INTRODUCTION

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Metalloprotease/disintegrin/cysteine-rich proteins (MDCs, also called ADAMs)¹ are membrane-anchored proteins with several domains including a metalloprotease domain, a disintegrin-like domain, a cysteine-rich sequence, an epidermal growth factor-like sequence, a transmembrane domain, and a short cytoplasmic domain (1). The biological functions of MDCs are not clear; however, we do know that fertilins (MDC-1 and -2) (2) are involved in sperm-egg binding and fusion (3), meltrins (MDC-12) (4) are involved in myoblast fusion during muscle development, and KUZ (a Drosophila MDC protein) (5) assists in neurogenesis. The MDC cytoplasmic domain has a proline-rich potential SH3 binding motif, suggesting that MDC-counter receptor interaction may induce signal transduction.

Integrins are a family of cell adhesion receptors that bind to a variety of ligands, including extracellular matrix proteins and other cell surface molecules (6-10). MDCs are potential ligands for integrins, since most snake venom disintegrins interact with integrins IIb3 and v3 (reviewed in Ref. 11 and references therein). However, little is known about the receptor specificity of MDCs, except that mouse egg integrin 61 has been proposed as a receptor for fertilin (2). Evans et al. (12) recently expressed recombinant fertilin fragments in bacteria as fusion proteins with maltose-binding protein (12). The recombinant fertilin- fragment has been shown to bind to the egg membrane to which sperm bind and to block sperm from binding to the egg. These results suggest that the disintegrin-like domains of MDCs may be properly folded in bacteria, that glycosylation of the disintegrin-like domain may not be required for interaction with receptors, and that a strategy using recombinant MDC proteins is a viable alternative to those using purified materials that are not easily available.

MDC-15 (metargidin) (13, 14) is the only known MDC that has an RGD sequence in its disintegrin-like domain. MDC-15 is widely expressed in various tissues and cells, including human umbilical vein endothelial cells and smooth muscle cells (14). In this study, we determined the receptor specificities of MDC-15 using the recombinant disintegrin-like domain of MDC-15, which was expressed in bacteria. We determined the receptor specificity of the recombinant protein using mammalian cells expressing different recombinant human integrins. We discovered that the recombinant disintegrin-like domain specifically interacts with integrin v3 but not with other RGD-dependent or independent integrins tested, including IIb3. This is in contrast to snake venom disintegrins, most of which bind to both IIb3 and v3 (Ref. 11 and references therein). We also found that the integrin specificity of the recombinant disintegrin-like domain is mediated by the RGD tripeptide and its flanking sequence, as in snake venom disintegrins. These results suggest that MDC-15 may be involved in v3-mediated cell-cell adhesion.

	EXPERIMENTAL PROCEDURES

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Monoclonal Antibodies and Cell lines-- mAb 15 (15) was a kind gift from M. H. Ginsberg (The Scripps Research Institute, La Jolla, CA), 2G12 (16) was from V. Woods (University of California at San Diego, La Jolla, CA), PL98DF6 (17) was from J. Ylanne (University of Helsinki, Helsinki, Finland), PT25-2 (18) was from M. Handa and Y. Ikeda (Keio University, Tokyo, Japan), and LM142 (to human v) and LM609 (to v3) (19) was from D. Cheresh (Scripps). Polyclonal anti-v cytoplasmic peptide antibody was purchased from Chemicon, Temecula, CA. M-21 human melanoma cells were provided by D. Cheresh.

Preparation of the GST Fusion Protein of the Disintegrin-like Domain of MDC-15 and of the 8-11th Type III Repeats of Fibronectin-- A cDNA fragment of about 270 nucleotides that encodes the disintegrin-like domain of MDC-15 (Met-420 to Glu-510) (13) was amplified by polymerase chain reaction with a human erythroleukemia (K562) cell cDNA library as a template using 5'-CGGGATCCATGGCTGCTTTC-TGCGG and 5'-CGGGATCCTTACTCGCCATCCCCTAGGCTG as primers. The cDNA fragment was subcloned into the BamHI site of a pGEX-2T vector (Amersham Pharmacia Biotech). Synthesis of the GST fusion protein of the disintegrin-like domain of MDC-15 was induced in Escherichia coli DH5 by adding 0.1 mM isopropyl-1-thio--D-galactopyranoside in culture medium as described previously (20). Protein was extracted from the bacterial suspension by sonication and purified using glutathione-agarose (Sigma) affinity chromatography.

Development of Chinese Hamster Ovary (CHO) Cells Expressing Different Human Integrins-- We developed CHO cells that express different human integrins. The cDNA constructs were transfected into CHO cells together with a neomycine-resistant gene. Those cell lines expressing human 2 (2-CHO) (23), human 3 (3-CHO) (24), human 4 (4-CHO) (25, 26), human 5 (5-CHO) (26, 27), human L2 (L2-CHO) (28), human v (v-CHO), human 3 (3-CHO), both human v and 3 (v3-CHO) (29), and human IIb3 (IIb3-CHO) (30) have been described in the cited references. The cDNA constructs for 6 (6-CHO) and 6 and 4 (for 64-CHO) were co-transfected into CHO cells with a neomycine gene. After selection with G-418, cells stably expressing human integrins were cloned by sorting to obtain high expressors.² The 2-, 3-, 4-, 5-, 6-, and v-CHO cells homogeneously expressed human 2, 3, 4, 5, and v/hamster 1 hybrids, respectively. The 3-CHO cells expressed human 3/hamster v hybrid.

Adhesion Assays-- Wells of 96-well Immulon-2 microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 100 µl of PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.4) containing substrates at a concentration of 20 µg/ml and were incubated overnight at 4 °C. The remaining protein binding sites were blocked by incubating with 1% bovine serum albumin (Calbiochem) for 1 h at room temperature. Cells (10⁵ cells/well) in 100 µl of Dulbecco's modified Eagle's medium were added to the wells and incubated at 37 °C for 1 h. After gently rinsing the wells three times with PBS to remove unbound cells, bound cells were quantified by measuring endogenous phosphatase activity (31).

Affinity Chromatography on D-15-- Purified D-15 was absorbed to glutathione-agarose (Sigma) after free glutathione was removed by gel filtration on a PD-10 column (Amersham). Cells were harvested with 3.5 mM EDTA in PBS and washed with PBS. Cells (about 5 × 10⁶) were then surface-labeled with ¹²⁵I using IODO-GEN (Pierce) (32), washed three times with PBS, and solubilized at 4 °C for 1 h in 1 ml of 10 mM Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl, 100 mM octylglucoside, 2.5 mM MnCl₂, and 1 mM phenylmethylsulfonyl fluoride (Sigma). The insoluble materials were removed by centrifugation at 15,000 × g for 10 min. The supernatant was then incubated with a small amount of underivatized agarose at 4 °C for 15 min to remove nonspecific binding material. The supernatant was incubated at 4 °C for 1 h with 200-500 µl of packed D-15-glutathione-agarose that had been equilibrated with 10 mM Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl, 25 mM octylglucoside, 2.5 mM MnCl₂, and 1 mM phenylmethylsulfonyl fluoride (washing buffer). The unbound materials were washed with 20 times the column volume of washing buffer, and the bound materials were eluted with 20 mM EDTA instead of 2.5 mM MnCl₂ in washing buffer; then, 0.5-ml fractions were collected. Twenty-µl aliquots from each fraction were analyzed by SDS-polyacrylamide gel electrophoresis using 7% polyacrylamide gel under nonreducing conditions followed by autoradiography.

Binding Recombinant GST Fusion Protein and Fibrinogen to CHO Cells-- Recombinant GST fusion protein and fibrinogen (Chromogenix, Stockholm, Sweden) were labeled with fluorescein isothiocyanate (FITC) (33). Cells were incubated with mouse IgG or mAb PT25-2 at 10 µg/ml for 30 min at 4 °C in Dulbecco's modified Eagle's medium. Then, FITC-labeled fibrinogen was added at a final concentration of 100 µg/ml, and the mixture was further incubated for 30 min at room temperature. After washing the cells once with PBS to remove unbound labeled protein, bound protein was quantified by flow cytometry in FACSCan (Beckton-Dickinson).

Other Methods-- Site-directed mutagenesis was carried out using the unique site elimination method (34). The presence of mutations was verified by DNA sequencing.

	RESULTS

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The Recombinant Disintegrin-like Domain of MDC-15-- To prove that MDC-15 mediates cell-cell adhesion through interaction with integrins, we expressed the disintegrin-like domain of MDC-15 (Met-420 to Glu-510), a putative integrin binding site (Fig. 1A), as a fusion protein with GST in bacteria (designated D-15). We obtained soluble D-15 and purified it using affinity chromatography on glutathione-agarose. Fig. 1B shows that the purified D-15 migrates as a monomer with a M_r of 36,000 under nonreducing conditions (lane 1), and the control, wt GST protein, migrates as a monomer with a M_r of approximately 26,000. The sizes of these proteins match the values calculated from the primary structures of these proteins (35,930 and 26,968, respectively). Although the D-15 preparation contained some degradation products, we used it for adhesion and binding assays without further purification.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   GST MDC7-15 disintegrin-like domain fusion proteins (wt and mutant) and wt GST. A cDNA fragment of the disintegrin-like domain of MDC-15 (residues 419-510; wt) was obtained by PCR amplification and subcloned into pGEX-2T vector. The GST fusion protein (D-15) and wt GST were synthesized in E. coli, extracted from the bacteria, and purified by affinity chromatography on glutathione-agarose. Bound protein was eluted using 5 mM reduced glutathione in 50 mM Tris-HCl, pH 7.5. Eluted materials were analyzed by SDS-gel electrophoresis using 10% gel and staining with Coomassie Blue. Lane 1, wt GST (calculated Mr, 26,968); lane 2, D-15 (calculated Mr, 35,930); lane 3, D-15/SGA mutant; lane 4, D-15/NKW mutant. TM, transmembrane; EGF, epidermal growth factor.

Adhesion to D-15 of CHO Cells Expressing Different Recombinant Integrins-- We determined whether D-15 supports integrin-mediated cell adhesion using CHO cells expressing different recombinant integrins. Parent CHO cells express 51 as a major integrin but do not express 2 or 3 integrins (29, 35). As shown in Fig. 2A, wt GST (the negative control) does not support adhesion to any of the cells used, but GST-FN (the positive control), which contains the central cell binding domain of rat fibronectin (the 8-11th type III repeats), supported all of the cell lines used. D-15 supported adhesion of 3-CHO cells (that express v3) and IIb3-CHO cells (that express both IIb3 and v3) but not parent CHO cells and cells expressing other exogenous integrins (including 21, 31, 41, 51, 61, 64, v1, and L2). These results indicate that D-15 interacts either with v3 or with both IIb3 and v3. Fig. 2B shows that both D-15 and GST-FN support maximum adhesion of 3-CHO cells at the coating concentration of 10 µg of protein/ml, suggesting that D-15 and GST-FN support adhesion of 3-CHO cells at comparable levels. Fig. 2C shows that adhesion to D-15 of 3-CHO cells is completely blocked by function-blocking anti-v3 mAb LM609, but adhesion to GST-FN is not. These results support the idea that adhesion to D-15 is mediated exclusively by v3, whereas adhesion to GST-FN is mediated by multiple receptors that include v3, v1, and 51.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Adhesion of cells expressing different integrins to the recombinant disintegrin-like domain of MDC-15. A, wells of 96-well microtiter plates were coated with 20 µg/ml (in PBS) D-15 (black column), GST-FN (white column), or wt GST (shaded column). Cells homogeneously expressing different human integrins were incubated in wells at 37 °C for 1 h. After rinsing the wells to remove unbound cells, bound cells were quantified using endogenous phosphatase activity. GST-FN and purified FN (human or bovine) gave almost identical results. B, adhesion of 3-CHO cells to D-15 and GST-FN was determined as a function of the substrate. The data suggest that the D-15 protein used in the above experiment (20 µg/ml in PBS) is a saturating concentration and that D-15 is comparable to GST-FN in supporting cell adhesion. C, the effect of anti-v3 mAb LM609 on adhesion of 3-CHO cells to D-15 and GST-FN was determined. Adhesion assays were performed as described above (A). A control mAb KH72 (to integrin 5) did not block adhesion of 3-CHO cells to D-15 (data not shown). LM609 and KH72 were used at X250 dilution of ascites.

Affinity Chromatography of Solubilized IIb3 and v3 Integrins on D-15 Immobilized to Agarose-- To determine whether D-15 interacts with v3 or both IIb3 and v3, we carried out affinity chromatography on immobilized D-15. Purified D-15 was coupled to glutathione-agarose and incubated with lysates of ¹²⁵I-labeled v3, IIb3, or parent CHO cells. Bound materials were eluted with 20 mM EDTA. Fig. 3 shows that two protein bands corresponding in size to v/IIb and 3 were eluted with lysates from v3- and IIb3-CHO cells, but very little radioactivity was eluted with the lysate of parent CHO cells. We analyzed the eluted materials by immunoprecipitation using antibodies specific to v, IIb, or 3. IIb3 was detected only in the unbound fraction with IIb3-CHO cells. v3 was detected in both the bound and unbound fractions with v3-CHO and IIb3-CHO cells. These results indicate that D-15 binds to solubilized v3 but not to solubilized IIb3.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Affinity chromatography on D-15 immobilized to agarose. A, lysates of surface 125I-labeled cells were incubated with D-15 immobilized to glutathione-agarose that had been equilibrated with buffer containing 2.5 mM MnCl2. The bound materials were eluted with 20 mM EDTA. Twenty-µl aliquots from the first four 0.5-ml fractions were analyzed by SDS-polyacrylamide gel electrophoresis using 7% polyacrylamide gel under nonreducing conditions. B, immunoprecipitation of eluted materials from D-15 agarose with IIb3-CHO (lanes 1-6) and v3-CHO (lanes 7-12) cells. Antibodies used were control antibody (nonimmune rabbit serum) (lanes 1 and 7), anti-3 mAb 15 (lanes 2 and 8), anti-human v LM142 (lane 9), anti-hamster v peptide antibody (lanes 3 and 10), anti-IIb PL98 DF6 (lanes 4 and 11), anti v3 LM609 (lanes 5 and 12), and anti-IIb3 2G12 (lane 6). The data suggest that v3 bound to D-15 agarose, but IIb3 did not.

v3-D-15 Interaction Is Dependent on the Tripeptide RGD and the Flanking Sequence in the Putative Integrin Binding Site-- To determine whether v3-D-15 interaction is RGD-dependent, we mutated the D-15 RGD sequence to SGA. The mutant protein (designated D-15/SGA) was expressed as a soluble monomer (Fig. 1B), purified using affinity chromatography, and used for adhesion assays. The mutation completely blocked adhesion of 3-CHO cells to the fusion protein (Fig. 4). Increasing the coating concentration of the D-15/SGA mutant did not reverse the effects of the mutation. These results indicate that D-15-v3 interaction is dependent on the RGD tripeptide in the putative integrin binding site.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Adhesion of 3-CHO cells to wt and mutant D-15 as a function of coating concentration of substrates. Clonal CHO cells expressing v3 (3-CHO) were incubated for 1 h at 37 °C with wt D-15, D-15/SGA mutant (in which the RGD sequence is mutated to SGA), or wt GST immobilized at different concentrations (up to 20 µg/ml coating concentration). Adhesion was measured as described in the legend to Fig. 2. The data suggest that adhesion to D-15 is dependent on the RGD sequence in the disintegrin-like domain.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of mutating the sequence flanking the RGD tripeptide in the recombinant disintegrin-like domain of MDC-15. A, 3-CHO cells (that express v3) and IIb3-CHO cells (that express both IIb3 and v3) were incubated with wt D-15 (black column) and D-15/NWK (white column) immobilized to wells of a 96-microtiter plate at 20 µg/ml coating concentrations. Adhesion was measured as described in the legend to Fig. 2. The data suggest that the D-15/NWK mutant (in which the RPTRGD sequence has been mutated to NWKRGD) binds to v3, but it is not clear whether it binds to IIb3. B, parent CHO cells and IIb3-CHO cells were first incubated with either mouse IgG (dotted line) or the activating anti-IIb3 mAb PT25-2 (solid) and then with FITC-labeled protein. FITC-labeled protein bound to cells was determined by flow cytometry. The data suggest that the D-15/NWK mutant and fibrinogen bind to IIb3-CHO cells in the presence of PT25-2, but wt D-15 does not. Fbg, fibrinogen.

Interaction between a Natural Human v3 Heterodimer and D-15-- Since we used a recombinant hamster v/human 3 hybrid integrin on CHO cells to study the receptor specificity of D-15, we determined whether the natural human v3 heterodimer recognize wt and mutant D-15. As shown in Fig. 6, M-21 human melanoma cells expressing v3 adhered to wt D-15 and D-15/NWK but not to D-15/SGA. Adhesion of M-21 cells to wt D-15 and D-15/NWK was completely blocked by LM609. These results indicate that the natural human v3 heterodimer, like a hybrid v3 on CHO cells, specifically recognizes D-15 in an RGD-dependent manner.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Adhesion of human v3 to D-15. M-21 melanoma cells were incubated in 96-well titer plates coated with D-15, D-15/SGA, D-15/NWK, wt GST, or GST-FN (at 20 µg/ml coating concentrations) in the presence and absence of function-blocking anti-v3 mAb LM609 (at ×250 dilution of ascites). A control mAb KH72 (to integrin 5) did not block adhesion of M-21 cells to D-15 (data not shown). The data indicate that natural human v3 heterodimer in M-21 cells recognizes D-15 in an RGD-dependent manner.

	DISCUSSION

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Although MDC-15 and other members of its family have been proposed to represent a new class of cell adhesion molecules based on deduced amino acid sequences, their function is unclear in most cases (see the Introduction). In this paper we have evaluated the receptor specificity of MDC-15 by studying its recombinant disintegrin-like domain. Although the recombinant fertilin- (MDC-2) fragment shows biological functions (12), it is possible that the native disintegrin-like domains of MDCs purified from mammalian cells or of recombinant MDCs expressed in eukaryotic systems may fold or be modified differently and show additional or different properties. In this study, we have shown that the recombinant disintegrin-like domain of MDC-15 specifically interacts with integrin v3 using adhesion assays and affinity chromatography. We also present evidence that the receptor recognition and specificity of the recombinant disintegrin-like domain of MDC-15 is determined by the RGD tripeptide and flanking sequence. Substitution of the RGD tripeptide to SGA completely blocks binding of D-15 to v3. Substitution of the sequence flanking the tripeptide RGD changes the receptor specificity (from v3 to both IIb3 and v3). Thus, the binding specificity of the recombinant disintegrin-like domain of MDC-15, like that of snake venom disintegrins and dendroaspin, an RGD-containing neurotoxin variant (38), is mediated by the RGD tripeptide and flanking sequence. NMR structural studies of the snake venom disintegrins kistrin and echistatin show that, in both cases, the region containing the RGD sequence is a loop structure exposed to the outside of the disintegrin molecule (40, 41). This peculiar structure probably supports the high affinity interaction of snake venom disintegrins with integrins (36, 37). It would be interesting to determine whether the structure of the recombinant disintegrin-like domain of MDC-15 is similar to those of snake venom disintegrins.

It should be noted that the disintegrin-like domain of MDC-15 is unique in its receptor specificity. Most snake venom disintegrins recognize both v3 and IIb3 (11). Barbourin is the only disintegrin that is specific to IIb3. There is no known snake venom disintegrin that is specific to v3. Thus, the RGD tripeptide and flanking sequence in the disintegrin-like domain of MDC-15 represent a unique v3-specific recognition sequence. The disintegrin-like domains of MDCs have additional Cys residues in the middle of the loops in their putative integrin binding sites (RGDC in MDC-15, for example). It has not been determined whether this Cys residue makes a disulfide link with another Cys residue. The topology of this region of the disintegrin-like domain is probably very different from that of the snake venom class P II disintegrins in that its loop structure is probably less flexible and its conformation more restricted, with an increase in restriction if the Cys residues flanking the RGD tripeptide are involved in a disulfide linkage (in the snake venom class P II disintegrins, the RGD sequence is positioned within an extended, flexible loop structure where there is only limited conformational restriction of the RGD sequence; see Ref. 42 for review). It is possible, therefore, that using short synthetic peptides (cyclic or linear) derived from the RGD and flanking sequences of the disintegrin-like domain of MDC-15 might provide different receptor specificities than those obtained in this study using recombinant or purified disintegrin-like domains.

The interaction of the disintegrin-like domain of MDC-15 with integrin v3 may be related to its biological functions. MDC-15 is not expressed in vivo in normal vessels but is up-regulated in lesions of atherosclerosis, where many macrophages are present (14). MDC-15 on cultured endothelial cells undergoes proteolytic processing (14), which appears to be associated with MDC activation (4, 43). It is possible that activated MDC-15 on endothelial cells interacts with v3 on leukocytes during atherogenesis through its exposed disintegrin-like domain. v3 has been shown to be involved in the progression of melanoma and the induction of neo-vascularization by tumor cells. v3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels (44, 45). It is possible that activated MDC-15 and v3 on endothelial cells interact with each other, leading to homotypic aggregation of endothelial cells during angiogenesis. Based on the wide distribution of MDC-15, MDC-15-v3 interaction may mediate cell-cell interactions in many other instances (e.g. metastasis). The snake venom metalloprotease/disintegrin jararhagin is known to block collagen-induced aggregation of platelets (46). It has been proposed that the inhibition of platelet response to collagen by jararhagin is mediated through the binding of jararhagin to the platelet 2-subunit via the disintegrin domain followed by proteolysis of the 1 subunit with loss of the integrin structure (conformation) necessary for the binding of macromolecular ligands (47). It has been hypothesized that a fragment of MDC-15 containing the metalloprotease and disintegrin-like domains is released from cultured endothelial cells (14). It is possible that the proteolytic fragment of MDC-15 containing the metalloprotease and disintegrin-like domains interacts with v3, as in the case of jararhagin and 21, and either modifies the function of the integrin or promotes degradation of the matrix proteins surrounding the cells that express v3. Therefore, the proposed v3-MDC-15 interaction may be of wide biological importance.