COMMUNICATION:
Identification of an Adhesion Site within the Syndecan-4 Extracellular Protein Domain*

(Received for publication, February 20, 1997, and in revised form, March 18, 1997)

Aidan J. McFall and Alan C. Rapraeger Dagger

From the Program in Cellular and Molecular Biology and Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, 53706-1532

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The syndecan family of cell surface proteoglycans regulates cell adhesion and growth factor signaling by binding components of the extracellular matrix and growth factors. To date, all known ligand interactions are via the covalently attached glycosaminoglycan chains. To assay for potential extracellular interactions via the core proteins directly, the recombinant extracellular domain of syndecan-4 (S4ED), one of the four syndecan family members, was tested as a substratum for the attachment of mammalian cells. Human foreskin fibroblasts bind to mouse S4ED, and both mouse and chicken S4ED can block this binding, with 50% inhibition observed between 0.1 and 1 × 10-7 M. The extracellular domain of another syndecan family member, syndecan-1, fails to compete for cell binding to mouse S4ED. Amino acids 56-109 of the 120-amino acid mouse S4ED compete fully, suggesting that the cell binding domain is within this region. The ability of syndecan-4 to interact with molecules at the cell surface via its core protein as well as its glycosaminoglycan chains may uniquely regulate the formation of cell surface signaling complexes following engagement of this proteoglycan with its extracellular ligands.


INTRODUCTION

Syndecan-4, also known as ryudocan or amphiglycan, is one of four members of the syndecan family of cell surface proteoglycans. These type I transmembrane proteins are unified by their homologous transmembrane and cytoplasmic domains and the covalent addition of glycosaminoglycan chains, predominantly heparan sulfate, to their extracellular domains. Their heparan sulfate chains bind components of the extracellular matrix, such as fibronectin and collagen, as well as heparin-binding growth factors such as the FGFs1 (1-4). In tissues, these proteoglycans show distinct expression patterns, although their expression can be overlapping and more than one syndecan family member may be found on a single cultured cell type (3, 5-7). The syndecans are present at the plasma membrane and are also in the medium of cultured cells as a result of shedding the extracellular domain from the cell surface (7, 8).

Despite the emphasis on the binding of ligands to their glycosaminoglycan chains, the syndecan core proteins are also likely to have important roles in the regulation of cell adhesion and cell morphology. Syndecan-1 expressed in Schwann cells co-aligns with actin filaments in response to antibody ligation, a process dependent on a specific tyrosine residue within the syndecan-1 cytoplasmic domain (9). Syndecan-1 expressed in a B cell line enables the cells to bind and spread on heparan sulfate ligands and on core protein-specific antibodies (10). Antibody-mediated spreading does not require the cytoplasmic domain of syndecan-1 or its glycosaminoglycan chains, suggesting that the signals that convey syndecan-1-specific cell spreading result from interactions between the transmembrane and/or extracellular protein domains of syndecan-1 and other cell surface or transmembrane molecules (10).

Engagement of cell surface proteoglycans, such as the syndecans, with other receptors may be a common growth factor and adhesion signaling mechanism. In FGF signaling, the high affinity signaling complex consists of a receptor tyrosine kinase(s), the FGF ligand, and heparan sulfate proteoglycans such as the syndecans (11-13). Similar multi-molecular complexes may assemble during cell adhesion. NG2, a cell surface chondroitin sulfate proteoglycan, cooperates with alpha 4beta 1 integrin in the development of focal adhesions (14). Simultaneous engagement of NG2 and alpha 4beta 1 signals melanoma cells to spread and form focal adhesions in a tyrosine kinase-dependent process. When either receptor is individually engaged, cells bind but fail to change shape. Similarly, the interaction between cell surface heparan sulfate proteoglycans and fibronectin stimulates focal adhesion formation but only in cooperation with integrins, such as the alpha 5beta 1 integrin (15, 16). In this assay, the engagement of proteoglycan can be replaced by activating protein kinase C, suggesting that adhesive interactions with cell surface proteoglycans can lead to intracellular signaling events (17). Syndecan-4, unlike the other syndecan family members, localizes to focal adhesions, implicating this proteoglycan as part of their signaling mechanism (18, 19). To better understand how syndecan core proteins might regulate the formation of multi-molecular cell adhesion and growth factor signaling complexes, studies were undertaken to identify interactions between the extracellular protein domain of syndecan-4, the most widely expressed of the syndecan family members, and other receptors at the cell surface.


EXPERIMENTAL PROCEDURES

Cell Culture

Neonatal human foreskin fibroblasts were cultivated in DMEM (Life Technologies, Inc.), supplemented with 10% calf serum (Hyclone), and used prior to 60 days in culture.

Plasmid Constructs

Dr. Merton Bernfield (Harvard Medical School, Boston, MA) kindly provided the cDNAs encoding mS4ED and mouse syndecan-1 (7, 20). Chicken syndecan-4 cDNA was kindly provided by Dr. Paul F. Goetinck (Massachusetts General Hospital, Charlestown, MA) (21). His-mS4ED was constructed by cloning mS4ED into the BamHI (5') and EcoRI (3') sites of the pET-30a vector (Novagen), Full-length and truncated mS4ED, cS4ED (nucleotides 114-482) and mS1ED (nucleotides 291-989) cDNAs were PCR amplified using primers containing restriction enzyme sites, BamHI (5') or EcoRI (3'), for cloning into pGEX-2T (Pharmacia Biotech Inc.). The EcoRI site is immediately preceded by the in-frame stop codon TAA. PCR amplified cDNAs were confirmed by sequencing.

Protein Sequence Analysis

S4ED protein alignment, calculation of amino acid similarity, and homology searches were performed with Genetics Computer Group, Inc. software (PileUp, Gap, FastA) and the National Center for Biotechnology Information network service (BLAST), which access GenBank and EMBL sequence data bases.

Production and Quantification of Recombinant S4ED

GST fusion proteins were isolated using glutathione-Sepharose beads according to the manufacturer's instructions for "bulk purification of fusion proteins" (Pharmacia) with the following exceptions. After induction, bacterial pellets were resuspended in phosphate-buffered saline (pH 7.4) containing 25% sucrose, 1 mM EDTA, 5 mM dithiothreitol, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Lysozyme (1 mg/ml) and 1% TX-100 were added, followed by sonication and DNase I (1500 NIH units) treatment prior to centrifugation. His-mS4ED protein was prepared similarly, except that purification was achieved by nickel chromatography (Qiagen) followed by imidazole elution. GST fusion proteins bound to glutathione beads were digested with 5 NIH units thrombin/mg fusion protein for 90 min at 4 °C, and thrombin was removed with benzamidine-Sepharose. Glutathione-eluted fusion proteins were quantified by absorbance at 280 nm. For GST and GST constructs containing mS4ED sequences, protein concentrations were calculated using the formula 1 A280 = 0.5 mg/ml. For GST-cS4ED and GST-mS1ED, 1 A280 = 0.4 mg/ml. His-mS4ED and thrombin-released peptides were quantified relative to a standard curve of thrombin-released mS4ED by Coomassie Blue or SYPRO®-Red (Molecular Probes) staining of proteins in polyacrylamide gels.

Substrata for Cell Binding Assays

High binding 96-well plates (Costar, 2585) were used for all cell binding assays. Recombinant proteins were diluted in HEPES/DMEM to 0.2 µM, unless otherwise noted, and incubated overnight at 4 °C. Plasma fibronectin was diluted in phosphate-buffered saline (pH 7.4) to approximately 45 nM (10 µg/ml) and plated for 2 h at 37 °C. For the experiment shown in Fig. 2B, the plating concentration of GST-mS4ED was 45 nM (1.75 µg/ml). Wells were blocked by incubating with HEPES/DMEM containing 1% heat-denatured BSA prior to the addition of cells.


Fig. 2. Human dermal fibroblasts bind to mS4ED substrata. A, cells were added to microtiter wells coated with 0.2 µM of various ligands. The extent of cell binding was quantified over time, as described under "Experimental Procedures." B, binding was performed as in A, except ligand coating concentrations were 0.045 µM. Data from triplicate samples (means ± S.E.) are shown: GST (open circle ), GST-mS4ED (bullet ), His-mS4ED (square ), mS4ED (black-square), and fibronectin (black-triangle).
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Cell Binding Assays

Cells were washed twice with HEPES/DMEM and treated with 25 µg/ml cycloheximide in HEPES/DMEM for 2 h prior to trypsinization. After washing with 20 mM Tris (pH 7.6) containing 1 mM EDTA, 150 mM NaCl, and 25 µg/ml cycloheximide, cells were trypsinized for 2 min at 37 °C by incubating with 0.25% trypsin dissolved in the same buffer. The trypsin was inactivated by washing cells with 0.5 mg/ml soybean trypsin inhibitor dissolved in HEPES/DMEM containing 25 µg/ml cycloheximide. For all experiments, 50,000 cells were added per well except for Fig. 2B, where 25,000 cells were added. Competing proteins were added to each well after blocking but prior to the addition of cells. Cell binding assays were performed at 37 °C in HEPES/DMEM containing 0.2% heat-denatured BSA and 25 µg/ml cycloheximide in a total volume of 100 µl/well. Nonadherent cells were removed by washing the wells three times with HEPES/DMEM. Fixed, adherent cells were quantified by colorimetric detection with 1% bromphenol blue (22).


RESULTS AND DISCUSSION

Expression of mS4ED in Bacteria

Recombinant mS4ED was engineered and expressed in bacteria, providing glycosylation-free core protein. Two independent expression vectors, each of which encodes mS4ED as a C-terminal fusion protein with distinct amino sequences, were used (Fig. 1A). One encodes a GST fusion protein containing the entire 120-amino acid mS4ED attached to the GST C terminus. This construct also contains a thrombin cleavage site, allowing isolation and testing of the mS4ED domain (Fig. 1A). As another means of ensuring that the activity of the fusion protein is due to mS4ED alone, a second fusion construct was used, namely, His-mS4ED (Fig. 1A) His-mS4ED is comprised of six histidines and an immunological tag (S·TagTM) at the N terminus of mS4ED. Polyacrylamide gel electrophoresis of the affinity purified protein products reveals one major protein band following purification of GST, GST-mS4ED, and thrombin-released mS4ED (Fig. 1B). Immunoblotting with GST-mS4ED serum, which recognizes both mS4ED and GST, confirmed the identity of the protein bands and demonstrated that little, if any, GST protein contaminated the thrombin-released mS4ED preparation (data not shown). The His-mS4ED preparation contains three major protein bands. Only the slowest migrating band of the three reacts with GST-mS4ED serum, thus identifying this protein as intact His-mS4ED (data not shown). The two smaller proteins may represent co-purifying proteins or degradation products. All proteins containing mS4ED sequence migrate 5-7 kDa higher than their calculated molecular mass. This is not unexpected, as deglycosylated, full-length syndecan-4 has been shown to migrate with an apparent molecular mass 10-15 kDa greater than its actual molecular mass (3, 23). Importantly, none of the minor contaminating bands in any one mS4ED preparation is visibly shared by all three mS4ED preparations (GST-mS4ED, His-mS4ED, and thrombin-released mS4ED).


Fig. 1. Expression of recombinant mS4ED. A, mS4ED was expressed in pGEX-2T or pET-30a for production of GST-mS4ED or His-mS4ED protein as described under "Experimental Procedures." The pGEX-2T vector encodes a thrombin cleavage site upstream of mS4ED, allowing thrombin-mediated release of the mS4ED peptide. Stippled bars, GST; striped bar, His + S·TagTM; solid bars, mS4ED. Arrow, thrombin cleavage site; arrowhead, amino acid position. B, affinity purified proteins, 8 µg each, were analyzed by polyacrylamide gel electrophoresis, followed by Coomassie Blue staining. Lane 1, GST; lane 2, GST-mS4ED; lane 3, His-mS4ED; lane 4, thrombin-released mS4ED.
[View Larger Version of this Image (45K GIF file)]

Human Foreskin Fibroblasts Bind to mS4ED Substrata

To detect interactions between recombinant mS4ED and other receptors at the cell surface, a solid phase cell binding assay was developed. Substrata were prepared by incubating microtiter wells with the various recombinant forms of mS4ED, including intact fusion proteins or thrombin-released peptide. After blocking, the individual substrata were assessed for their ability to support cell binding over time (Fig. 2A). Human foreskin fibroblasts, pretreated with cycloheximide prior to suspension with trypsin, bind avidly to all substrata containing mS4ED but fail to bind to a substratum composed of GST alone. Binding is maximal by 60 min at 37 °C, and greater than 75% of the input cells bind to GST-mS4ED. Binding occurs despite the presence of cycloheximide, suggesting that binding is via a trypsin-resistant cell surface receptor.

To compare the binding of mS4ED to that of a known adhesive ligand, microtiter wells were coated with fibronectin. Cell binding was monitored over time and quantified (Fig. 2B). At equimolar plating concentrations, the extent of cell binding to GST-mS4ED and fibronectin was comparable, although the kinetics of binding to GST-mS4ED were slightly slower.

Cell Binding to mS4ED Is Specific

To assess the specificity and affinity of cell binding, soluble proteins were evaluated for their ability to compete in the binding assay. Binding to GST-mS4ED is reduced in a dose-dependent manner by GST-mS4ED (Fig. 3A). The IC50 in this experiment is 0.13 µM, suggesting that the interaction between mS4ED and its putative cell surface receptor(s) is of moderate affinity, similar to that of some integrins for their ligands (24). Thrombin-released mS4ED peptide competes as effectively or better than the GST-mS4ED fusion protein (Fig. 3B). Six independent experiments yielded an average IC50 of 0.04 µM for thrombin-released mS4ED, with a range of 0.01-0.1 µM (Fig. 4). The extracellular domain of another syndecan family member, syndecan-1, was also evaluated for its ability to compete for binding of cells to a GST-mS4ED substratum. Neither full-length GST-mS1ED nor thrombin-released mS1ED compete for binding when added at 13 and 3.5 µM, respectively (Fig. 3, A and B). Interestingly, chicken syndecan-4 extracellular domain (cS4ED) competes as well as mS4ED (cloned from mouse) for binding of cells to GST-mS4ED (Fig. 3A), suggesting that the cell binding domain of mS4ED is conserved across species.


Fig. 3. Cell binding to mS4ED is specific. A, increasing amounts of soluble GST fusion proteins were added to wells coated with GST-mS4ED ligand. Cell binding was quantified after 2 h as described under "Experimental Procedures." B, increasing amounts of thrombin-released syndecan extracellular domains were also tested in this assay. Data from triplicate samples (means ± S.E.) are shown: GST-mS4ED (black-square), GST-cS4ED (black-triangle), GST-mS1ED (bullet ), mS4ED (open circle ), and mS1ED (square ).
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Fig. 4. The mS4ED56-109 fragment contains the cell binding domain. Increasing amounts of soluble proteins were added to wells coated with the GST-mS4ED ligand. Cell binding was quantified after 2 h as described under "Experimental Procedures." All IC50 values were determined from triplicate dilution series. For thrombin-cleaved mS4ED amino acids 1-120, 1-80, 81-120, and 56-109, at least two separate experiments were performed with similar results. ND, not determined; NC, no competition at the concentration shown in the parentheses; striped box, signal sequence; solid boxes, amino acids 56-109 of mS4ED; open boxes, syndecan-4 core protein; stippled box, syndecan-4 transmembrane domain; vertical lines, glycosaminoglycan chains; arrowheads, amino acid positions.
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Although cells from both human and mouse species bind to GST-mS4ED, not all cell types bind, suggesting that the receptor(s) for mS4ED may be expressed in a cell type-specific manner. Cells that bind avidly to GST-mS4ED include human foreskin fibroblasts, mouse aortic endothelial cells, and mouse NIH 3T3 and Swiss 3T3 fibroblast cell lines. Cells that fail to bind include mouse NMuMG epithelial cells and the human Raji and ARH-77 B cell lines (data not shown).

The S4ED56-109 Fragment Contains the Cell Binding Domain

A domain containing the cell binding activity was identified by testing truncated mS4ED peptides as competitors in the cell binding assay. Complementary DNAs encoding mS4ED peptides were created by PCR amplification and were expressed as GST fusion proteins. IC50 values were calculated for both intact GST fusion proteins and their thrombin-released peptides (Fig. 4). Thrombin-released mS4ED and mS4ED56-109 compete equally well for cell binding to GST-mS4ED, demonstrating that amino acids 56-109 can fully replace the cell binding activity of the full-length mS4ED. Further, thrombin-released mS4ED1-80 and mS4ED81-120 compete 100-fold less effectively than mS4ED56-109. These two peptides together contain all 120 amino acids that constitute mS4ED but bisect amino acids 56-109, demonstrating that the cell binding domain is not reiterated within the mS4ED sequence and that the binding activity does not reside solely in either half of the mS4ED56-109 domain.

Some thrombin-released peptides compete better than the GST fusion proteins from which they were derived, suggesting that proximity to GST can sterically hinder the cell binding domain. For example, GST-mS4ED and thrombin-released mS4ED compete with similar effectiveness, whereas GST-mS4ED56-109 competes 1000 times less effectively than thrombin-released mS4ED56-109. Importantly, thrombin-released mS4ED and thrombin-released mS4ED56-109 compete identically.

Alignment of Vertebrate Syndecan-4 Extracellular Domains

An alignment of the cloned syndecan-4 extracellular domains reveals the positioning and degree of conservation of amino acids 56-109 (Fig. 5). Although chicken and mouse are only 34% identical within this domain, pairwise alignment of the sequences indicate similarity values ranging from 62 to 70%. The S4ED56-109 domain begins 15 amino acids C-terminal to the last possible glycosaminoglycan acceptor site in the extracellular domain of syndecan-4. This distinguishes the cell binding domain spatially from the glycosaminoglycan acceptor sites. Syndecan-4 is expressed as a heparan sulfate proteoglycan and to a lesser extent as a hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate chains (3, 18, 21, 25). Because heparin and chondroitin sulfate A, B, and C are unable to compete for binding of cells to GST-mS4ED (up to 100 µg/ml, data not shown), it seems unlikely that the glycosaminoglycan decoration of endogenous syndecan-4 would compete directly for cell binding domain interactions. Because no other proteins currently in the data base share significant homology to this region of syndecan-4, the interaction between amino acids 56-109 and the cell surface may be unique to syndecan-4.


Fig. 5. Alignment of the cloned S4ED protein sequences. Amino acid 1 is immediately downstream of the predicted signal peptidase cleavage site (23). Amino acid 120 is the final residue of mS4ED and is the third amino acid from the transmembrane domain in all complete cDNAs of syndecan-4. M, mouse; R, rat; H, human; C, chicken. Amino acids 56-109, which contain the cell binding domain, are boxed.
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The cell binding activity of the syndecan-4 core protein constitutes a new class of interactions for the syndecans, namely, the association of the extracellular core protein domain of a syndecan and other cell surface molecules. This domain may interact with molecules on adjacent cells to mediate cell-cell adhesion or potentially function as a shed or matrix-embedded adhesive ligand. Because syndecan-4 is the major heparan sulfate proteoglycan to localize to focal adhesions, it is intriguing to speculate that the interactions mediated by the cell binding domain of syndecan-4 may regulate its localization to or the formation of such adhesion structures (18, 19). Given the propensity of the syndecans to homodimerize (26, 27) and potentially multimerize in response to ligand engagement (28), it seems equally likely that the syndecan-4 cell binding domain might regulate the formation of other multi-molecular cell surface signaling complexes.


FOOTNOTES

*   This work was supported by Grant HD21881 from the National Institutes of Health.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: Dept. of Pathology and Laboratory Medicine, University of Wisconsin-Madison, 1300 University Ave., Madison, WI 53706-1532. Tel.: 608-262-7577; Fax: 608-262-2327.
1   The abbreviations used are: FGF, fibroblast growth factor; S4ED, syndecan-4 extracellular domain; mS4ED, mouse S4ED; cS4ED, chicken S4ED; mS1ED, mouse syndecan-1 extracellular domain; GST, glutathione-S-transferase; His, PET-30a vector-encoded His + S·TagTM protein; DMEM, Dulbecco's modified Eagle's medium; HEPES/DMEM, HEPES-buffered DMEM (pH 7.4); BSA, bovine serum albumin; PCR, polymerase chain reaction.

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