FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1993894490> ?p ?o ?g. }

• W1993894490 endingPage "32163" @default.
• W1993894490 startingPage "32156" @default.
• W1993894490 abstract "Syndecans are cell surface proteoglycans involved in cell adhesion and motility. Syndecan-4 is an important component of focal adhesions and is involved in cytoskeletal reorganization. Previous work has shown that the syndecan-4 ectodomain can support cell attachment. Here, three vertebrate syndecan-4 ectodomains were compared, including that of the zebrafish, and we have demonstrated that the cell binding activity of the syndecan-4 ectodomain is conserved. Cell adhesion to the syndecan-4 ectodomain appears to be a characteristic of mesenchymal cells. Comparison of syndecan-4 ectodomain sequences led to the identification of three conserved regions of sequence, of which the NXIP motif is important for cell binding activity. We have shown that cell adhesion to the syndecan-4 ectodomain involves β1 integrins in several cell types. Syndecans are cell surface proteoglycans involved in cell adhesion and motility. Syndecan-4 is an important component of focal adhesions and is involved in cytoskeletal reorganization. Previous work has shown that the syndecan-4 ectodomain can support cell attachment. Here, three vertebrate syndecan-4 ectodomains were compared, including that of the zebrafish, and we have demonstrated that the cell binding activity of the syndecan-4 ectodomain is conserved. Cell adhesion to the syndecan-4 ectodomain appears to be a characteristic of mesenchymal cells. Comparison of syndecan-4 ectodomain sequences led to the identification of three conserved regions of sequence, of which the NXIP motif is important for cell binding activity. We have shown that cell adhesion to the syndecan-4 ectodomain involves β1 integrins in several cell types. Syndecans are type 1 transmembrane heparan sulfate proteoglycans involved in cell adhesion, spreading, and cell migration (for reviews, see Refs. 1Oh E.S. Couchman J.R. Mol. Cells. 2004; 17: 181-187PubMed Google Scholar, 2Couchman J.R. Nat. Rev. Mol. Cell Biol. 2003; 4: 926-937Crossref PubMed Scopus (332) Google Scholar, 3Tkachenko E. Rhodes J.M. Simons M. Circ. Res. 2005; 96: 488-500Crossref PubMed Scopus (354) Google Scholar, 4Couchman J.R. Chen L. Woods A. Int. Rev. Cytol. 2001; 207: 113-150Crossref PubMed Scopus (120) Google Scholar). They consist of a short, highly conserved cytoplasmic domain, a single transmembrane domain, and a longer ectodomain that bears heparan sulfate side chains. Syndecan-4 is a focal adhesion component and is involved in focal adhesion formation in conjunction with the α5β1 integrin. Fibroblasts form focal adhesions in response to fibronectin fragments containing the integrin binding (RGD-containing) and heparin binding domains. Integrin engagement is not sufficient to stimulate this process, and the heparan sulfate binding occurs through syndecan-4 (5Woods A. Couchman J.R. Johansson S. Höök M. EMBO J. 1986; 5: 665-670Crossref PubMed Scopus (322) Google Scholar, 6Woods A. Couchman J.R. Mol. Biol. Cell. 1994; 5: 183-192Crossref PubMed Scopus (276) Google Scholar, 7Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (328) Google Scholar). The cytoplasmic domain of syndecan-4 can interact with a range of proteins. As with all syndecans, the C2 region of the cytoplasmic domain of syndecan-4 contains a PDZ binding site, and PDZ proteins, such as GIPC (GAIP-interacting protein-C terminus) and syntenin, have been shown to bind (8Grootjans J.J. Zimmermann P. Reekmans G. Smets A. Degeest G. Durr J. David G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13683-13688Crossref PubMed Scopus (337) Google Scholar, 9Gao Y.H. Li M. Chen W.Z. Simons M. J. Cell. Physiol. 2000; 184: 373-379Crossref PubMed Scopus (151) Google Scholar). The dimeric syndecan-4 cytoplasmic domain is stabilized by phosphatidylinositol 4,5-bisphosphate interactions with the V region (10Lee D. Oh E.S. Woods A. Couchman J.R. Lee W. J. Biol. Chem. 1998; 273: 13022-13029Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In turn, protein kinase Cα binds and is persistently activated (11Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 12Couchman J.R. Vogt S. Lim S.T. Lim Y. Oh E.S. Prestwich G.D. Theibert A. Lee W. Woods A. J. Biol. Chem. 2002; 277: 49296-49303Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13Keum E. Kim Y. Kim J. Kwon S. Lim Y. Han I. Oh E.S. Biochem. J. 2004; 378: 1007-1014Crossref PubMed Scopus (71) Google Scholar), and syndecan-4 is able to recruit protein kinase Cα to focal adhesions (14Lim S.T. Longley R.L. Couchman J.R. Woods A. J. Biol. Chem. 2003; 278: 13795-13802Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The V region can also interact with α-actinin and syndesmos with potential linkage to the cytoskeleton (15Greene D.K. Tumova S. Couchman J.R. Woods A. J. Biol. Chem. 2003; 278: 7617-7623Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 16Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar), whereas overexpression of rat syndecan-4 results in a reduction in cell migration (17Longley R.L. Woods A. Fleetwood A. Cowling G.J. Gallagher J.T. Couchman J.R. J. Cell Sci. 1999; 112: 3421-3431Crossref PubMed Google Scholar). Evidence suggests that the syndecan-4 ectodomain may have biological activity (18McFall A.J. Rapraeger A.C. J. Biol. Chem. 1997; 272: 12901-12904Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 19McFall A.J. Rapraeger A.C. J. Biol. Chem. 1998; 273: 28270-28276Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Syndecan-4 fusion proteins expressed in prokaryotic systems support fibroblast attachment when used as substrates in adhesion assays. This activity was mapped to a 54-residue region of the murine syndecan-4 ectodomain between the GAG attachment sites and the transmembrane domain. This putative cell binding domain was shown to completely inhibit fibroblast attachment to the full-length syndecan-4 ectodomain substratum when used in competition assays (18McFall A.J. Rapraeger A.C. J. Biol. Chem. 1997; 272: 12901-12904Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 19McFall A.J. Rapraeger A.C. J. Biol. Chem. 1998; 273: 28270-28276Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Syndecan-1 ectodomain may also possess important biological activity. MDA-MB-231 mammary carcinoma cells overexpressing syndecan-1 seeded on cognate antibodies resulted in spreading in an α5β3-dependent manner after exogenous integrin activation by manganese ions (20Beauvais D.M. Rapraeger A.C. Exp. Cell Res. 2003; 286: 219-232Crossref PubMed Scopus (122) Google Scholar). This effect was observed with constructs expressing cytoplasmic domain-null and heparan sulfate-null forms of syndecan-1 and could only be compromised when amino acids 88–252 of the ectodomain had been deleted (21Beauvais D.M. Burbach B.J. Rapraeger A.C. J. Cell Biol. 2004; 167: 171-181Crossref PubMed Scopus (198) Google Scholar). Syndecan-1 ectodomain can regulate ARH77 mammary carcinoma migration into collagen gels; this effect was mapped to a five-residue sequence AVAAV in murine syndecan-1. However, when this hydrophobic sequence was deleted, only matrix invasion was affected, and syndecan-1-mediated spreading and binding was unaffected by this mutation (22Langford J.K. Yang Y. Kieber-Emmons T. Sanderson R.D. J. Biol. Chem. 2005; 280: 3467-3473Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Recently, recombinant syndecan-2 ectodomain has been shown to promote capillary tube formation in microvascular endothelial cells when incorporated into matrigel (23Fears C.Y. Gladson C.L. Woods A. J. Biol. Chem. 2006; 281: 14533-14536Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Here we have shown that the cell adhesion activity of the syndecan-4 ectodomain is conserved across vertebrate evolution and that adhesion activity is restricted to mesenchymal and leukocytic cells. Epithelial cell lines appear not to interact with this protein. We have also identified a conserved NXIP motif within the syndecan-4 ectodomain that is important for cell adhesion. Finally, we have also shown evidence that the cellular response to the syndecan-4 ectodomain involves β1 integrins. Cell Culture—Rat embryo fibroblasts (REFs) 2The abbreviations used are: REF, rat embryo fibroblast; FCS, fetal calf serum; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate. and syndecan-4 null fibroblasts (24Ishiguro K. Kadomatsu K. Kojima T. Muramatsu H. Tsuzuki S. Nakamura E. Kusugami K. Saito H. Muramatsu T. J. Biol. Chem. 2000; 275: 5249-5252Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) were routinely grown and maintained in α minimal essential medium (Cambrex) in the presence of 5% fetal calf serum (FCS). Swiss 3T3, Madin-Darby canine kidney cells, T47D, Ovcar3, mouse skin endothelial cells, MCF7, and COS7 cells were grown in Dulbecco's modified Eagle's medium (Cambrex) supplemented with 10% FCS. HEK293 and Jurkat cells were cultured in Dulbecco's modified Eagle's/F-12 (Invitrogen) media with 10% FCS, whereas K562 were grown in RPMI 1640 medium (Invitrogen) with 5% FCS. Cloning of Zebrafish Syndecan-4—Plasmids and primers are listed in supplemental Table S1. The expressed sequence tag clone IMAGp998K2410802Q3 corresponding to the zebrafish homologue of syndecan-4 was obtained from Deutsches Ressourcenzentrum für Genomschung GmbH (Berlin, Germany) and sequenced by conventional methods. The 5′-end of zebrafish syndecan-4 cDNA was obtained using 5′-RACE (Invitrogen). Briefly, zebrafish syndecan-4 cDNA was prepared by reverse transcription using primer ZebSDC4rev, and this was used as a template in sequential PCR reactions, using primer Zeb4gsp1 in the primary PCR and Zeb4gsp2 in the secondary PCR. The blunt-ended PCR products were phosphorylated using T4 polynucleotide kinase (Invitrogen) and ligated into the EcoRV site of pBluescript II KS(±) (Stratagene) using standard procedures. Using the newly generated 5′ sequence, the full-length gene sequence of the zebrafish syndecan-4 was amplified by reverse transcription-PCR from 24-h zebrafish embryo RNA using oligo(dT) and PCR primers Zeb4PstI and Zeb4BamHI. The resultant PCR product was digested with BamHI and PstI and ligated into the corresponding sites of pBluescript II KS(±) to construct the pBSZebSDC4pr plasmid. Glutathione S-Transferase (GST) Ectodomain Fusion Proteins; Construct Generation, Protein Expression, and Purification—Plasmids containing the full-length syndecan-4 cDNAs from human, mouse, and zebrafish were used as templates for PCR. The human, mouse, and zebrafish ectodomain sequences were amplified using the primer pairs S4petA and S4petB, MouEDfor and MouEDrev, and ZebEDfor and ZebEDrev, respectively. PCR products were kinased and digested with BamHI prior to ligation into the PshAI and BamHI sites of the bacterial expression vector pET41(a+) (Novagen). Human syndecan-4 ectodomain mutants were made using a PCR-based approach. Primer pairs SNKVSMfor and SNKVSMrev, NEVfor and NEVrev, or NHIPfor and NHIPrev were used to amplify linear pGSThS4ED. The primer positions were designed such that the PCR product from each respective primer pair would lack the coding sequence for the amino acid sequences 125SNKVSM130, 109NEV111, or 87NHIP90. PCR products were digested with DpnI and column-purified (Qiagen) prior to ligation and transformation. Successful deletion was verified by DNA sequencing. Alanine-scanning mutations of the NHIP motif were prepared using a similar method. pGSThS4ED-NHIP was used as the PCR template, and primer NHIPrev was used with primers NHIPN-A, NHIPH-A, NHIPI-A, and NHIPP-A to yield plasmids pGSThS4EDN-A, pGSThS4EDH-A, pGSThS4EDI-A, and pGSThS4EDP-A. GST fusion proteins containing either the cell binding domain (Pro76–Ser152) or the remaining N-terminal fragment (Glu24–Gly75) of the syndecan-4 ectodomain were made as follows. Human syndecan-4 sequence coding from Pro76–Ser152 was PCR-amplified using primers CBDa and CBDb and cloned into the PshAI and BamHI sites of pET41(a+) to make the plasmid pGSTHS4EDcbd. Using the same PCR mutagenesis protocol as above, a stop codon was introduced into the human syndecan-4 ectodomain coding sequence after Gly75. pGSTHS4ED was used as the template in conjunction with primers S4ectfrfor and S4ectfrrev, and the resultant PCR product was digested and ligated as above to produce pGSTHS4EDfr. Plasmids were transformed into the Escherichia coli BL21 strain (Promega), and the bacteria were grown to an A600 of 0.6 at 37 °C prior to the addition of 0.25 ml/liter 1 m isopropyl 1-thio-β-d-galactopyranoside (Calbiochem) followed by a further 3-h incubation. Bacteria were resuspended in phosphate-buffered saline (PBS), lysed in a sonicator, and GST syndecan-4 ectodomain fusion proteins were purified on columns of glutathione-Sepharose 4B (GE Healthcare) as described in the manufacturer's protocol. Cell Adhesion Assay—Adhesion assays were performed using 24-well plates. The wells were coated either with 0.25 ml of the various syndecan-4 ectodomain fusion proteins at concentrations up to 10 μg/ml or with 5% bovine serum albumin (BSA) overnight at 4 °C, and human plasma fibronectin (10 μg/ml) was purified according the method described in Ref. 25Miekka S.I. Ingham K.C. Menache D. Thromb. Res. 1982; 27: 1-14Abstract Full Text PDF PubMed Scopus (246) Google Scholar. The wells were washed once with PBS and blocked using 1% w/v BSA for 1 h at 37°C followed by two washes with PBS. Cells were detached using trypsin/EDTA and suspended in serum-containing medium. The cells were sedimented and resuspended in serum-free medium to a concentration of 2.5 × 105 cells/ml, and 0.2 ml of cell suspension/well was used in the assay. The non-adherent cell lines K562 and Jurkat were incubated for 15 min in serum-free medium containing 10 ng/ml phorbol 12-myristate 13-acetate (PMA) prior to seeding on substrates. The blocking antibodies AIIb2, Ha2/5 (Chemicon), and LM609 (Chemicon) were used at a concentration of 10 μg/ml; REF, Swiss 3T3, COS7, and Jurkat cells (PMA-treated as above) were incubated at 4 °C for 30 min in serum-free medium in the presence or absence of antibody and then seeded on substrate at 37 °C. Unless stated otherwise, the cells were allowed to adhere for 1 h at 37 °C, after which time the medium was removed and replaced with serum-free medium containing 15 μm calcein (Invitrogen) and incubated for a further 20 min. The wells were washed twice with PBS, and cell-bound fluorescence was measured using a Fluostar Galaxy Plate reader (BMG Lab Technologies) at 485 nm excitation and 520 nm emission. Immunofluorescence Microscopy—Coverslips were coated overnight with the appropriate substrate, and cells were seeded in serum-free conditions and allowed to spread for 1 h. The cells were then fixed for 20 min in 4% paraformaldehyde in PBS followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min. Thereafter, the cells were stained using conventional procedures with AlexaFluor 568-conjugated phalloidin (Molecular Probes) for F-actin and to detect focal adhesions, anti-vinculin (Sigma) followed by AlexaFluor 488-conjugated goat anti-mouse IgG (Molecular Probes) was used. The samples were analyzed on a Provis AX module fluorescence microscope (Olympus; objectives were UPlanApo 40 × 1.0 oil iris and UPlanApo 60 × 1.4 oil). Images were collected using a SPOT Insight Mono digital camera and processed in Adobe Photoshop. Zebrafish Gene Cloning and Sequencing—To compare ectodomain properties across a spectrum of vertebrates, the zebrafish syndecan-4 was identified from a BLAST search of the I.M.A.G.E. Consortium (LLNL) zebrafish cDNA clone collection with the human syndecan-4 sequence. Clone IMAGp998K2410802Q3 contained a partial sequence of the zebrafish syndecan-4 homologue. The clone was truncated at the 5′-end, and 5′-RACE was used to obtain the entire zebrafish SDC4 (zSDC4) mRNA sequence (Fig. 1A), which is deposited in the EMBL data base under accession number AM260521. zSDC4 has a predicted 17-amino-acid signal sequence principally comprising hydrophobic residues. The N terminus of the mature protein is well conserved with other syndecan-4 sequences, as is the frequency and distribution of the three potential GAG substitution sites. Consistent with other syndecan sequences, there is little sequence homology between the ectodomains of syndecan-4 proteins across the vertebrates (Fig. 1B); however, the transmembrane and cytoplasmic domains of zSDC4 share considerable homology with syndecan-4 sequences from other species (Fig. 1C). Divergent Vertebrate Syndecan-4 Ectodomains Support Mesenchymal Cell Adhesion—Bacterially expressed mouse syndecan-4 ectodomain can support cell adhesion when used as a substratum for attachment assays (18McFall A.J. Rapraeger A.C. J. Biol. Chem. 1997; 272: 12901-12904Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 19McFall A.J. Rapraeger A.C. J. Biol. Chem. 1998; 273: 28270-28276Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). GST fusion proteins containing the ectodomains of human (GSThS4ED), mouse (GSTmS4ED), and zebrafish (GSTzS4ED) syndecan-4 were prepared (Fig. 2, A and B). In each case, the ectodomain protein did not include either the signal sequence or the four amino acids immediately proximal to the transmembrane domain (Fig. 2A). Cleavage of the GST from the fusion proteins was unsuccessful, because syndecan-4 ectodomains aggregate to form insoluble complexes. The GST fusion proteins, however, remained dimeric and soluble (Fig. 2B). Cell attachment assays were performed using the three GST ectodomains, human plasma fibronectin, GST, and BSA, as substrates. REFs attached well to fibronectin and failed to adhere to either BSA or GST. As reported previously (18McFall A.J. Rapraeger A.C. J. Biol. Chem. 1997; 272: 12901-12904Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), the GSTmS4ED also supported cell attachment and equivalent activity was observed for the human and zebrafish ectodomains (Fig. 2C). This effect was not restricted to REFs, because Swiss 3T3 and mouse skin endothelial cells also exhibited similar levels of attachment to each syndecan-4 ectodomain. Further analysis revealed that, although a number of mesenchymal cell lines adhered to syndecan-4 ectodomain, all of the epithelial cell lines (except endothelial cells) failed to do so (Fig. 2C and Table 1). Leukocytic cell lines were variable; Jurkat cells adhered to the syndecan-4 ectodomains after PMA stimulation, whereas K562 cells were unresponsive.TABLE 1Attachment to GSTS4ED species is restricted to mesenchymal and leukocytic cells. Adhesion assays were performed on cells seeded on the various substrata (10 μg/ml) as describedCell lineMorphologyAdhesion on fibronectinAdhesion on GSThS4EDAdhesion on GSTmS4EDAdhesion on GSTzS4EDREFFibroblastic✓✓✓✓COS7Fibroblastic✓✓✓✓MEFFibroblastic✓✓✓✓S4KOMef (-/-)Fibroblastic✓✓✓✓Swiss3T3Fibroblastic✓✓✓✓SendEndothelial✓✓✓✓K562Lymphoblastic✓aAfter PMA stimulation.XaAfter PMA stimulation.XaAfter PMA stimulation.XaAfter PMA stimulation.JurkatLymphoblastic✓aAfter PMA stimulation.✓aAfter PMA stimulation.✓aAfter PMA stimulation.✓aAfter PMA stimulation.CHOKIEpithelial✓XXXMDCKEpithelial✓XXXT47DEpithelial✓XXXOvcarEpithelial✓XXXHEK293Epithelial✓XXXMCF7Epithelial✓XXXa After PMA stimulation. Open table in a new tab Cell Spreading in Response to the Syndecan-4 Ectodomain—It was evident from the attachment assays that cells not only attached to the different syndecan-4 ectodomains but could also spread in response to these substrates. Fibroblasts, including REFs and Swiss 3T3 cells, all showed a similar spread morphology after 1 h of adhesion to the three forms of the syndecan-4 ectodomain (Fig. 3A). Spreading was not as extensive as that observed when REFs were seeded on fibronectin, whereas cells failed to spread on either BSA or GST (Fig. 3A). REFs did not organize actin stress fibers when spread on the ectodomain species. The actin was more cortical in organization, in contrast to fibronectin-mediated adhesion (Fig. 3B). In addition, REFs did not form focal adhesions during spreading on any form of the syndecan-4 ectodomain (Fig. 3C and data not shown). Vinculin-containing focal adhesions were observed in cells spread under equivalent conditions on fibronectin. In further experiments, it was demonstrated that allowing increased time for adhesion did not result in focal adhesion acquisition on syndecan-4 ectodomains (data not shown). The NXIP Motif Is Important for Syndecan-4 Ectodomain Activity—Syndecan-4 ectodomain from mammals and fish elicited similar responses from cells when used as substrata in adhesion assays. In turn, this suggested the presence of conserved features within the syndecan-4 ectodomain sequence supporting cell adhesion. Previous work had mapped the cell binding activity of the murine S4ED to a 54-amino-acid region between the GAG attachment sites and transmembrane domain. Consistent with this, a GST fusion protein containing the equivalent sequence of human syndecan-4 supported REF cell attachment. A second fusion protein containing the remaining N-terminal portion of the ectodomain did not support cell adhesion (supplemental Fig. S2, A and B). A multiple alignment of the equivalent regions from all syndecan-4 sequences (Fig. 1B) revealed little sequence homology between species. Three small regions were identifiable as conserved, NXIP, NEV, and SNKVSM. Using the human syndecan-4 ectodomain (GSThS4ED) as the template, mutants with in-frame deletions of these three putative motifs were prepared (Fig. 4A). REF cell attachment at substrate-coating concentrations (>10 μg/ml) were essentially identical (Fig. 4B). However, at lower concentrations, cell attachment and spreading were much reduced in response to the mutant protein in which the NXIP motif had been deleted (Fig. 4, B and C). GSThS4ED lacking either the NEV or SNKVSM motif supported normal levels of REF cell attachment. To ensure that the wild-type and deletion mutant fusion proteins coated the plates with equal efficiency, enzyme-linked immunosorbent assay (using an antibody to GST) showed that all of the proteins coated the tissue culture plastic equivalently (supplemental Fig. S3A). There was also no evidence to suggest that GSThS4EDΔNHIP was any more unstable than the wild-type fusion protein (Fig. 4A). No further compromise of cell attachment or spreading on the GSThS4EDΔNHIP was obtained from additional deletions of the NEV conserved motif or the 4-amino-acid proline-rich region immediately preceding the NHIP sequence. Neither substrate showed any reduction in cell attachment properties (supplemental Fig. S3B). Identical results to REFs in terms of adhesion were seen with Swiss 3T3 and Jurkat cells to each of these deletion mutants (data not shown). To further characterize the NXIP motif, mutants of GSThS4ED were prepared in which each residue of the motif were substituted with an alanine residue (Fig. 5A). Of these single amino acid substitutions, the Ile→Ala mutant alone showed compromised cell attachment and spreading properties (Fig. 5, C and D). As described previously, all of the alanine scanning mutants had similar coating properties to wild-type GST syndecan-4 ectodomain fusion protein (supplemental Fig. S3C), and these mutants were as stable as the wild-type protein (Fig. 5B). Cell Adhesion to the Syndecan-4 Ectodomain Is Integrin-dependent—Cell adhesion and spreading responses to the syndecan ectodomain could have resulted from association between the exogenous recombinant syndecan-4 protein and endogenously expressed syndecan-4. However, because syndecan-4 null fibroblasts adhered to all three forms of syndecan-4 ectodomain fusion protein, this was effectively ruled out. As shown in Fig. 3C, cells seeded on the syndecan-4 ectodomain spread with actin organized in a cortical distribution. This was suggestive of integrin involvement and was further supported by the use of EDTA, where REF failed to attach to the human form of the syndecan-4 ectodomain (Fig. 6A). Because Jurkat cell attachment to syndecan-4 ectodomains required phorbol ester pretreatment (Fig. 6B and Table 1), this also suggested a role for integrins. Integrin involvement was confirmed with the β1 integrin-blocking antibodies AIIb2, which inhibited PMA-induced adhesion of Jurkat cells to GSThS4ED (Fig. 6B) and Ha2/5 and which compromised REF and Swiss 3T3 fibroblast attachment (Fig. 6C). The Ha2/5 antibody also compromised REF attachment to the GST fusion protein containing only the cell binding domain of syndecan-4 (supplemental Fig. S2C). The αVβ3 integrin-blocking antibody LM609 had no effect on the adhesion of COS7 cells to GSThS4ED but did block adhesion to serumcoated substrate (Fig. 6D). Syndecan ectodomains are highly divergent in primary sequence, although there are small regions of homology among syndecan-4 sequences, including sites of glycosaminoglycan substitution. One of the most obvious roles for the syndecan ectodomain is to support and orient heparan sulfate and, where present, chondroitin sulfate chains. Syndecan-4 ectodomain is the only syndecan family member where cell binding activity has been observed when used as a substrate in adhesion assays (18McFall A.J. Rapraeger A.C. J. Biol. Chem. 1997; 272: 12901-12904Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 19McFall A.J. Rapraeger A.C. J. Biol. Chem. 1998; 273: 28270-28276Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We have shown here that it is a conserved feature of the syndecan-4 ectodomain being shared almost identically in terms of adhesion and spreading characteristics between zebrafish, mouse, and human versions. Although the actin cytoskeleton of fibroblasts adherent to the syndecan-4 ectodomain is well organized, this type of adhesion does not culminate in the formation of focal adhesions. Analysis of the syndecan-4 ectodomain sequences revealed three small motifs common across the vertebrates. Of these, only one (the NXIP motif) appears to be important, because its deletion compromises both attachment and spreading of fibroblasts. However, this may be a complex situation, because deletion of the NXIP motif from the human syndecan-4 ectodomain did not ablate cell attachment but merely reduced it. Consistent with this was the finding from alanine-scanning mutations that the isoleucine, although critical, was the only residue identified by single amino acid substitutions to affect cell attachment to the syndecan-4 ectodomain. Quite likely, there are surrounding sequences that may represent a secondary site for adhesion, or there may be a more distant site not yet identified. Future structural work should provide information on the orientation of this NXIP motif and its relationships to other features of syndecan structure. Although the syndecan-1 and -2 ectodomains also carry cell adhesion sites, the NXIP motif is not common with these and, as such, may have distinct roles. In this regard, the finding that only fibroblast and some leukocytic cells are able to adhere to the syndecan-4 ectodomain is of interest. Syndecan-4, although abundant in these cell types, is not restricted to them and is also found in epithelial cells. However, the ectodomain does not promote epithelial cell adhesion, with the exception of vascular endothelial cells. In contrast, syndecan-1 ectodomain promotes epithelial cell adhesion mediated by integrins (20Beauvais D.M. Rapraeger A.C. Exp. Cell Res. 2003; 286: 219-232Crossref PubMed Scopus (122) Google Scholar). It has been shown here that cell adhesion to the syndecan-4 ectodomain is sensitive to the blockade of the β1 integrin but not αVβ3 integrin. This is consistent with experiments showing the requirement for divalent cations as well as the finding that Jurkat adhesion to the syndecan-4 ectodomain requires activation of protein kinase C through phorbol ester treatment. This is a known property of Jurkat integrins (26Faull R.J. Kovach N.L. Harlan J.M. Ginsberg M.H. J. Exp. Med. 1994; 179: 1307-1316Crossref PubMed Scopus (134) Google Scholar). Once again, however, it reveals that adhesion to syndecan-4 ectodomain through integrins may be complex, because many cell types, including epithelial cells, possess β1 integrins. Altogether this suggests that mesenchymal cells and Jurkat cells possess an intermediate that is responsible for the bridging between syndecan-4 and the β1 integrin. The identity of this intermediate remains unknown but could be predicted to be absent in epithelial cells. In the case of syndecan-1 ectodomain, MDA-MB-231 cell adhesion is dependent on the αVβ3 integrin, whereas in B82L fibroblasts, adhesion is dependent on the αVβ5 integrin (21Beauvais D.M. Burbach B.J. Rapraeger A.C. J. Cell Biol. 2004; 167: 171-181Crossref PubMed Scopus (198) Google Scholar, 27McQuade K.J. Beauvais D.M. Burbach B.J. Rapraeger A.C. J. Cell Sci. 2006; 15: 2445-2456Crossref Scopus (89) Google Scholar). Because the adhesion of fibroblasts to syndecan-4 ectodomains is a well conserved function, demonstrable in lower vertebrates, this appears to be an important function of syndecan-4 worthy of further exploration. It may, in part, explain the special relationship that syndecan has with integrins in adhesion to extracellular molecules such as fibronectin. Of the four vertebrate syndecan family members, only syndecan-4 has the ability to become incorporated into focal adhesions, but it is not yet understood whether this involves the ectodomain site identified here. The present experiments report on the activity of syndecan-4 in trans, a feature that has also been suggested to occur in Xenopus development with respect to syndecan-2 (28Kramer K.L. Yost H.J. Dev. Cell. 2002; 2: 115-124Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). So far, syndecan-4 regulation of focal adhesion formation has been suggested to be in cis but should now be further investigated. We thank Drs. N. Vargesson and B. Leitinger of Imperial College for the zebrafish RNA and the T47-D, Ovcar-3, HEK293, and Jurkat cell lines. In addition, we are grateful to Prof. T. Muramatsu and Dr. K. Ishiguro of Nagoya University for syndecan-4 null mice and to Dr. A. Woods (University of Alabama at Birmingham) for establishing fibroblast cultures from them. Download .pdf (.27 MB) Help with pdf files" @default.
• W1993894490 created "2016-06-24" @default.
• W1993894490 creator A5051382096 @default.
• W1993894490 creator A5089583715 @default.
• W1993894490 date "2006-10-01" @default.
• W1993894490 modified "2023-09-29" @default.
• W1993894490 title "A Conserved NXIP Motif Is Required for Cell Adhesion Properties of the Syndecan-4 Ectodomain" @default.
• W1993894490 cites W1529514920 @default.
• W1993894490 cites W1590696423 @default.
• W1993894490 cites W1972287742 @default.
• W1993894490 cites W1973064894 @default.
• W1993894490 cites W1973603507 @default.
• W1993894490 cites W1974604649 @default.
• W1993894490 cites W1984484904 @default.
• W1993894490 cites W1988176449 @default.
• W1993894490 cites W1993440705 @default.
• W1993894490 cites W1996549773 @default.
• W1993894490 cites W1998715260 @default.
• W1993894490 cites W2013762859 @default.
• W1993894490 cites W2038279507 @default.
• W1993894490 cites W2045891730 @default.
• W1993894490 cites W2054280794 @default.
• W1993894490 cites W2064813013 @default.
• W1993894490 cites W2065156837 @default.
• W1993894490 cites W2072871114 @default.
• W1993894490 cites W2073355679 @default.
• W1993894490 cites W2097509210 @default.
• W1993894490 cites W2122306511 @default.
• W1993894490 cites W2123128528 @default.
• W1993894490 cites W2125240547 @default.
• W1993894490 cites W2126652156 @default.
• W1993894490 cites W2141941210 @default.
• W1993894490 cites W2148230934 @default.
• W1993894490 cites W2168535042 @default.
• W1993894490 cites W2171091522 @default.
• W1993894490 cites W34966227 @default.
• W1993894490 doi "https://doi.org/10.1074/jbc.m605553200" @default.
• W1993894490 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16936286" @default.
• W1993894490 hasPublicationYear "2006" @default.
• W1993894490 type Work @default.
• W1993894490 sameAs 1993894490 @default.
• W1993894490 citedByCount "45" @default.
• W1993894490 countsByYear W19938944902012 @default.
• W1993894490 countsByYear W19938944902013 @default.
• W1993894490 countsByYear W19938944902014 @default.
• W1993894490 countsByYear W19938944902015 @default.
• W1993894490 countsByYear W19938944902016 @default.
• W1993894490 countsByYear W19938944902018 @default.
• W1993894490 countsByYear W19938944902020 @default.
• W1993894490 countsByYear W19938944902021 @default.
• W1993894490 countsByYear W19938944902022 @default.
• W1993894490 countsByYear W19938944902023 @default.
• W1993894490 crossrefType "journal-article" @default.
• W1993894490 hasAuthorship W1993894490A5051382096 @default.
• W1993894490 hasAuthorship W1993894490A5089583715 @default.
• W1993894490 hasBestOaLocation W19938944901 @default.
• W1993894490 hasConcept C1009742 @default.
• W1993894490 hasConcept C107038049 @default.
• W1993894490 hasConcept C138885662 @default.
• W1993894490 hasConcept C1491633281 @default.
• W1993894490 hasConcept C170493617 @default.
• W1993894490 hasConcept C185592680 @default.
• W1993894490 hasConcept C32276052 @default.
• W1993894490 hasConcept C54355233 @default.
• W1993894490 hasConcept C65001120 @default.
• W1993894490 hasConcept C70721500 @default.
• W1993894490 hasConcept C78458016 @default.
• W1993894490 hasConcept C85789140 @default.
• W1993894490 hasConcept C86803240 @default.
• W1993894490 hasConcept C95444343 @default.
• W1993894490 hasConceptScore W1993894490C1009742 @default.
• W1993894490 hasConceptScore W1993894490C107038049 @default.
• W1993894490 hasConceptScore W1993894490C138885662 @default.
• W1993894490 hasConceptScore W1993894490C1491633281 @default.
• W1993894490 hasConceptScore W1993894490C170493617 @default.
• W1993894490 hasConceptScore W1993894490C185592680 @default.
• W1993894490 hasConceptScore W1993894490C32276052 @default.
• W1993894490 hasConceptScore W1993894490C54355233 @default.
• W1993894490 hasConceptScore W1993894490C65001120 @default.
• W1993894490 hasConceptScore W1993894490C70721500 @default.
• W1993894490 hasConceptScore W1993894490C78458016 @default.
• W1993894490 hasConceptScore W1993894490C85789140 @default.
• W1993894490 hasConceptScore W1993894490C86803240 @default.
• W1993894490 hasConceptScore W1993894490C95444343 @default.
• W1993894490 hasIssue "43" @default.
• W1993894490 hasLocation W19938944901 @default.
• W1993894490 hasOpenAccess W1993894490 @default.
• W1993894490 hasPrimaryLocation W19938944901 @default.
• W1993894490 hasRelatedWork W1510886429 @default.
• W1993894490 hasRelatedWork W1554961657 @default.
• W1993894490 hasRelatedWork W1993894490 @default.
• W1993894490 hasRelatedWork W2025806834 @default.
• W1993894490 hasRelatedWork W2065156837 @default.
• W1993894490 hasRelatedWork W2070467043 @default.
• W1993894490 hasRelatedWork W2077938409 @default.
• W1993894490 hasRelatedWork W2129273432 @default.
• W1993894490 hasRelatedWork W253058985 @default.
• W1993894490 hasRelatedWork W319757775 @default.

• next