkine-induced neuronal migration was inhibited strongly by
chondroitin sulfate E and moderately by chondroitin sulfates B, C, and
D (Fig. 7). Similar inhibitory effects by chondroitin sulfates were
observed for pleiotrophin-induced neuronal migration (data not shown;
data partly shown in Ref. 11).

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Fig. 7.
Effects of various glycosaminoglycans on
midkine-induced neuronal migration. Cortical neurons were analyzed
by Boyden chamber cell migration assay using membranes coated with 33 µg/ml of midkine in the presence of various concentrations of
glycosaminoglycans. Neurons were cultured in the presence of heparin
(HR), heparan sulfate (HS), chondroitin sulfate A
(CSA), chondroitin sulfate B (CSB), chondroitin
sulfate C (CSC), chondroitin sulfate D (CSD),
chondroitin sulfate E (CSE), and keratan sulfate
(KS).
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|
 |
DISCUSSION |
In this study, we demonstrated that midkine binds to PTP
. The
characteristics of binding of midkine to PTP
were indistinguishable from those of pleiotrophin (10), suggesting that PTP
is a common receptor of midkine and pleiotrophin. Here, the C-terminal half of
midkine was revealed to be sufficient for the binding. The C-terminal
half domain of midkine exhibits various activities: strong
heparin-binding activity, neurite promoting activity, and tissue
plasminogen activator enhancing activity (21, 22). On the other hand,
specific functions have not been found for the N-terminal half of
midkine, although it weakly binds to heparin (21, 22, 26).
NMR spectroscopy indicated that there are two heparin-binding sites in
the C-terminal half domain: Cluster I, which is composed of
Lys76, Arg78, and Lys99, and
Cluster II, which is composed of Lys83, Lys84,
and Arg86 (23). On the other hand, in the N-terminal half
domain, the basic amino acids do not form clusters which are expected
to interact with the sulfate groups on heparin (23, 26). Our data
showed that PTP
-midkine binding was significantly affected by the
mutation of Arg78, but not by mutations of
Lys83, Lys84, or Lys83 + Lys84. Here, mutation of Arg78 resulted in loss
of high affinity binding between midkine and PTP
(Fig. 3), and the
chondroitin sulfate portion of PTP
plays an essential role in
formation of the high affinity binding site (Fig. 4). Therefore, it
seems that Arg78 of midkine is involved in binding to
chondroitin sulfate on PTP
. In support of this idea, various
chondroitin sulfate preparations differentially affected midkine-PTP
binding (Fig. 5). Among various chondroitin sulfate species, there was
a significant difference in the inhibiting activity. This finding
suggested that there must be a specific structural motif of chondroitin
sulfate that strongly inhibits midkine-PTP
binding. However, the
nature of this structure is not known at present because commercially
available chondroitin sulfate samples contain considerable
heterogeneity. Nevertheless, it is possible to speculate that
Arg78 of midkine recognizes a specific structure of
chondroitin sulfate on the PTP
molecule, which is also present in
chondroitin sulfates C, D, and E, but not in chondroitin sulfate A. An
oversulfated structure is one of the candidates; however, the fine
structure of chondroitin sulfate chains of PTP
must be determined to
further clarify this point. A similar finding was reported for
DSD-1-PG, a chondroitin sulfate proteoglycan expressed in the rodent
central nervous system, that is recognized by a monoclonal antibody
473HD (27). DSD-1-PG exhibited neurite outgrowth-promoting activity, which was blocked by 473HD or by chondroitinase ABC digestion of this
proteoglycan (27). The binding of 473HD to DSD-1-PG was inhibited by
chondroitin sulfates C and D, but not by chondroitin sulfates A or B
(27, 28), suggesting that a specific structural motif of chondroitin
sulfate plays an important physiological function in the brain.
Chondroitinase ABC-treated PTP
showed markedly reduced binding
affinity to midkine. Mutations of midkine at Arg78,
Lys83, and Lys84 did not influence binding to
the chondroitinase ABC-treated PTP
, suggesting that these amino
acids do not play an essential role in binding to the core glycoprotein
portion of PTP
. In summary, there seems to be a hierarchy with three
steps in the binding between PTP
and midkine: 1) low affinity
binding between midkine and core glycoprotein portion of PTP
(Kd = ~8 nM); 2) medium affinity
binding between midkine and PTP
bearing general structure of
chondroitin sulfate (Kd = ~3 nM); and
3) high affinity binding between midkine and PTP
bearing a specific structural motif of chondroitin sulfate (Kd = ~0.6
nM), which involves a specific contribution of
Arg78 of midkine.
Boyden chamber cell migration assay indicated that the mutation of
Arg78 of midkine significantly reduced the neuronal
migration-inducing activity of this factor (Fig. 6). In contrast,
mutations of Lys83 and Lys84 did not influence
this activity. These observations suggested that the high affinity
binding of midkine and PTP
is important for the neuronal
migration-inducing activity. Here, heparin strongly inhibited midkine-
and pleiotrophin-induced neuronal migration, and only the
substrate-bound forms of these factors exhibit this activity (11, 17),
which is consistent with the finding that PTP
exhibits very low
affinity to soluble pleiotrophin (Fig. 2). In contrast, plasminogen
activator-enhancing activity of midkine was markedly reduced by double
mutation of Lys83 and Lys84, but not by the
single mutation of Arg78, Lys83, or
Lys84 (22). The soluble forms of midkine and pleiotrophin
enhance plasminogen activator activity. However, it has been suggested that enzymatic dimerization of midkine and pleiotrophin induced by
heparin-like oligosaccharides (presumably endogenous heparan sulfate)
is required for plasminogen activator-enhancing activity (17). Here,
exogenously added heparin could substitute for endogenous heparan
sulfate (17). Taken together, these two activities of midkine and
pleiotrophin are thought to be mediated by distinct receptors.
Neurite-promoting activity of midkine was also markedly reduced by
mutation of Arg78, while mutations of Lys83 and
Lys84 were less effective (22). These observations
suggested that the neurite-promoting and the neuronal
migration-inducing activities of midkine are mediated at least partly
by the same or similar receptor(s).
Midkine binds to a syndecan family heparan sulfate proteoglycan,
ryudocan, with high affinity (29). Pleiotrophin/heparin-binding growth-associated molecule binds to N-syndecan, which is
thought to be another pleiotrophin receptor involved in
pleiotrophin-induced neurite extension (30). It would be helpful to
examine the binding of syndecan family proteoglycans with midkine
mutants to determine the physiological significance of these interactions.
 |
ACKNOWLEDGEMENT |
We thank Akiko Kodama for secretarial assistance.
 |
FOOTNOTES |
*
This work was supported by grants from the Ministry of
Education, Science, Sports, and Culture of Japan and from CREST of Japan Science and Technology Corporation.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: Division of
Molecular Neurobiology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki 444-8585, Japan. Tel.:
81-564-55-7590; Fax: 81-564-55-7595; E-mail: madon{at}nibb.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
PTP, protein-tyrosine phosphatase;
RPTP, receptor-like protein-tyrosine
phosphatase;
BSA, bovine serum albumin;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
ELISA, enzyme-linked immunosorbent assay.
 |
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