Specific Interaction of the Recombinant Disintegrin-like Domain of MDC-15 (Metargidin, ADAM-15) with Integrin alpha vbeta 3*

Xi-Ping Zhang, Tetsuji Kamata, Kenji Yokoyama, Wilma Puzon-McLaughlin, and Yoshikazu TakadaDagger

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

MDC-15 (ADAM-15, metargidin), a membrane-anchored metalloprotease/disintegrin/cysteine-rich protein, is expressed on the surface of a wide range of cells and has an RGD tripeptide in its disintegrin-like domain. MDC-15 is potentially involved in cell-cell interactions through its interaction with integrins. We expressed a recombinant MDC-15 disintegrin-like domain as a fusion protein with glutathione S-transferase (designated D-15) in bacteria and examined its binding function to integrins using mammalian cells expressing different recombinant integrins. We found that D-15 specifically interacts with alpha vbeta 3 but not with the other integrins tested (alpha 2beta 1, alpha 3beta 1, alpha 4beta 1, alpha 5beta 1, alpha 6beta 1, alpha 6beta 4, alpha vbeta 1, alpha IIbbeta 3, and alpha Lbeta 2). Mutation of the tripeptide RGD to SGA totally blocked binding of D-15 to alpha vbeta 3, suggesting that D-15-alpha vbeta 3 interaction is RGD-dependent. When the sequence RPTRGD is mutated to NWKRGD, D-15 is recognized by both alpha IIbbeta 3 and alpha vbeta 3, suggesting that the receptor binding specificity is mediated by the sequence flanking the RGD tripeptide, as in snake venom disintegrins. These results indicate that the disintegrin-like domain of MDC-15 functions as an adhesion molecule and may be involved n alpha vbeta 3-mediated cell-cell interactions.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha IIbbeta 3 and alpha vbeta 3 (reviewed in Ref. 11 and references therein). However, little is known about the receptor specificity of MDCs, except that mouse egg integrin alpha 6beta 1 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-beta 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 alpha vbeta 3 but not with other RGD-dependent or independent integrins tested, including alpha IIbbeta 3. This is in contrast to snake venom disintegrins, most of which bind to both alpha IIbbeta 3 and alpha vbeta 3 (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 alpha vbeta 3-mediated cell-cell adhesion.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha v) and LM609 (to alpha vbeta 3) (19) was from D. Cheresh (Scripps). Polyclonal anti-alpha 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 DH5alpha by adding 0.1 mM isopropyl-1-thio-beta -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.

A cDNA fragment of about 1100 nucleotides that encodes the 8-11th type III repeats of rat fibronectin (Ala-1356 to Thr-1720) was amplified by polymerase chain reaction with rat fibronectin cDNA (provided by J. Schwarzbauer, Princeton University, NJ) as a template using 5'-CGGGATCCGCCGTCCCTCCTCCCACG-3' and 5'-CGGGATCCTTAGGTCACTGCAGTCTGAAC-3' as primers. The cDNA fragment was subcloned into the BamHI site of a pGEX-2T vector. We expressed the GST fusion protein of rat fibronectin (designated GST-FN) in bacteria and purified it as described above. The GST-FN preparation has a major band with a Mr of about 65,000 (approximately 80% of the total) and some minor protein bands (degradation products) (data not shown), which is consistent with the Mr of 65,773 calculated from the primary structure of GST-FN.

Absorbance at 280 nm was measured to determine the concentration of purified proteins using A280 = 1.356 for D-15, A280 = 1.281 for GST-FN, and A280 = 1.567 for wild-type (wt) GST. The extinction coefficient for each protein was calculated from the amino acid sequence by counting the number of Tyr, Trp, and Cys residues and using the following values for molar extinction. For Tyr, epsilon 280 = 1400; for Trp, epsilon 280 = 5600; and for Cys, epsilon 280 = 127 for each disulfide bond (2 Cys residues) (21, 22).

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 alpha 2 (alpha 2-CHO) (23), human alpha 3 (alpha 3-CHO) (24), human alpha 4 (alpha 4-CHO) (25, 26), human alpha 5 (alpha 5-CHO) (26, 27), human alpha Lbeta 2 (alpha Lbeta 2-CHO) (28), human alpha v (alpha v-CHO), human beta 3 (beta 3-CHO), both human alpha v and beta 3 (alpha vbeta 3-CHO) (29), and human alpha IIbbeta 3 (alpha IIbbeta 3-CHO) (30) have been described in the cited references. The cDNA constructs for alpha 6 (alpha 6-CHO) and alpha 6 and beta 4 (for alpha 6beta 4-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 alpha 2-, alpha 3-, alpha 4-, alpha 5-, alpha 6-, and alpha v-CHO cells homogeneously expressed human alpha 2, alpha 3, alpha 4, alpha 5, and alpha v/hamster beta 1 hybrids, respectively. The beta 3-CHO cells expressed human beta 3/hamster alpha 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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Abstract
Introduction
Procedures
Results
Discussion
References

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 Mr of 36,000 under nonreducing conditions (lane 1), and the control, wt GST protein, migrates as a monomer with a Mr 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.


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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 alpha 5beta 1 as a major integrin but do not express beta 2 or beta 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 beta 3-CHO cells (that express alpha vbeta 3) and alpha IIbbeta 3-CHO cells (that express both alpha IIbbeta 3 and alpha vbeta 3) but not parent CHO cells and cells expressing other exogenous integrins (including alpha 2beta 1, alpha 3beta 1, alpha 4beta 1, alpha 5beta 1, alpha 6beta 1, alpha 6beta 4, alpha vbeta 1, and alpha Lbeta 2). These results indicate that D-15 interacts either with alpha vbeta 3 or with both alpha IIbbeta 3 and alpha vbeta 3. Fig. 2B shows that both D-15 and GST-FN support maximum adhesion of beta 3-CHO cells at the coating concentration of 10 µg of protein/ml, suggesting that D-15 and GST-FN support adhesion of beta 3-CHO cells at comparable levels. Fig. 2C shows that adhesion to D-15 of beta 3-CHO cells is completely blocked by function-blocking anti-alpha vbeta 3 mAb LM609, but adhesion to GST-FN is not. These results support the idea that adhesion to D-15 is mediated exclusively by alpha vbeta 3, whereas adhesion to GST-FN is mediated by multiple receptors that include alpha vbeta 3, alpha vbeta 1, and alpha 5beta 1.


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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 beta 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-alpha vbeta 3 mAb LM609 on adhesion of beta 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 alpha 5) did not block adhesion of beta 3-CHO cells to D-15 (data not shown). LM609 and KH72 were used at X250 dilution of ascites.

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


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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 alpha IIbbeta 3-CHO (lanes 1-6) and alpha vbeta 3-CHO (lanes 7-12) cells. Antibodies used were control antibody (nonimmune rabbit serum) (lanes 1 and 7), anti-beta 3 mAb 15 (lanes 2 and 8), anti-human alpha v LM142 (lane 9), anti-hamster alpha v peptide antibody (lanes 3 and 10), anti-alpha IIb PL98 DF6 (lanes 4 and 11), anti alpha vbeta 3 LM609 (lanes 5 and 12), and anti-alpha IIbbeta 3 2G12 (lane 6). The data suggest that alpha vbeta 3 bound to D-15 agarose, but alpha IIbbeta 3 did not.

alpha vbeta 3-D-15 Interaction Is Dependent on the Tripeptide RGD and the Flanking Sequence in the Putative Integrin Binding Site-- To determine whether alpha vbeta 3-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 beta 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-alpha vbeta 3 interaction is dependent on the RGD tripeptide in the putative integrin binding site.


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Fig. 4.   Adhesion of beta 3-CHO cells to wt and mutant D-15 as a function of coating concentration of substrates. Clonal CHO cells expressing alpha vbeta 3 (beta 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.

It has been reported that receptor specificity of snake venom disintegrins is defined by the sequence flanking the RGD tripeptide (36-38). To determine whether receptor specificity is determined by the sequence flanking the RGD tripeptide in D-15, we replaced the sequence RPTRGD with NWKRGD, which has been reported to support high affinity binding to alpha IIbbeta 3 in a phage display system (39). The mutant, designated D-15/NWK, was also expressed as a soluble monomer in bacteria (Fig. 1B), purified by affinity chromatography, and used for adhesion assays. Fig. 5A shows that D-15/NWK supports adhesion of both beta 3-CHO and alpha IIbbeta 3-CHO cells, suggesting that the mutant interacts with alpha vbeta 3; however, it is not clear whether the mutant binds to alpha IIbbeta 3 (beta 3-CHO cells express alpha vbeta 3, and alpha IIbbeta 3-CHO cells express both alpha vbeta 3 and alpha IIbbeta 3). To clarify this point, we examined binding of FITC-labeled wt D-15 and D-15/NWK mutant to cells expressing recombinant alpha IIbbeta 3. Since CHO cells express an inactive form of alpha IIbbeta 3, we activated the integrin using the anti-alpha IIbbeta 3 mAb PT25-2 (30). As shown in Fig. 5B, D-15/NWK and fibrinogen (a positive control) bound to alpha IIbbeta 3-CHO, but wt D-15 did not. These results indicate that the specificity of the recombinant disintegrin-like domain of MDC-15 is dependent on the sequence flanking the RGD tripeptide in the putative integrin binding site and confirm that wt D-15 binds to alpha vbeta 3 but not to alpha IIbbeta 3.


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Fig. 5.   Effects of mutating the sequence flanking the RGD tripeptide in the recombinant disintegrin-like domain of MDC-15. A, beta 3-CHO cells (that express alpha vbeta 3) and alpha IIbbeta 3-CHO cells (that express both alpha IIbbeta 3 and alpha vbeta 3) 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 alpha vbeta 3, but it is not clear whether it binds to alpha IIbbeta 3. B, parent CHO cells and alpha IIbbeta 3-CHO cells were first incubated with either mouse IgG (dotted line) or the activating anti-alpha IIbbeta 3 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 alpha IIbbeta 3-CHO cells in the presence of PT25-2, but wt D-15 does not. Fbg, fibrinogen.

Interaction between a Natural Human alpha vbeta 3 Heterodimer and D-15-- Since we used a recombinant hamster alpha v/human beta 3 hybrid integrin on CHO cells to study the receptor specificity of D-15, we determined whether the natural human alpha vbeta 3 heterodimer recognize wt and mutant D-15. As shown in Fig. 6, M-21 human melanoma cells expressing alpha vbeta 3 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 alpha vbeta 3 heterodimer, like a hybrid alpha vbeta 3 on CHO cells, specifically recognizes D-15 in an RGD-dependent manner.


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Fig. 6.   Adhesion of human alpha vbeta 3 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-alpha vbeta 3 mAb LM609 (at ×250 dilution of ascites). A control mAb KH72 (to integrin alpha 5) did not block adhesion of M-21 cells to D-15 (data not shown). The data indicate that natural human alpha vbeta 3 heterodimer in M-21 cells recognizes D-15 in an RGD-dependent manner.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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-beta (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 alpha vbeta 3 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 alpha vbeta 3. Substitution of the sequence flanking the tripeptide RGD changes the receptor specificity (from alpha vbeta 3 to both alpha IIbbeta 3 and alpha vbeta 3). 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 alpha vbeta 3 and alpha IIbbeta 3 (11). Barbourin is the only disintegrin that is specific to alpha IIbbeta 3. There is no known snake venom disintegrin that is specific to alpha vbeta 3. Thus, the RGD tripeptide and flanking sequence in the disintegrin-like domain of MDC-15 represent a unique alpha vbeta 3-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 alpha vbeta 3 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 alpha vbeta 3 on leukocytes during atherogenesis through its exposed disintegrin-like domain. alpha vbeta 3 has been shown to be involved in the progression of melanoma and the induction of neo-vascularization by tumor cells. alpha vbeta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels (44, 45). It is possible that activated MDC-15 and alpha vbeta 3 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-alpha vbeta 3 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 alpha 2-subunit via the disintegrin domain followed by proteolysis of the beta 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 alpha vbeta 3, as in the case of jararhagin and alpha 2beta 1, and either modifies the function of the integrin or promotes degradation of the matrix proteins surrounding the cells that express alpha vbeta 3. Therefore, the proposed alpha vbeta 3-MDC-15 interaction may be of wide biological importance.

    ACKNOWLEDGEMENTS

We thank D. Cheresh, M. H. Ginsberg, M. Handa, Y. Ikeda, V. Woods, and J. Ylanne for valuable reagents.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM47157 and GM49899. This is publication 10574-VB from The Scripps Research Institute.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 Dept. of Vascular Biology, VB-1, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7122; Fax: 619-784-7323; E-mail: takada{at}scripps.edu.

1 The abbreviations used are: MDC, metalloprotease/disintegrin/cysteine-rich protein; mAb, monclonal antibody; GST, glutathione S-transferase; CHO, Chinese hamster ovary; FN, fibronectin; FITC, fluorescent isothiocyanate; wt, wild-type; PBS, phosphate-buffered saline.

2 X.-P. Zhang, W. Puzon-McLaughlin, and Y. Takada, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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