Solution structure of the dimeric cytoplasmic domain of syndecan-4.

The syndecans, transmembrane proteoglycans which are involved in the organization of cytoskeleton and/or actin microfilaments, have important roles as cell surface receptors during cell-cell and/or cell-matrix interactions. Since previous studies indicate that the function of the syndecan-4 cytoplasmic domain is dependent on its oligomeric status, the conformation of the syndecan-4 cytoplasmic domain itself is important in the understanding of its biological roles. Gel filtration results show that the syndecan-4 cytoplasmic domain (4L) itself forms a dimer stabilized by ionic interactions between peptides at physiological pH. Commensurately, the NMR structures demonstrate that syndecan-4L is a compact intertwined dimer with a symmetric clamp shape in the central variable V region with a root-mean-square deviation between backbone atom coordinates of 0.95 A for residues Leu(186)-Ala(195). The molecular surface of the 4L dimer is highly positively charged. In addition, no intersubunit NOEs in membrane proximal amino acid resides (C1 region) have been observed, demonstrating that the C1 region is mostly unstructured in the syndecan-4L dimer. Interestingly, two parallel strands of 4L form a cavity in the center of the dimeric twist similar to our previously reported 4V structure. The overall topology of the central variable region within the 4L structure is very similar to that of 4V complexed with the phosphatidylinositol 4,5-bisphosphate; however, the intersubunit interaction mode is affected by the presence of C1 and C2 regions. Therefore, we propose that although the 4V region in the full cytoplasmic domain has a tendency for strong peptide--peptide interaction, it may not be enough to overcome the repulsion of the C1 regions of syndecan-4L.     

Identification of a novel Ezrin-binding site in syndecan-2 cytoplasmic domain

ERM (Ezrin/Radixin/Moesin) proteins are crosslinkers between plasma membrane proteins and the actin cytoskeleton, thereby involved in the formation of cell adhesion sites. Earlier work showed that Ezrin links syndecan-2 to the actin cytoskeleton. Here we provide evidence that the Ezrin N-terminal domain binds to the syndecan-2 cytoplasmic domain with an estimated K(D) of 0.71 microM and without the requirement of other proteins. We also studied the regions in the syndecan-2 cytoplasmic domain implicated in the binding to Ezrin. By truncating the syndecan-2 cytoplasmic domain and by oligopeptide competition assays we show that the Ezrin-binding sequence is not located in the positively charged juxtamembrane region (RMRKK), but in the neighboring sequence DEGSYD. We therefore conclude that the consensus sequence for Ezrin binding is unique among membrane proteins, suggesting a distinct regulation.     

Critical amino acids in syndecan-4 cytoplasmic domain modulation of turkey satellite cell growth and development

Syndecan-4 is composed of a core protein and covalently attached glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains. The core protein is divided into extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain has two conserved regions and a variable region in the middle. The Ser residue in the conserved region 1 and the Tyr residue in the variable region are important in regulating protein kinase C alpha (PKCα) membrane localization and focal adhesion formation. The objective of the current study was to investigate the role of syndecan-4 Ser and Tyr residues in combination with the GAG and N-glycosylated chains in turkey satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Site-directed mutagenesis was used to generate Ser and Tyr mutants with or without GAG and N-glycosylated chains. The wild type and mutant syndecan-4 constructs were transfected into turkey satellite cells. The over-expression of Ser and Tyr mutants increased cell proliferation and differentiation and decreased membrane localization of PKCα. Furthermore, Ser mutants enhanced cellular responsiveness to FGF2. The results from this study are the first demonstration of a role of syndecan-4 cytoplasmic domain Ser and Tyr residues in regulating satellite cell proliferation, differentiation, and the modulation of cellular responsiveness to FGF2.     

The PDZ-binding domain of syndecan-2 inhibits LFA-1 high-affinity conformation

Syndecans are cell membrane proteoglycans that can modulate the activity and dynamics of some growth factor receptors and integrins. Here, we show the down-regulation of integrin lymphocyte function-associated antigen-1 (LFA-1) and inhibition of adhesion of Jurkat T cells transfected with syndecan-2. The PDZ-binding domain in the cytoplasmic region of syndecan-2 was necessary to block the LFA-1 high-affinity conformation, and to reduce cellular adhesion. A second cytoplasmic motif comprising tyrosines 179 and 191, and serines 187 and 188 contributed also to reduce LFA-1 function and cellular adhesion. Inhibition of the LFA-1 high-affinity conformation by syndecan-2 was independent of the expression of the talin head domain and RhoA, Rac1 and Cdc42 GTPases. These results demonstrate the importance of PDZ-binding domain of syndecan-2 for controlling LFA-1 affinity and cell adhesion.     

Fibroblast growth factor 2 and protein kinase C alpha are involved in syndecan-4 cytoplasmic domain modulation of turkey myogenic satellite cell proliferation

Syndecan-4 core protein is composed of extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain functions in transmitting signals into the cell through the protein kinase C alpha (PKCα) pathway. The glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains attached to the extracellular domain influence cell proliferation. The current study investigated the function of syndecan-4 cytoplasmic domain in combination with GAG and N-glycosylated chains in turkey muscle cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Syndecan-4 or syndecan-4 without the cytoplasmic domain and with or without the GAG and N-glycosylated chains were transfected or co-transfected with a small interfering RNA targeting syndecan-4 cytoplasmic domain into turkey muscle satellite cells. The overexpression of syndecan-4 mutants increased cell proliferation but did not change differentiation. Syndecan-4 mutants had increased cellular responsiveness to FGF2 during proliferation. Syndecan-4 increased PKCα cell membrane localization, whereas the syndecan-4 mutants decreased PKCα cell membrane localization compared to syndecan-4. However, compared to the cells without transfection, syndecan-4 mutants increased cell membrane localization of PKCα. These data indicated that the syndecan‐4 cytoplasmic domain and the GAG and N-glycosylated chains are critical in syndecan-4 regulating satellite cell proliferation, responsiveness to FGF2, and PKCα cell membrane localization.     

A role for syndecan-1 in coupling fascin spike formation by thrombospondin-1

An important role of cell matrix adhesion receptors is to mediate transmembrane coupling between extracellular matrix attachment, actin reorganization, and cell spreading. Thrombospondin (TSP)-1 is a modulatory component of matrix expressed during development, immune response, or wound repair. Cell adhesion to TSP-1 involves formation of biochemically distinct matrix contacts based on stable fascin spikes. The cell surface adhesion receptors required have not been identified. We report here that antibody clustering of syndecan-1 proteoglycan specifically transduces organization of cortical actin and fascin bundles in several cell types. Transfection of COS-7 cells with syndecan-1 is sufficient to stimulate cell spreading, fascin spike assembly, and extensive protrusive lateral ruffling on TSP-1 or on syndecan-1 antibody. The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation. Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1. These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure.     

Regulation of inositol phospholipid binding and signaling through syndecan-4

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains a central region unlike that of any other vertebrate or invertebrate syndecan core protein with a cationic motif that binds inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)(2)) and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. Affinity was much reduced for 3-phosphorylated inositides while no binding of diacylglycerol was detected. Syndecan-2 cytoplasmic domain had negligible affinity for any lipid examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P(2). Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down-regulator of syndecan-4 signaling. Similarly, phosphorylation of serine 183 in syndecan-4 cytoplasmic domain reduced PtdIns(4,5)P(2) binding affinity by over 100-fold, although interaction could still be detected by nuclear magnetic resonance spectroscopy. Only protein kinase Calpha was up-regulated in activity by the combination of syndecan-4 and PtdIns(4,5)P(2), with all other isoforms tested showing minimal response. This is consistent with the codistribution of syndecan-4 with the alpha isoform of protein kinase C in focal adhesions.     

Functional role of syndecan-1 cytoplasmic V region in lamellipodial spreading, actin bundling, and cell migration

Cell protrusions contribute to cell motility and migration by mediating the outward extension and initial adhesion of cell edges. In many cells, these extensions are supported by actin bundles assembled by the actin cross-linking protein, fascin. Multiple extracellular cues regulate fascin and here we focus on the mechanism by which the transmembrane proteoglycan, syndecan-1, specifically activates lamellipodial cell spreading and fascin-and-actin bundling when clustered either by thrombospondin-1, laminin, or antibody to the syndecan-1 extracellular domain. There is almost no knowledge of the signaling mechanisms of syndecan-1 cytoplasmic domain and we have tested the hypothesis that the unique V region of syndecan-1 cytoplasmic domain has a crucial role in these processes. By four criteria--the activities of N-cadherin/V region chimeras, syndecan-1 deletion mutants, or syndecan-1 point mutants, and specific inhibition by a membrane-permeable TAT-V peptide--we demonstrate that the V region is necessary and sufficient for these cell behaviors and map the molecular basis for its activity to multiple residues located across the V region. These activities correlate with a V-region-dependent incorporation of cell-surface syndecan-1 into a detergent-insoluble form. We also demonstrate functional roles of syndecan-1 V region in laminin-dependent C2C12 cell adhesion and three-dimensional cell migration. These data identify for the first time specific cell behaviors that depend on signaling through the V region of syndecan-1.     

Syndecan-4 cytoplasmic domain regulation of turkey satellite cell focal adhesions and apoptosis

Syndecan-4 is a cell membrane proteoglycan composed of a transmembrane core protein and substituted glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains. The core protein has three domains: extracellular, transmembrane and cytoplasmic domains. The GAG and N-glycosylated chains and the cytoplasmic domain of syndecan-4, especially the amino acids: Ser(178) and Tyr(187) are critical in regulation of turkey satellite cell growth and development. How these processes are regulated is still unknown. The objective of the current study was to determine whether the syndecan-4 GAG and N-glycosylated chains and the cytoplasmic domain functions through modulating focal adhesion formation and apoptosis. Twelve mutant clones were generated: a truncated syndecan-4 without the cytoplasmic domain with or without GAG and N-glycosylated chains, and Ser(178) and Tyr(187) mutants with or without GAG and N-glycosylated chains. The wild type syndecan-4 and all of the syndecan-4 mutants were transfected into turkey myogenic satellite cells after which cell apoptosis and focal adhesion formation were measured. Syndecan-4 increased cell membrane localization of β1 integrin and the activity of focal adhesion kinase (FAK) whereas the cytoplasmic domain mutation decreased the phosphorylation of FAK. However, syndecan-4 and syndecan-4 mutants did not influence cell apoptosis. They also had no effect on vinculin or paxillin-containing focal adhesion formation. These results suggested that the syndecan-4 cytoplasmic domain plays an important role in regulating FAK activity and β1 integrin cell membrane localization but not cell apoptosis and vinculin or paxillin-containing focal adhesion formation.     

Structural basis of syndecan-4 phosphorylation as a molecular switch to regulate signaling

The syndecan transmembrane proteoglycans are involved in the organization of the actin cytoskeleton and have important roles as cell surface receptors during cell-matrix interactions. We have shown that the syndecan-4 cytoplasmic domain (4L) forms oligomeric complexes that bind to and stimulate PKCalpha activity in the presence of PtdIns(4,5)P2, emphasizing the importance of multimerization in the regulation of PKCalpha activation. Oligomerization of the cytoplasmic domain of syndecan-4 is regulated either positively by PtdIns(4,5)P2 or negatively by phosphorylation of serine 183. Phosphorylation results in reduced PKCalpha activity by inhibiting PtdIns(4,5)P2-dependent oligomerization of the syndecan-4 cytoplasmic domain. Data from NMR and gel-filtration chromatography show that the phosphorylated cytoplasmic domain (p-4L) exists as a dimer, similar to 4L, but not as higher-order oligomers. NMR analysis showed that the overall conformation of p-4L is a compact intertwined dimer with an unusually symmetric clamp shape, and its molecular surface is mostly positively charged. The two parallel strands form a cavity in the center of the dimeric twist. An especially marked effect of phosphorylation of the syndecan-4 cytoplasmic domain is a dramatic conformational change near the C2 region that ablates an interaction site with the PDZ domain of syntenin. Wound healing studies further suggest that syndecan-4 phosphorylation might influence cell migration behavior. We conclude that the phosphorylation (Ser183) of syndecan-4 can play a critical role as a molecular switch to regulate its functions through conformational change.     

Functional analysis of the interface regions involved in interactions between the central cytoplasmic loop and the C-terminal tail of adenylyl cyclase

The mammalian adenylyl cyclase is a membrane-bound enzyme that is predicted to have 12 trans-membrane spans. Between membrane spans 6 and 7 there is a large cytoplasmic loop, which, along with the C-terminal tail, makes up the catalytic site of the enzyme. Crystal structures of these soluble cytoplasmic domains have identified the regions that are involved in interactions with each other. The functional consequences of these interactions in the full-length membrane-embedded enzymes have not been established. In this study, we analyzed the role of various interaction regions within the central cytoplasmic loop (C1) and the C-terminal tail (C2) on basal, Galphas-, forskolin-, and Mn(2+)-stimulated activities of adenylyl cyclases 2 and 6 (AC2 and AC6). We tested synthetic peptides encoding the different interface surfaces of both the C1 and C2 domain on different activities of membrane-bound AC2 and AC6 expressed in insect cells. We found the C1-alpha2-beta2-beta3 and C2-beta2'-beta3' regions to be involved in stimulation by Galphas and forskolin but not in the basal or Mn(2+)-stimulated activities. Both the C1-beta4-beta5-alpha4 region and the C2-alpha3'-beta4' region play a role in the Galphas- and forskolin-stimulated activities as well as in basal activity, because the peptides encoding these regions inhibit basal activity by 30%. In contrast, the C2-alpha2' region peptide inhibits both basal and Mn(2+)-stimulated activity by >50%. These results suggest that the different stimulated activities may involve distinct interface interactions in the intact enzyme and, consequently, the distinct mechanisms by which Mn(2+) activates the enzyme as compared with Galphas and forskolin, leading to the possibility that the full-length adenylyl cyclase may have multiple catalytically competent configurations.     

A peptide derived from tenascin-C induces beta1 integrin activation through syndecan-4

Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated beta1 integrin-mediated cell adhesion of nonadherent and adherent cell types, by inducing activation of beta1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced beta1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated beta1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced beta1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of beta1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase Calpha inhibitor G?6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in beta1 integrin necessary for its functional activation. A lateral interaction of beta1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change.     

Syndecan-2 cytoplasmic domain regulates colon cancer cell migration via interaction with syntenin-1

The cell surface heparan sulfate proteoglycan, syndecan-2, is crucial for the tumorigenic activity of colon cancer cells. However, the role played by the cytoplasmic domain of the protein remains unclear. Using colon cancer cells transfected with various syndecan-2-encoding genes with deletions in the cytoplasmic domain, it was shown that syndecan-2-induced migration activity requires the EFYA sequence of the C-terminal region; deletion of these residues abolished the rise in cell migration seen when the wild-type gene was transfected and syndecan-2 interaction with syntenin-1, suggesting that syntenin-1 functioned as a cytosolic signal effector downstream from syndecan-2. Colon cancer cells transfected with the syntenin-1 gene showed increased migratory activity, whereas migration was decreased in cells in which syntenin-1 was knock-down using small inhibitory RNA. In addition, syntenin-1 expression potentiated colon cancer cell migration induced by syndecan-2, and syndecan-2-mediated cell migration was reduced when syntenin-1 expression diminished. However, syntenin-1-mediated migration enhancement was not noted in colon cancer cells transfected with a gene encoding a syndecan-2 mutant lacking the cytoplasmic domain. Furthermore, in line with the increase in cell migration, syntenin-1 mediated Rac activation stimulated by syndecan-2. Together, the data suggest that the cytoplasmic domain of syndecan-2 regulates colon cancer cell migration via interaction with syntenin-1.     

Syndecan-4 promotes the retention of phosphatidylinositol 4,5-bisphosphate in the plasma membrane

Although phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates syndecan-4 function, the potential influence of syndecan-4 on PIP₂ remains unknown. GFP containing PIP₂-binding-PH domain of phospholipase Cδ (GFP-PHδ) was used to monitor PIP₂. Syndecan-4 overexpression in COS-7 cells enhanced membrane translocation of GFP-PHδ, while the opposite was observed when syndecan-4 was knocked-down. PIP₂ levels were higher in total phospholipids extracted from rat embryo fibroblasts expressing syndecan-4. Syndecan-4-induced membrane targeting of GFP-PHδ was further enhanced by phosphoinositide-3-kinase inhibitor, but not by phospholipase C (PLC) inhibitor. Besides, both ionomycin and epidermal growth factor caused dissociation of GFP-PHδ from plasma membrane, an effect that was significantly delayed by syndecan-4 over-expression. Collectively, these data suggest that syndecan-4 promotes plasma membrane retention of PIP₂ by negatively regulating PLC-dependent PIP₂ degradation.     

Molecular interplays involved in the cellular uptake of octaarginine on cell surfaces and the importance of syndecan-4 cytoplasmic V domain for the activation of protein kinase Cα

Arginine-rich cell-penetrating peptides (CPPs) are promising carriers for the intracellular delivery of various bioactive molecules. However, many ambiguities remain about the molecular interplays on cell surfaces that ultimately lead to endocytic uptake of CPPs. By treatment of cells with octaarginine (R8), enhanced clustering of syndecan-4 on plasma membranes and binding of protein kinase Cα (PKCα) to the cytoplasmic domain of syndecan-4 were observed; these events potentially lead to the macropinocytic uptake of R8. The cytoplasmic V domain of syndecan-4 made a significant contribution to the cellular uptake of R8, whereas the cytoplasmic C1 and C2 domains were not involved in the process.     

Syndecan-1 expression in epithelial cells is induced by transforming growth factor beta through a PKA-dependent pathway

Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans bind and modulate a wide variety of biological molecules through their heparan sulfate (HS) moiety. Although all syndecans contain the ligand binding HS chains, they likely perform specific functions in vivo because their temporal and spatial expression patterns are different. However, how syndecan expression is regulated has yet to be clearly defined. In this study, we examined how syndecan-1 expression is regulated in epithelial cells. Our results showed that among several bioactive agents tested, only forskolin and three isoforms of TGFbeta (TGFbeta1-TGFbeta3) significantly induced syndecan-1, but not syndecan-4, expression on various epithelial cells. Steady-state syndecan-1 mRNA was not increased by TGFbeta treatment and cycloheximide did not inhibit syndecan-1 induction by TGFbeta, indicating that TGFbeta induces syndecan-1 in a post-translational manner. However, TGFbeta induction of syndecan-1 was inhibited by transient expression of a dominant-negative construct of protein kinase A (PKA) and by specific inhibitors of PKA. Further (i) syndecan-1 cytoplasmic domains were Ser-phosphorylated when cells were treated with TGFbeta and this was inhibited by a PKA inhibitor, (ii) PKA was co-immunoprecipitated from cell lysates by anti-syndecan-1 antibodies, (iii) PKA phosphorylated recombinant syndecan-1 cytoplasmic domains in vitro, and (iv) expression of a syndecan-1 construct with its invariant Ser(286) replaced with a Gly was not induced by TGFbeta. Together, these findings define a regulatory mechanism where TGFbeta signals through PKA to phosphorylate the syndecan-1 cytoplasmic domain and increases syndecan-1 expression on epithelial cells.     

PKCgamma regulates syndecan-2 inside-out signaling during xenopus left-right development

The transmembrane proteoglycan syndecan-2 cell nonautonomously regulates left-right (LR) development in migrating mesoderm by an unknown mechanism, leading to LR asymmetric gene expression and LR orientation of the heart and gut. Here, we demonstrate that protein kinase C gamma (PKCgamma) mediates phosphorylation of the cytoplasmic domain of syndecan-2 in right, but not left, animal cap ectodermal cells. Notably, both phosphorylation states of syndecan-2 are obligatory for normal LR development, with PKCgamma-dependent phosphorylated syndecan-2 in right ectodermal cells and nonphosphorylated syndecan-2 in left cells. The ectodermal cells contact migrating mesodermal cells during early gastrulation, concurrent with the transmission of LR information. This precedes the appearance of monocilia and is one of the earliest steps of LR development. These results demonstrate that PKCgamma regulates the cytoplasmic phosphorylation of syndecan-2 and, consequently, syndecan-2-mediated inside-out signaling to adjacent cells.     

Synbindin, A novel syndecan-2-binding protein in neuronal dendritic spines

Dendritic spines are small protrusions on the surface of dendrites that receive the vast majority of excitatory synapses. We previously showed that the cell-surface heparan sulfate proteoglycan syndecan-2 induces spine formation upon transfection into hippocampal neurons. This effect requires the COOH-terminal EFYA sequence of syndecan-2, suggesting that cytoplasmic molecules interacting with this sequence play a critical role in spine morphogenesis. Here, we report a novel protein that binds to the EFYA motif of syndecan-2. This protein, named synbindin, is expressed by neurons in a pattern similar to that of syndecan-2, and colocalizes with syndecan-2 in the spines of cultured hippocampal neurons. In transfected hippocampal neurons, synbindin undergoes syndecan-2-dependent clustering. Synbindin is structurally related to yeast proteins known to be involved in vesicle transport. Immunoelectron microscopy localized synbindin on postsynaptic membranes and intracellular vesicles within dendrites, suggesting a role in postsynaptic membrane trafficking. Synbindin coimmunoprecipitates with syndecan-2 from synaptic membrane fractions. Our results show that synbindin is a physiological syndecan-2 ligand on dendritic spines. We suggest that syndecan-2 induces spine formation by recruiting intracellular vesicles toward postsynaptic sites through the interaction with synbindin.     

Identification of an invasion regulatory domain within the core protein of syndecan-1

Among the four members of the syndecan family there exists a high level of divergence in the ectodomain core protein sequence. This has led to speculation that these core proteins bear important functional domains. However, there is little information regarding these functions, and thus far, the biological activity of syndecans has been attributed largely to their heparan sulfate chains. We have previously demonstrated that cell surface syndecan-1 inhibits invasion of tumor cells into three-dimensional gels composed of type I collagen. Inhibition of invasion is dependent on the syndecan heparan sulfate chains, but a role for the syndecan-1 ectodomain core protein was also indicated. To more closely examine this possibility and to map the regions of the ectodomain essential for syndecan-1-mediated inhibition of invasion, a panel of syndecan-1 mutational constructs was generated, and each construct was transfected individually into myeloma tumor cells. The anti-invasive effect of syndecan-1 is dramatically reduced by deletion of an ectodomain region close to the plasma membrane. Further mutational analysis identified a stretch of 5 hydrophobic amino acids, AVAAV (amino acids 222-226), critical for syndecan-1-mediated inhibition of cell invasion. This invasion regulatory domain is 26 amino acids from the start of the transmembrane domain. Importantly, this domain is functionally specific because its mutation does not affect syndecan-1-mediated cell binding to collagen, syndecan-1-mediated cell spreading, or targeting of syndecan-1 to specific cell surface domains. This invasion regulatory domain may play an important role in inhibiting tumor cell invasion, thus explaining the observed loss of syndecan-1 in some highly invasive cancers.