Macrophage Stimulating Protein (MSP) Binds to Its Receptor via the MSP beta  Chain*

(Received for publication, November 7, 1996, and in revised form, April 30, 1997)

Ming-Hai Wang Dagger , Felix M. Julian §, Richard Breathnach , Paul J. Godowski par , Toyohiro Takehara **Dagger Dagger , Wataru Yoshikawa **§§, Michio Hagiya **¶¶ and Edward J. Leonard

From the Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, the § Antibody Department, Immunotech 13276 Marseille Cedex 09, the  Inserm U211, Institut de Biologie, 44035 Nantex Cedex 01, France, the par  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


ABSTRACT

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 alpha  and beta  chains, and a complex including the first two kringles and IgG Fc (MSP-NK2). Radioiodinated MSP and MSP beta  chain both bound specifically to RON. The Kd of 1.4 nM for MSP beta  chain is higher than the reported Kd range of 0.6-0.8 nM for MSP. Pro-MSP, MSP alpha  chain, and MSP-NK2 did not bind. Only MSP stimulated RON autophosphorylation. Although the beta  chain bound to RON and partially inhibited MSP-induced RON phosphorylation in kidney 293 cells, it did not induce RON phosphorylation. Pro-MSP, MSP alpha  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 beta  chain, in contrast to structurally similar hepatocyte growth factor, in which the receptor binding site is in the alpha  chain. However, full activation of RON requires binding of the complete MSP disulfide-linked alpha beta chain heterodimer.


INTRODUCTION

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 alpha  chain and a 25-kDa beta  chain (calculated from amino acid composition). The alpha  chain contains a N-terminal hairpin loop followed by four kringle domains. The beta  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).

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 alpha  chain and 150-kDa transmembrane beta  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).

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 alpha  and beta  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.


MATERIALS AND METHODS

Reagents

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 alpha  and beta  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 beta  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 alpha  chain, MSP beta  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)]

Cells

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).

Radiolabeling of MSP, MSP alpha  and beta  Chains, and MSP-NK2

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.

Absorption of Radiolabeled MSP beta  Chain by MDCK-RE7 and 3T3/STK Cells

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 alpha  and beta  chain. We concluded that about 30% of the recombinant pro-MSP preparation did not have a disulfide link between its alpha  and beta  chain, which results in free alpha  and beta  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 alpha  and beta  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.

Binding of 125I-MSP or Its Subunits to Cells

Binding of 125I-MSP, MSP alpha , MSP beta , 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 beta , 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 beta , 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.

Detection of Tyrosine Phosphorylation of RON

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).

Assay for Macrophage Shape Change

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.

Cell Migration Assay

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 alpha  or beta  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.

Cell Proliferation Assay

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 alpha  or beta  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.


RESULTS

Absorption of Free MSP beta  Chain by MDCK-RE7 or 3T3/STK Cells

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 alpha  and beta  chain, which resulted in free alpha  and beta  chain after specific cleavage by kallikrein of the pro-MSP R-V bond at the alpha beta chain junction (data not shown). We took advantage of this finding to determine if free alpha  or beta  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 beta  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 beta  chain was absorbed by MDCK-RE7 cells, and about 50% was absorbed by 3T3/STK cells. In contrast, the level of MSP alpha  chain did not change. Absorption by these cells suggested that the MSP beta  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 beta  chain lines is due to absorption of free beta  chain by transfected cells.
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Assay for Binding of 125I-pro-MSP, MSP, and Its Subunits to Cells Expressing RON or STK

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 beta  chain but not by MSP alpha  chain. On a molar basis, MSP is more potent than the free beta  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 beta  chain to MDCK-RE7 cells is shown in Fig. 4. Binding of the MSP beta  chain to the RON receptor was specific; either unlabeled MSP or MSP beta  chain inhibited binding of 125I-MSP beta  chain. From Fig. 4C, we estimated a Kd for binding of the MSP beta  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 alpha  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 alpha  chain, or MSP beta  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.
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Fig. 4. Specific binding of 125I-MSP beta  chain to MDCK-RE7 cells. Cells were equilibrated for 3 h at 0 °C with different concentrations of labeled beta  chain. Nonspecific binding was determined by equilibration with a 30-fold excess of unlabeled beta  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.
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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.
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Induction of RON Tyrosine Phosphorylation by MSP and Its Subunits

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 beta  chain (Fig. 6A). No phosphorylated proteins were observed in cells treated with pro-MSP, MSP alpha  chain, or MSP-NK2 protein. Interestingly, although MSP beta  chain binds to RON, it failed to induce RON autophosphorylation, indicating that MSP beta  chain alone is insufficient to activate RON. Experiments were also designed to study if MSP alpha  chain, MSP beta  chain, or NK2 protein could modulate RON phosphorylation. Fig. 6B shows that high concentrations of MSP beta  chain could partially inhibit MSP-induced tyrosine phosphorylation of RON. No inhibition was observed by MSP alpha  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.
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Effect of MSP Subunits on MSP-induced Cell Shape Change and Migration

To see if MSP subunits can induce cell shape change or migration, mouse peritoneal resident macrophages were used. Fig. 7 shows that MSP alpha , MSP beta  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 beta , 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.
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Effect of MSP Subunits on MSP-stimulated Cell Proliferation

BK-1 keratinocytes were used to assess if MSP alpha  chain, MSP beta  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 beta  chain.

Table I. Effects of MSP and MSP subunits, alone or in combination, on proliferation of murine BK-1 keratinocytes

The results represent one of two similar experiments.

Stimulus Cell number at 5 days, ×10-4 Ratioa

Medium 16.7  ± 0.07
MSP (2 nM) 38.9  ± 0.17 2.3
MSP alpha  chain (2 nM) 15.3  ± 0.02 0.9
MSP beta  chain (2 nM) 18.9  ± 0.02 1.1
NK2 (2 nM) 18.6  ± 0.15 1.1
MSP + alpha  (30 nM) 38.1  ± 0.31 2.3
MSP + alpha  (100 nM) 37.0  ± 0.45 2.2
MSP + beta  (30 nM) 35.6  ± 0.4 2.1
MSP + beta  (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.


DISCUSSION

We have presented several lines of evidence that the MSP beta  chain binds to RON. 1) Metabolically labeled free beta  chain, but not alpha  chain, was specifically absorbed by cells expressing the RON receptor. 2) 125I-beta chain bound to RON in intact cells in a specific and saturable manner. 3) Not only unlabeled MSP but also beta  chain competitively inhibited binding of 125I-MSP to RON in intact cells. Thus, in contrast to the beta  chain of HGF/SF, which does not bind to its receptor (Met) (27), the beta  chain of MSP appears to contain the primary binding site for the RON receptor. The beta  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 beta  chain might form the binding site for RON. To obtain clues about the MSP binding domain, we have begun modeling of the MSP beta  chain for comparison with the published model of the HGF beta  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 alpha  chain, fused to IgG Fc. We found that neither MSP-NK2 nor MSP alpha  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 beta  chain data combined with the fact that free alpha  chain does not bind to RON support the conclusion that MSP binds to its receptor via the beta  chain.

We have shown that although the MSP beta  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 alpha beta chain disulfide-linked heterodimeric ligand. Although HGF differs from MSP in that the primary binding site resides in the alpha  chain, two or three amino acid substitutions in the beta  chain are sufficient to reduce biological activity to less than 2% that of wild type HGF (31). Thus, for both MSP and HGF the alpha beta 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 beta  chain surface loop arginines2; a single arginine on the N domain hairpin loop of the alpha  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 alpha  chain. This model would account for primary binding by MSP beta  or HGF-alpha , and the requirement for binding by the alpha beta chain heterodimer for optimal receptor activation. The model should be readily testable by mutagenesis studies.


FOOTNOTES

*   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.
Dagger    To whom correspondence should be addressed: Div. of Pulmonary Medicine, Wayne State University, VA Medical Center, B-4365, 4646 John Rd., Detroit, MI 48201. Tel.: 313-576-4498; Fax: 313-576-3431.
Dagger Dagger    Present address: Amgen Ltd., 2-31-1 Nihonbashi Hamacho, Tokyo 103 Japan.
§§   Tetsuseikai Hospital, Esebi-Cho, Shijou-nawate, Osaka 575 Japan.
¶¶   Takara Shuzo Co., Biomedical Group, Seta 3-4-1, Otsu, Shiga, 520-21 Japan.
1   The abbreviations used are: MSP, macrophage stimulating protein; HGF, hepatocyte growth factor; SF, scatter factor; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; MDCK, Madin-Darby canine kidney.
2   M. Miller, A. Danilkovitch, and E. J. Leonard, unpublished data.

ACKNOWLEDGEMENTS

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.


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