(Received for publication, November 7, 1996, and in revised form, April 30, 1997)
From the Laboratory of Immunobiology, National Cancer Institute,
Frederick Cancer Research and Development Center, Frederick,
Maryland 21702, the § Antibody Department, Macrophage stimulating protein (MSP) is a 78-kDa
disulfide-linked heterodimer belonging to the plasminogen-related
kringle protein family. MSP activates the RON receptor protein-tyrosine kinase, which results in cell migration, shape change, or
proliferation. A structure-activity study of MSP was performed using
pro-MSP, MSP, MSP Macrophage stimulating protein (MSP)1
was originally purified from human plasma, based on its activity for
murine resident peritoneal macrophages (1). It is a 78-kDa
heterodimeric protein composed of a disulfide-linked 53-kDa The receptor for MSP was recently identified as the human RON gene
product (18), a transmembrane receptor protein-tyrosine kinase cloned
from a human keratinocyte cDNA library (19). The murine STK gene
cloned from hematopoietic stem cells of bone marrow is the homologue of
human RON (20, 21). The RON gene encodes a 190-kDa heterodimeric
protein composed of a 40-kDa extracellular In this work, we initiated a structure-activity study of MSP to
identify functionally important domains that interact with the RON
receptor. Five purified recombinant proteins were used, including
pro-MSP, MSP, MSP Human mature plasma MSP was purified as described
(1). Human recombinant single chain pro-MSP was derived from CHO cells transfected with human MSP cDNA and purified in two steps by
S-Sepharose and anti-MSP IgG affinity column chromatography. MSP
Madin-Darby canine kidney (MDCK) cells transfected
with a human RON cDNA (clone RE7) (18), NIH3T3 cells transfected
with murine STK cDNA (21), and CHO-K1 cells transfected with human MSP cDNA (clone 18) (8) were as described. Human kidney 293 cells
were from ATCC (Rockville, MD). Murine keratinocyte cell line PAM212,
BK-1, and MK308 were provided by Dr. A. Dlugosz (NCI, Bethesda, MD).
Cells were cultured in Dulbecco's modified Eagle's medium with 10%
fetal bovine serum at 37 °C in a humidified incubator containing 5%
CO2 in air. Mouse peritoneal resident macrophages were
obtained from C3H/HeN mice by lavage of the peritoneal cavity with 15 ml of sterile RPMI 1640 medium containing 0.5% fetal bovine serum (8).
10 µg of each protein in 15 µl of 0.1 M
borate buffer, pH 8.5, were added to 250 µCi of
125I-labeled Bolton-Hunter reagent (25) and equilibrated on
ice for 60 min. The reaction was terminated by the addition of 0.2 M, pH 8.5, glycine borate buffer. The reaction mixture was
then applied to an Excellulose GF-5 desalting column (Pierce)
equilibrated with phosphate-buffered saline containing 0.25% gelatin.
Iodinated protein was eluted with 2 ml of phosphate-buffered
saline-gelatin buffer and counted in a gamma counter (Gamma 5500, Beckman). The specific activity of the labeled proteins was about 200 Ci/mmol.
CHO-MSP18 cells were incubated with 100 µCi of
[35S]cysteine in cysteine-free Dulbecco's modified
Eagle's medium without fetal bovine serum for 56 h. Under these
conditions, all the recombinant protein is 35S-labeled
single chain pro-MSP (8). 35S-pro-MSP in culture
supernatants was then converted into two chain mature MSP with 50 nM kallikrein, a serine protease that specifically cleaves
pro-MSP at the Arg483-Val484 bond (8). The
concentrations of MSP were determined by a specific sandwich
enzyme-linked immunosorbent assay (26). Immunoprecipitation and
SDS-PAGE under nonreducing conditions of cleaved
35S-pro-MSP showed not only disulfide-linked mature MSP but
also free Binding of 125I-MSP, MSP A suspension
of 5 × 106 cells in 1 ml of binding buffer was
incubated with 5 nM MSP at 37 °C for different time
intervals. Cell were then equilibrated for 30 min in 200 µl of lysis
buffer (50 mM Tris buffer, pH 7.4, 1% Triton X-100, 1%
Nonidet P-40, 150 mM NaCl, 2 mM EDTA, 100 µM vanadate, 20 µg/ml leopeptin, 20 µg/ml aprotinin,
and 50 µg/ml soybean trypsin inhibitor). Lysate proteins were
precipitated with monoclonal antibody ID2 to RON or rabbit anti-STK
serum coupled to protein G-Sepharose beads. Samples were dissolved in
sample buffer with 2-mercaptoethanol, separated on a 7.5%
polyacrylamide gel by SDS-PAGE, and transferred to Immobilon-P
(Millipore). Membranes were blocked with 1% bovine serum albumin in
0.15 M pH 7.6 Tris buffer with 0.5% Tween 20, then
incubated with 0.2 µg/ml anti-phosphotyrosine antibody overnight, followed by goat anti-mouse IgG conjugated with horseradish peroxidase. The horseradish peroxidase reaction was developed with ECL detection reagents. In some experiments, the membrane was treated with
SDS/2-mecaptoethanol erasure buffer and reprobed with rabbit anti-RON
serum as described (18).
Murine peritoneal
resident macrophages (5 × 105/ml) were incubated in 1 ml of serum-free RPMI 1640 medium in 24-well tissue culture plates.
MSP, MSP subunits, or their different combinations were added. After
incubation at 37 °C for 45 min, cells were photographed.
The assay was done as described (18).
Bottom wells of a chemotaxis chamber were filled in triplicate with 30 µl of RPMI 1640 medium containing different amounts of MSP or MSP
subunits and then covered with a polycarbonate membrane coated with
mouse collagen IV. Upper wells were filled with 45 µl of cell
suspension (2 × 106/ml in RPMI 1640 medium). To see
the effect of MSP subunits on MSP-induced migration, cells were first
mixed with 5 or 30 nM of MSP The experiments were performed as
described (15). BK-1 cells at a concentration of 105/ml of
a serum-free medium (equal volumes of keratinocyte serum free-medium,
Eagle's minimum essential medium, and CHO-SF medium) were seeded at
100 µl/well in a 96-well culture plate. MSP, MSP In the course of studying pro-MSP conversion into mature
MSP, we noticed that about 30% of our metabolically
35S-labeled recombinant pro-MSP lacked the disulfide link
between its
We tested for binding of
radiolabeled pure MSP and its subunits to murine keratinocyte BK1 and
MK308 cells, which express 10,000-15,000 STK receptors/cell (15). Fig.
3 shows that in both cell lines, specific binding of
125I-MSP was inhibited in a
concentration-dependent manner by unlabeled MSP
We next studied tyrosine phosphorylation of RON induced
by pro-MSP, MSP, and its subunits in kidney 293 and MDCK-RE7 cells. After precipitation of proteins with ID2 anti-RON, Western blot with
monoclonal antibody 4G10 to phosphotyrosine showed that only MSP-induced tyrosine phosphorylation of the 150-kDa RON
To see if MSP subunits can induce cell shape change or
migration, mouse peritoneal resident macrophages were used. Fig.
7 shows that MSP
BK-1 keratinocytes were used to assess if MSP Table I.
Effects of MSP and MSP subunits, alone or in combination, on
proliferation of murine BK-1 keratinocytes
Department of Cell
Genetics, Genentech, Inc., South San Francisco, California 94080, and ** Toyobo Co., Ltd., Ohtsu, Shiga, 520-02, Japan.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
chains, and a complex including the first
two kringles and IgG Fc (MSP-NK2). Radioiodinated MSP and MSP
chain both bound specifically to RON. The Kd of 1.4 nM for MSP
chain is higher than the reported
Kd range of 0.6-0.8 nM for MSP.
Pro-MSP, MSP
chain, and MSP-NK2 did not bind. Only MSP stimulated
RON autophosphorylation. Although the
chain bound to RON and
partially inhibited MSP-induced RON phosphorylation in kidney 293 cells, it did not induce RON phosphorylation. Pro-MSP, MSP
chain,
or MSP-NK2 failed to activate RON, consistent with their inability to
bind to the RON receptor. Functional studies showed that only MSP
induced cell migration, and shape change in resident macrophages, and
growth of murine keratinocytes. Our data indicate that the primary
receptor binding domain is located in a region of the MSP
chain, in
contrast to structurally similar hepatocyte growth factor, in which the
receptor binding site is in the
chain. However, full activation of
RON requires binding of the complete MSP disulfide-linked
chain
heterodimer.
chain
and a 25-kDa
chain (calculated from amino acid composition). The
chain contains a N-terminal hairpin loop followed by four kringle
domains. The
chain has a serine protease-like domain but is devoid
of enzymatic activity due to amino acid substitutions in the catalytic
triad. MSP belongs to the kringle protein family that includes
plasminogen (2) and hepatocyte growth factor/scatter factor (HGF/SF)
(3, 4). MSP is synthesized mainly by liver cells (5, 6), circulates in
blood as a biologically inactive single chain precursor (7), and is
cleaved by members of the kallikrein family (8, 9) or by trypsin-like
enzymes located on macrophage surfaces (7). Recent functional studies
have revealed that in addition to induction of macrophage shape change,
chemotactic migration (10), and phagocytosis of C3bi-coated
erythrocytes (1), MSP has other activities. These include inhibition of
expression of inducible nitric oxide synthase mRNA in endotoxin or
cytokine-stimulated macrophages (11), induction of interleukin-6
production and differentiation of megakaryocytes (12), suppression of
colony formation of human bone marrow cells induced by Steel factor
plus granulocyte macrophage-stimulating factor (13), increase in beat
frequency of nasal epithelium cilia (14), and stimulation in
vitro of proliferation of certain epithelial cell lines
(15-17).
chain and 150-kDa
transmembrane
chain with intrinsic tyrosine kinase activity (21).
This property places the product of the RON/STK gene into a subfamily
of receptor tyrosine kinases that includes proto-oncogene MET and SEA
(22, 23). These receptors share many unique structural properties
including a putative proteolytic cleavage site, similar location of
cysteine residues in their extracellular domain, and two conserved
tyrosines in the C-terminal tail (19, 20, 22, 23). Studies of the
signaling pathways of RON have shown that tyrosine-phosphorylated RON
associates in vivo with intracellular signal transducers,
including Grb-2-Sos and phosphatidylinositol 3-kinase (17, 24).
and
chains, and the MSP N terminus (including
the first two kringles) fused to human IgG Fc. We report the binding
capacity of MSP and its subunits to RON receptor in intact cells. We
also analyzed the capacity of MSP and its subunits to induce receptor
phosphorylation and consequent cellular responses.
Reagents
and
chains were obtained from kallikrein-treated pro-MSP and
purified on a CM-Sepharose column. An N-terminal segment of recombinant
MSP that included the first two kringles (MSP-NK2) fused with human IgG
Fc (16) was produced at Genentech, Inc. (San Francisco, CA). The purity
of the above reagents was evaluated by SDS-PAGE under reducing and
nonreducing conditions (Fig. 1). Rabbit IgG antibodies
against a synthetic C-terminal peptide of RON
chain were as
described (18). Mouse monoclonal antibody to phosphotyrosine (4G10) was
from Upstate Biotechnology Inc. (Lake Placid, NY). Goat anti-mouse or
rabbit IgG conjugated with horseradish peroxidase and enhanced ECL
detection reagents were from Amersham Corp. RPMI 1640 and Dulbecco's
modified Eagle's medium were from Life Technologies, Inc.
Bolton-Hunter reagent was from NEN Life Science Products. Protein
G-Sepharose was from Pharmacia Biotech Inc.
Fig. 1.
SDS-PAGE of recombinant human pro-MSP, human
serum MSP, recombinant MSP chain, MSP
chain, and MSP-NK2 (fused
with IgG Fc). Proteins (1.5-4 µg) were dissolved in sample
buffer with or without 2-mercaptoethanol and separated in 10%
polyacrylamide gel and stained with Coomassie Blue.
[View Larger Version of this Image (74K GIF file)]
and
Chains, and
MSP-NK2
Chain by MDCK-RE7 and 3T3/STK
Cells
and
chain. We concluded that about 30% of the
recombinant pro-MSP preparation did not have a disulfide link between
its
and
chain, which results in free
and
chain after
specific R-V bond cleavage. For the absorption assay, MDCK-RE7 or
3T3/STK cells (6 × 106 in 0.5 ml of RPMI 1640 medium)
were equilibrated with 0.5 nM 35S-MSP mixtures
at 0 °C for 2 h. Supernatants were collected, and rabbit
anti-MSP IgG was added to precipitate the remaining MSP as well as MSP
and
chains; this was followed by the addition of protein
G-Sepharose. After extensive washing with 0.1 M Tris buffer, pH 7.6, containing 0.15 M NaCl and 0.5% Tween 20, samples were separated on a 12% gel by SDS-PAGE under reducing
conditions. The gel were treated with Enlightning for 20 min, dried at
75 °C, and exposed to film with an intensifying screen.
, MSP
, or
MSP-NK2 protein to MDCK-RE7, BK-1, MK308, and other cells was carried
out as described (15). In steady-state binding assays, 3 × 105 cells were equilibrated in duplicate with increasing
amounts of 125I-labeled MSP, MSP
, or MSP-NK2 in binding
buffer (RPMI 1604 medium, pH 7.4, with 20 mM Hepes, and 100 µg/ml cytochrome c) in a total volume of 200 µl.
Nonspecific binding was determined in parallel equilibrations with a
30-fold excess of unlabeled MSP, MSP
, or MSP-NK2. After 3 h at
0 °C, cells were pelleted through an oil cushion (18). The tips of
tubes containing cells were cut. Radioactivity in supernatants and tips
was counted in a gamma counter. For estimation of
Kd, a we used a linear regression to generate a
straight line through Scatchard plot data points.
or
chain or MSP-NK2
and then added to top wells. After a 3-h incubation at 37 °C, the
chamber was disassembled, and the membranes were dried in air. The
migrated cells were stained and counted with an image analyzer. The
results were expressed as the percentage of input cells that
migrated.
or
chains,
MSP-NK2, or their different combinations were added. Cells without
stimulation served as control. After incubation for 5 days, cells were
stained and lysed in 1% SDS buffer. Color intensity was measured at
570 nM in an enzyme-linked immunosorbent assay plate
reader. Absorbance was converted into cell number by reference to a
standard curve derived from stained cell concentration.
Absorption of Free MSP Chain by MDCK-RE7 or 3T3/STK
Cells
and
chain, which resulted in free
and
chain
after specific cleavage by kallikrein of the pro-MSP R-V bond at the
chain junction (data not shown). We took advantage of this finding to determine if free
or
chain binds to the MSP receptor (human RON or murine STK) using an absorption assay. When
35S-labeled pro-MSP was cleaved by kallikrein and then
equilibrated with RON-expressing MDCK-RE7 cells or 3T3/STK cells as
absorbents, MSP
chain in recovered supernatants from both MDCK-RE7
and 3T3/STK cells was significantly reduced, as analyzed by SDS-PAGE
(Fig. 2). By densitometric comparison with
nontransfected control cells, about 80% of fluid phase MSP
chain
was absorbed by MDCK-RE7 cells, and about 50% was absorbed by 3T3/STK
cells. In contrast, the level of MSP
chain did not change.
Absorption by these cells suggested that the MSP
chain might bind
to the RON receptor.
Fig. 2.
SDS-PAGE under reducing conditions of
35S-labeled pro-MSP after partial cleavage by kallikrein,
followed by equilibration at 0 °C for 2 h with MDCK-RE7 or
NIH3T3/STK cells or nontransfected control cells. The decrease in
intensity of the chain lines is due to absorption of free
chain
by transfected cells.
[View Larger Version of this Image (37K GIF file)]
chain
but not by MSP
chain. On a molar basis, MSP is more potent than the
free
chain as a competitive inhibitor of labeled MSP binding.
Pro-MSP did not compete with MSP for RON, as previously reported (15).
Binding of 125I-MSP
chain to MDCK-RE7 cells is shown in
Fig. 4. Binding of the MSP
chain to the RON receptor
was specific; either unlabeled MSP or MSP
chain inhibited binding
of 125I-MSP
chain. From Fig. 4C, we
estimated a Kd for binding of the MSP
chain of
about 1.7 nM, higher than the Kd values
of 0.6 to 0.8 for binding of MSP to the RON receptor (17). On the other
hand, we did not detect specific binding of the MSP
chain to the
RON receptor (data not shown). The relatively low binding of
125I-MSP-NK2 to the cell surface was unaffected by
unlabeled MSP or MSP-NK2, indicating that the interaction of labeled
MSP-NK2 with MDCK-RE7 cells is nonspecific (Fig. 5).
Fig. 3.
Competitive inhibition of
125I-MSP binding to murine keratinocytes by unlabeled MSP
and its subunits. A, BK-1 cells. B, Mk308 cells.
Suspensions of 5 × 105 cells in 200 µl of binding
buffer were equilibrated for 3 h at 0 °C with 1 nM
125I-MSP in the presence of different concentrations of
unlabeled MSP, MSP chain, or MSP
chain. Cell-bound
radioactivity was measured. Nonspecific binding was measured in a
50-fold excess of unlabeled MSP. Specific binding was calculated by
subtracting values for nonspecific binding from the total binding. Each
value represents the mean ± S.E. of duplicates. One of three
experiments.
[View Larger Version of this Image (24K GIF file)]
Fig. 4.
Specific binding of 125I-MSP chain to MDCK-RE7 cells. Cells were equilibrated for 3 h at
0 °C with different concentrations of labeled
chain. Nonspecific
binding was determined by equilibration with a 30-fold excess of
unlabeled
chain in A or unlabeled MSP in B.
Scatchard plot for A is shown in C. The results
from one of two experiments are shown.
[View Larger Version of this Image (17K GIF file)]
Fig. 5.
Absence of specific binding of
125I-MSP-NK2 to MDCK-RE7 cells. Equilibration
conditions were as described for the experiment illustrated in Fig. 4.
Nonspecific binding was determined by equilibration with a 30-fold
excess of unlabeled MSP-NK2 or MSP. The results from one of two
experiments are shown.
[View Larger Version of this Image (18K GIF file)]
chain (Fig. 6A). No phosphorylated proteins were
observed in cells treated with pro-MSP, MSP
chain, or MSP-NK2
protein. Interestingly, although MSP
chain binds to RON, it failed
to induce RON autophosphorylation, indicating that MSP
chain alone
is insufficient to activate RON. Experiments were also designed to
study if MSP
chain, MSP
chain, or NK2 protein could modulate
RON phosphorylation. Fig. 6B shows that high concentrations
of MSP
chain could partially inhibit MSP-induced tyrosine
phosphorylation of RON. No inhibition was observed by MSP
chain or
MSP-NK2 protein.
Fig. 6.
Induction of autophosphorylation of the RON
receptor by pro-MSP, MSP and its subunits. Cells (3 × 106/ml) were incubated at 37 °C for 15 min with 5 nM protein in 1 ml of serum-free RPMI 1640 medium. Lysates
were immunoprecipitated with monoclonal anti-RON antibody. Samples were
loaded on 7.5% polyacrylamide gel under reducing conditions. After
transfer of proteins to Immobilon-P, the membrane was probed with
antibodies to phosphotyrosine (4G10), and developed with ECL.
A, MDCK-RE7 cells. B, kidney 293 cells. The
Control lane is for cells stimulated with 5 nM
HGF/SF.
[View Larger Version of this Image (42K GIF file)]
, MSP
or MSP-NK2 protein did not
induce morphological changes in resident macrophages. In combination
with MSP, none of these three subunits inhibited the biological effect
of MSP on macrophages. Likewise, except for a statistically
insignificant effect of MSP
, the addition of MSP subunits to
macrophages did not inhibit their migration toward MSP as a
chemoatractant (data not shown).
Fig. 7.
Stimulation of resident peritoneal macrophage
shape change by 1 nM MSP, MSP subunits, or combinations of
MSP and MSP subunit. For combination experiments, 1 nM
MSP was mixed with the indicated subunit (30 nM). Cells
were incubated for 45 min and then photographed.
[View Larger Version of this Image (114K GIF file)]
chain, MSP
chain, or MSP-NK2 protein at high concentration could
affect MSP-induced cell proliferation. Table I shows
that only MSP increased cell number after 5 days in culture, compared
with the medium control. In combination experiments, none of the
fragments affected MSP-induced proliferation, except for a small
inhibition by 100 nM MSP
chain.
Stimulus
Cell number at 5 days,
×10
4
Ratioa
Medium
16.7 ± 0.07
MSP (2 nM)
38.9 ± 0.17
2.3
MSP
chain (2 nM)
15.3 ± 0.02
0.9
MSP
chain (2 nM)
18.9 ± 0.02
1.1
NK2 (2 nM)
18.6 ± 0.15
1.1
MSP +
(30 nM)
38.1 ± 0.31
2.3
MSP +
(100 nM)
37.0 ± 0.45
2.2
MSP +
(30 nM)
35.6 ± 0.4
2.1
MSP +
(100 nM)
28.2 ± 0.1
1.7
MSP + NK2 (30 nM)
36.3 ± 0.3
2.2
MSP + NK2 (100 nM)
36.0 ± 0.2
2.2
a
Observed cell numbers divided by medium control cell
number.
We have presented several lines of evidence that the MSP chain
binds to RON. 1) Metabolically labeled free
chain, but not
chain, was specifically absorbed by cells expressing the RON receptor.
2) 125I-
chain bound to RON in intact cells in a
specific and saturable manner. 3) Not only unlabeled MSP but also
chain competitively inhibited binding of 125I-MSP to RON in
intact cells. Thus, in contrast to the
chain of HGF/SF, which does
not bind to its receptor (Met) (27), the
chain of MSP appears to
contain the primary binding site for the RON receptor. The
chain is
the serine protease domain of kringle proteins (28). In HGF/SF and MSP,
amino acid substitutions in the catalytic triad have eliminated the
protease activity. It is possible that residues in the modified
substrate pocket of the MSP
chain might form the binding site for
RON. To obtain clues about the MSP binding domain, we have begun
modeling of the MSP
chain for comparison with the published model
of the HGF
chain (29). MSP has Asp and Asn in the binding pocket, the corresponding locations of which are Gly in
HGF.2 Substitution of these candidate
residues, as well as two surface loop arginines, are now being made to
evaluate their significance for receptor binding.
There is one report that in RON cDNA-transfected COS-1 cells,
MSP-NK2 stimulated RON phosphorylation (16). MSP-NK2 is a recombinant
protein comprising the first two kringles of the MSP chain, fused
to IgG Fc. We found that neither MSP-NK2 nor MSP
chain bound to RON
on intact cells including kidney 293 and MDCK-RE7 cells. We cannot
explain the reported activity of MSP-NK2, especially because the source
of the MSP-NK2 was the same. However, our
chain data combined with
the fact that free
chain does not bind to RON support the
conclusion that MSP binds to its receptor via the
chain.
We have shown that although the MSP chain binds to RON, it does not
cause biological activity or induce phosphorylation of the receptor,
except for a small amount at high ligand concentrations. It is
generally accepted that ligand binding to growth factor receptors is
associated with receptor oligomerization and autophosphorylation (30).
Receptor oligomerization may be mediated by interaction of ligand
pairs. In this context, our results indicate that receptor oligomerization requires an intact
chain disulfide-linked
heterodimeric ligand. Although HGF differs from MSP in that the primary
binding site resides in the
chain, two or three amino acid
substitutions in the
chain are sufficient to reduce biological
activity to less than 2% that of wild type HGF (31). Thus, for both
MSP and HGF the
chain heterodimer is required to fully activate their respective receptors. An HGF dimer, formed by noncovalent interactions between kringles 2 and 3 of the protein pair, has been
suggested as the moiety that induces dimerization and activation of MET
(29). This idea is supported by a report of nonconvalent kringle-kringle interactions (32). Although MSP and HGF have different
primary receptor binding regions, they may have a similar structural
basis for receptor activation by dimer formation through kringle
interactions. We plan to express and purify selected kringle regions of
MSP and to determine their effects when added together with intact MSP
to target cells. If receptor activation requires ligand dimerization by
kringle interaction, the result could be no effect on MSP binding but
inhibition of receptor phosphorylation.
An interesting alternative mechanism for ligand-induced receptor
dimerization is suggested by the crystal structure of human growth
hormone and the extracellular domain of its receptor (33). The complex
comprises one ligand molecule per two receptors. Two structurally
unrelated regions of the ligand interact with similar binding surfaces
of the two receptors. It has been proposed that receptor dimerization
occurs by a sequential mechanism, because human growth hormone binds to
a second receptor only if it has bound to the first receptor (34). This
is consistent with the fact that the contact surface for the binding
site of the receptor I is about 30% larger than that for receptor II.
The authors suggest that ligand binding to receptor II is stabilized by
interaction between the two receptor domains near their C terminus. If
this model applied to the RON receptor, the candidate region for
binding to receptor I might be a cluster of chain surface loop
arginines2; a single arginine on the N domain hairpin loop
of the
chain (29) might mediate binding to receptor II. The Arg
cluster density for the corresponding regions of HGF is reversed, which
is consistent with receptor binding by the
chain. This model would
account for primary binding by MSP
or HGF-
, and the requirement
for binding by the
chain heterodimer for optimal receptor
activation. The model should be readily testable by mutagenesis
studies.
We thank Dr. Teizo Yoshimura for providing the cDNA that was used for the production of recombinant pro-MSP. We also thank Dr. C. Ronsin for expressing the RON-GST fusion protein used for generation of monoclonal antibody ID2 and Dr. Maria Miller for helpful discussions about molecular structure and for calling our attention to the model of growth factor receptor dimerization.