Vitronectin's Basic Domain is a Syndecan Ligand which Functions in transto Regulate Vitronectin Turnover

During the process of tissue remodeling, vitronectin (Vn) is deposited in the extracellular matrix where it plays a key role in the regulation of pericellular proteolysis and cell motility. In previous studies we have shown that extracellular levels of vitronectin are controlled by receptor-mediated endocytosis and that this process is dependent upon vitronectin binding to sulfated proteoglycans. We have now identified vitronectin's 12 amino acid “basic domain” which is contained within the larger 40 amino acid heparin binding domain, as a syndecan binding site. Recombinant vitronectins representing wild type vitronectin (rVn) and vitronectin with the basic domain deleted (rVnΔ347–358) were prepared in a baculoviral expression system. The rVn as well as a glutathione S-transferase (GST) fusion protein, consisting of vitronectin's 40 amino acid heparin binding domain (GST-VnHBD), exhibited dose dependent binding to HT-1080 cell surfaces, which was attenuated following deletion of the basic domain. In addition, GST-VnHBD supported both HT-1080 and dermal fibroblast cell adhesion, which was also dependent upon the basic domain. Similarly, ARH-77 cells transfected with syndecans -1, -2, or -4, but not Glypican-1, adhered to GST-VnHBD coated wells, while adhesion of these same cells was lost following deletion of the basic domain. HT-1080 cells were unable to degrade rVnΔ347–358. Degradation of rVnΔ347–358 was completely recovered in the presence of GST-VnHBD but not in the presence of GST-VnHBDΔ347–358. These results indicate that turnover of soluble vitronectin requires ligation of vitronectin's basic domain and that this binding event can work in transto regulate vitronectin degradation.     

Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin. It inhibits the adhesion of U937 cells to vitronectin by competing with the urokinase receptor (uPAR; CD87) on these cells for binding to the same domain. Although the inhibitor also blocks integrin-mediated cell adhesion, the molecular basis of this effect is unclear. In this study, the effect of the inhibitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 blocked the adhesion of all of these cells to vitronectin-coated wells, it did not block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recombinant SMB (which does not contain the RGD sequence), and this adhesion was again blocked by the inhibitor. These results affirm that PAI-1 can inhibit both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recombinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events.     

Plasminogen activator inhibitor type I stabilizes vitronectin-dependent adhesions in HT-1080 cells

Polyclonal antibodies against plasminogen activator inhibitor type-I (PAI-1) caused rapid retraction and rounding of substrate-attached HT- 1080 cells. The kinetics and extent of antibody-mediated cell rounding were not dependent on either urokinase or plasmin activity. Cells adherent to vitronectin-coated substrates detached within 2 h of antibody addition. Cells adherent to fibronectin were unaffected by the antibodies. Immunoblotting of substrate-attached material indicated that HT-1080 cells deposited PAI-1 into vitronectin, but not fibronectin, dependent contacts. These data suggest that the antibody- mediated cell rounding resulted from a steric disruption of vitronectin- dependent adhesions, indicating that the binding site on vitronectin for PAI-1 is near, but does not overlap, the binding site for vitronectin receptor. The accumulation of PAI-1 into vitronectin- dependent adhesion sites correlated temporally with the preferential degradation of fibronectin from the substrate. HT-1080 cells adherent to either fibronectin or vitronectin were able to activate exogenous plasminogen to plasmin. Plasmin levels were increased 200% on cells adherent to fibronectin and 100% on cells adherent to vitronectin. In the presence of a neutralizing antibody against PAI-1, vitronectin adherent cells activated plasminogen to the same extent as fibronectin adherent cells. Plasmin levels of 200% above baseline were associated with retraction of cells from the substrate. The ability of vitronectin adherent cells to activate exogenous plasmin was completely blocked in the presence of neutralizing antibodies against urokinase. These data represent the first demonstration that vitronectin-associated PAI-1 regulates urokinase in focal contact areas.     

Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit

The large carboxy-terminal globular domain (G domain; residues 2,110-3,060) of the A chain of murine-derived laminin has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. This study was conducted to define the potential sequence(s) originating from the G domain of laminin with any of these functional activities. A series of peptides were synthesized from the G domain, termed GD peptides, each approximately 20 amino acids long and containing multiple positively charged amino acids. In direct 3H-heparin binding assays, peptides GD-1 and GD-2 bound high levels of 3H-heparin, while peptides GD-3 and GD-4 bound lower levels of 3H-heparin, and GD-5 bound essentially no 3H-heparin. The binding of 3H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1.0 M. Four of the peptides, GD-1, GD-2, GD-3, and GD-4, directly promoted the adhesion and spreading of HT-1080 human fibrosarcoma cells as well as the outgrowth of neurites from chick spinal cord and dorsal root ganglia neurons. In addition, solutions of these peptides or antibodies generated against these peptides inhibited laminin-mediated HT-1080 cell adhesion. Antibodies against the beta 1 integrin subunit inhibited HT-1080 cell adhesion and neurite outgrowth on surfaces adsorbed with peptides GD-3 and GD-4. Therefore, laminin appears to have multiple, independent sequences in the G domain that serve a similar cell adhesion promoting function for different cell types. Furthermore, these results suggest that the sequences comprising peptides GD-3 and GD-4 use an integrin as a receptor, of which the beta 1 integrin subunit is a component for these various cell types.     

Localization of urokinase type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the alphavbeta5 integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the alphavbeta5 integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either alphavbeta5 or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the beta1 integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of alphavbeta3, alphavbeta5, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the alphavbeta3 or alphavbeta5 integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.     

Vitronectin regulates the synthesis and localization of urokinase-type plasminogen activator in HT-1080 cells.

The effect of extracellular matrix composition on the location, amount, and activity of cell-associated urokinase-type plasminogen activator was tested using HT-1080 cells adherent to either fibronectin or vitronectin. Specific immunoprecipitation of newly synthesized urokinase indicated that cells adherent to fibronectin synthesized 2-3-fold more urokinase than cells adherent to vitronectin. Complexes of urokinase and plasminogen activator inhibitor type 1 (PAI-1) were detected in cell layers of vitronectin-adherent but not fibronectin-adherent cells. Inhibition of PAI-1 using a neutralizing monoclonal antibody resulted in a 3-fold increase in urokinase enzymatic activity on vitronectin adherent cells. Urokinase activity on fibronectin adherent cells was only slightly increased following PAI-1 neutralization. Examination of both HT-1080 and normal human fibroblast cells by immunofluorescent microscopy localized urokinase-type plasminogen activator to discrete, focal areas underneath cells adherent to vitronectin. Urokinase was not detectable by immunofluorescence on cells adherent to fibronectin. The addition of exogenous prourokinase to locate urokinase receptors on adherent HT-1080 cells indicated that the focal localization of cell-surface urokinase resulted from the clustering of urokinase receptors following adhesion to vitronectin but not fibronectin-coated substrates. These results suggest that vitronectin can contribute to the control of cell-surface plasmin activity by regulating the synthesis of urokinase and directing the localization of urokinase receptors.     

Localization of Urokinase Type Plasminogen Activator to Focal Adhesions Requires Ligation of Vitronectin Integrin Receptors

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the α_vβT₅ integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the α_vβT₅ integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either α_vβT₅ or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the βT₁ integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of α_vβT₃, α_vβT₅, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the α_vβT₃ or α_vβT₅ integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell-matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.     

Induction of fibronectin matrix assembly in human fibrosarcoma cells by dexamethasone

Previous studies have suggested that the assembly of fibronectin into the extracellular matrix of cultured fibroblasts is mediated by specific matrix assembly receptors that recognize a binding site in the amino terminus of the fibronectin molecule (McKeown-Longo, P.J., and D.F. Mosher, 1985, J. Cell Biol., 100:364-374). In the presence of dexamethasone, human fibrosarcoma cells (HT-1080) acquired the ability to specifically bind exogenous plasma fibronectin and incorporate it into a detergent-insoluble extracellular matrix. Dexamethasone-induced fibronectin binding to HT-1080 cells was time dependent, dose dependent, and inhibited by cycloheximide. Saturation binding curves indicated that dexamethasone induced the appearance of 7.7 X 10(4) matrix assembly receptors per cell. The induced receptors exhibited a dissociation constant (KD) for soluble fibronectin of 5.0 X 10(-8) M. In parallel experiments, normal fibroblasts exhibited 4.1 X 10(5) receptors (KD = 5.3 X 10(-8) M) per cell. In the presence of cycloheximide, the induced fibronectin-binding activity on HT-1080 cells returned to uninduced levels within 12 h. In contrast, fibronectin-binding activity on normal fibroblasts was stable in the presence of cycloheximide for up to 54 h. The first-order rate constant (Kt = 2.07 X 10(-4) min-1) for the transfer of receptor-bound fibronectin to extracellular matrix was four- to fivefold less than that for normal fibroblasts (Kt = 1.32 X 10(-3) min-1). Lactoperoxidase-catalyzed iodination of HT-1080 monolayers indicated that a 48,000-mol-wt cell surface protein was enhanced with dexamethasone. The results from these experiments suggest that dexamethasone induces functional matrix assembly receptors on the surface of HT-1080 cells; however, the rate of incorporation of fibronectin into the matrix is much slower than that of normal fibroblasts.     

Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading

Vitronectin is a multi-functional protein found predominantly as a monomer in blood and as an oligomer in the extracellular matrix. We have dissected the minimal regions of vitronectin protein needed for effective integrin dependent cell adhesion and spreading. A fragment of vitronectin containing the RGD integrin binding site showed similar binding affinity as that of full vitronectin protein to purified integrin αvβ3 but had diminished cell adhesion and spreading function in vivo. We demonstrate that the oligomeric state of the protein is responsible for this effect. We provide compelling evidence for the involvement of the heparin binding domain of vitronectin in the oligomerization process and show that such oligomerization reinforces the activity of vitronectin in cell adhesion and spreading.

Structured summary

MINT-7905703: Vn (uniprotkb:P04004) and Vn (uniprotkb:P04004) bind (MI:0407) by molecular sieving (MI:0071)     

The uPA receptor and the somatomedin B region of vitronectin direct the localization of uPA to focal adhesions in microvessel endothelial cells.

Vitronectin is a plasma protein which can deposit into the extracellular matrix where it supports integrin and uPA dependent cell migration. In earlier studies, we have shown that the plasma protein, vitronectin, stimulates focal adhesion remodeling by recruiting urokinase-type plasminogen activator (uPA) to focal adhesion sites [Wilcox-Adelman, S. A., Wilkins-Port, C. E., McKeown-Longo, P. J., 2000. Localization of urokinase-type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell. Adhes. Commun.7, 477-490]. In the present study, we used a variety of vitronectin constructs to demonstrate that the localization of uPA to adhesion sites requires the binding of both vitronectin integrin receptors and the uPA receptor (uPAR) to vitronectin. A recombinant fragment of vitronectin containing the connecting sequence (VN(CS)) was able to support integrin-dependent adhesion, spreading and focal adhesion assembly by human microvessel endothelial cells. Cells adherent to this fragment were not able to localize uPA to focal adhesions. A second recombinant fragment containing both the amino-terminal SMB domain and the CS domain was able to restore the localization of uPA to adhesion sites. This fragment, which contains a uPAR binding site, also resulted in the localization of uPAR to adhesion sites. uPAR blocking antibodies as well as phospholipase C treatment of cells inhibited uPA localization to adhesion sites confirming a role for uPAR in this process. The SMB domain alone was unable to direct either uPAR or uPA to adhesion sites in the absence of the CS domain. Our results indicate that vitronectin-dependent localization of uPA to adhesion sites requires the sequential binding of vitronectin integrins and uPAR to vitronectin.     

Structural requirements for neural cell adhesion molecule-heparin interaction.

Two biological domains have been identified in the amino terminal region of the neural cell adhesion molecule (NCAM): a homophilic-binding domain, responsible for NCAM-NCAM interactions, and a heparin-binding domain (HBD). It is not known whether these two domains exist as distinct structural entities in the NCAM molecule. To approach this question, we have further defined the relationship between NCAM-heparin binding and cell adhesion. A putative HBD consisting of two clusters of basic amino acid residues located close to each other in the linear amino acid sequence of NCAM has previously been identified. Synthetic peptides corresponding to this domain were shown to bind both heparin and retinal cells. Here we report the construction of NCAM cDNAs with targeted mutations in the HBD. Mouse fibroblast cells transfected with the mutant cDNAs express NCAM polypeptides with altered HBD (NCAM-102 and NCAM-104) or deleted HBD (HBD-) at levels similar to those of wild-type NCAM. Mutant NCAM polypeptides purified from transfected cell lines have substantially reduced binding to heparin and fail to promote chick retinal cell attachment. Furthermore, whereas a synthetic peptide that contains both basic amino acid clusters inhibits retinal-cell adhesion to NCAM-coated dishes, synthetic peptides in which either one of the two basic regions is altered to contain only neutral amino acids do not inhibit this adhesion. These results confirm that this region of the NCAM polypeptide does indeed mediate not only the large majority of NCAM's affinity for heparin but also a significant portion of the cell-adhesion-mediating capability of NCAM.     

Vitronectin interaction with glycosaminoglycans. Kinetics, structural determinants, and role in binding to endothelial cells

Vitronectin (VN) is a high affinity heparin-binding protein. The physiological role of this binding has hitherto received little attention, and its molecular determinants are subject to controversy. In this study, we characterized vitronectin interaction with heparin, heparin analogues, bacterial extracts, and cell surface glycosaminoglycans. As assessed by (i) fluorescence assays, (ii) precipitation with heparin-Sepharose beads, or (iii) Western blotting with antibodies against VN(347-361) (the heparin-binding site), we demonstrate an exposure of the VN heparin-binding site in multimeric but not monomeric vitronectin. Through its heparin-binding site, vitronectin also bound other glycosaminoglycans and Staphylococcus aureus extracts. The kinetics of heparin binding to vitronectin were complex. After a fast association phase (tau = 0.3 s), a slow conversion of an unstable to a stable heparin-vitronectin complex (tau = 180 s) occurred. Heparin binding kinetics and transition to a stable complex were mimicked by VN(347-361), demonstrating that this area is the fully functional heparin-binding site of vitronectin. Multimeric vitronectin bound to endothelial cells. This binding was blocked by soluble heparin and was not observed when endothelial cells were pretreated with glycosaminoglycan-removing enzymes. Glycosaminoglycan-dependent interaction of endothelial cells with multimeric vitronectin might be a relevant mechanism for removal of multimeric vitronectin from plasma. Conversion of an unstable to a stable glycosaminoglycan-vitronectin complex is likely to be relevant for association with endothelial cells under flow conditions.     

The Heparin-Binding Activity of Secreted Modular Calcium-Binding Protein 1 (SMOC-1) Modulates Its Cell Adhesion Properties

Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.     

Amino acid sequence of the vitronectin receptor alpha subunit and comparative expression of adhesion receptor mRNAs

The amino acid sequence of the vitronectin receptor alpha subunit deduced from cDNA is presented. The sequence defines a 1047-amino-acid polypeptide precursor with a putative signal sequence, a large extracellular domain with several sites homologous to calcium binding sites in other proteins, a transmembrane domain, and a 32-amino-acid cytoplasmic domain. The 7-kilobase vitronectin receptor alpha subunit mRNA was found to be expressed in all cell lines examined, including endothelial cells, K562 and HEL leukemia cells, and osteosarcoma cells. In the two leukemia cell lines, the expression of the vitronectin receptor mRNA, as well as that of the fibronectin receptor, was enhanced in the presence of phorbol ester, a treatment known to increase the adhesiveness of these cells. The HEL cells were the only ones among the cell lines tested that also contained the mRNA of the platelet adhesion receptor alpha subunit, glycoprotein IIb. The expression of glycoprotein IIb was slightly enhanced by treatment of the cells with phorbol ester. These results complete the partial cDNA sequence of the vitronectin receptor alpha subunit published previously (Suzuki, S., Argraves, W. S., Pytela, R., Arai, H., Krusius, T., Pierschbacher, M. D., and Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. U.S.A., 83, 8614-8618), confirm that the vitronectin receptor, and not IIb, is expressed in endothelial cells, and show that changes in the level of its expression correlate with changes in cell adhesiveness.     

Amalgam is a ligand for the transmembrane receptor neurotactin and is required for neurotactin-mediated cell adhesion and axon fasciculation in Drosophila

Neurotactin (NRT), a member of the cholinesterase-homologous protein family, is a heterophilic cell adhesion molecule that is required for proper axon guidance during Drosophila development. In this study, we identify amalgam (AMA), a member of the immunoglobulin superfamily, as a ligand for the NRT receptor. Using transfected Schneider 2 cells and embryonic primary cultures, we demonstrate that AMA is a secreted protein. Furthermore, AMA is necessary for NRT-expressing cells both to aggregate with themselves and to associate with embryonic primary culture cells. Aggregation assays performed with truncated NRT molecules reveal that the integrity of the cholinesterase-like extracellular domain was not required either for AMA binding or for adhesion, with only amino acids 347-482 of the extracellular domain being necessary for both activities. Moreover, the NRT cytoplasmic domain is required for NRT-mediated adhesion, although not for AMA binding. Using an ama-deficient stock, we find that ama function is not essential for viability. Pupae deficient for ama do exhibit defasciculation defects of the ocellar nerves similar to those found in nrt mutants.     

The Membrane-proximal Region of the E-Cadherin Cytoplasmic Domain Prevents Dimerization and Negatively Regulates Adhesion Activity

Cadherins are transmembrane glycoproteins involved in Ca²⁺-dependent cell–cell adhesion. Deletion of the COOH-terminal residues of the E-cadherin cytoplasmic domain has been shown to abolish its cell adhesive activity, which has been ascribed to the failure of the deletion mutants to associate with catenins. Based on our present results, this concept needs revision. As was reported previously, leukemia cells (K562) expressing E-cadherin with COOH-terminal deletion of 37 or 71 amino acid residues showed almost no aggregation. Cells expressing E-cadherin with further deletion of 144 or 151 amino acid residues, which eliminates the membrane-proximal region of the cytoplasmic domain, showed E-cadherin–dependent aggregation. Thus, deletion of the membrane-proximal region results in activation of the nonfunctional E-cadherin polypeptides. However, these cells did not show compaction. Chemical cross-linking revealed that the activated E-cadherin polypeptides can be cross-linked to a dimer on the surface of cells, whereas the inactive polypeptides, as well as the wild-type E-cadherin polypeptide containing the membrane-proximal region, can not. Therefore, the membrane-proximal region participates in regulation of the adhesive activity by preventing lateral dimerization of the extracellular domain.     

An amino-terminal domain of Enterococcus faecalis aggregation substance is required for aggregation, bacterial internalization by epithelial cells and binding to lipoteichoic acid

Aggregation substance (AS), a plasmid-encoded surface protein of Enterococcus faecalis, plays important roles in virulence and antibiotic resistance transfer. Previous studies have suggested that AS-mediated aggregation of enterococcal cells could involve the binding of this protein to cell wall lipoteichoic acid (LTA). Here, a method to purify an undegraded form of Asc10, the AS of the plasmid pCF10, is described. Using this purified protein, direct binding of Asc10 to purified E. faecalis LTA was demonstrated. Equivalent binding of Asc10 to LTA purified from INY3000, an E. faecalis strain that is incapable of aggregation, was also observed. Surprisingly, mutations in a previously identified aggregation domain from amino acids 473 to 683 that abolished aggregation had no effect on LTA binding. In frame deletion analysis of Asc10 was used to identify a second aggregation domain located in the N-terminus of the protein from amino acids 156 to 358. A purified Asc10 mutant protein lacking this domain showed reduced LTA binding, while a purified N-terminal fragment from amino acids 44-331 had high LTA binding. Like the previously described aggregation domain, the newly identified Asc10((156-358)) aggregation domain was also required for efficient internalization of E. faecalis into HT-29 enterocytes. Thus, Asc10 possess two distinct domains required for aggregation and eukaryotic cell internalization: an N-terminal domain that promotes binding to LTA and a second domain located near the middle of the protein.     

Structural Requirements for Outside-In and Inside-Out Signaling by Drosophila Neuroglian,a Member of the L1 Family of Cell Adhesion Molecules

Expression of the Drosophila cell adhesion molecule neuroglian in S2 cells leads to cell aggregation and the intracellular recruitment of ankyrin to cell contact sites. We localized the region of neuroglian that interacts with ankyrin and investigated the mechanism that limits this interaction to cell contact sites. Yeast two-hybrid analysis and expression of neuroglian deletion constructs in S2 cells identified a conserved 36-amino acid sequence that is required for ankyrin binding. Mutation of a conserved tyrosine residue within this region reduced ankyrin binding and extracellular adhesion. However, residual recruitment of ankyrin by this mutant neuroglian molecule was still limited to cell contacts, indicating that the lack of ankyrin binding at noncontact sites is not caused by tyrosine phosphorylation. A chimeric molecule, in which the extracellular domain of neuroglian was replaced with the corresponding domain from the adhesion molecule fasciclin II, also selectively recruited ankyrin to cell contacts. Thus, outside-in signaling by neuroglian in S2 cells depends on extracellular adhesion, but does not depend on any unique property of its extracellular domain. We propose that the recruitment of ankyrin to cell contact sites depends on a physical rearrangement of neuroglian in response to cell adhesion, and that ankyrin binding plays a reciprocal role in stabilizing the adhesive interaction.     

Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides

The neural cell adhesion molecule (N-CAM) plays an integral role in cell interactions during neural development, with the binding of heparan sulfate proteoglycan to the amino-terminal region of N-CAM being required for N-CAM function. In the present study we have used synthetic peptides (HBD-1 and HBD-2), derived from the primary amino acid sequence of rat N-CAM, to identify the region of N-CAM that binds heparan sulfate. The 28 amino acid HBD-1 synthetic peptide was shown to bind both [3H]heparin and dissociated retinal cells. Retinal cells also attach to a substratum of HBD-2 peptide, but fail to bind to a control peptide containing a scrambled amino acid sequence of HBD-2. The HBD-2 peptide also inhibits retinal cell adhesion to N-CAM, demonstrating the physiological importance of the amino acid sequence encoded by the HBD peptide. These data therefore permit the localization of a heparin binding domain to a 17 amino acid region of immunoglobulin-like loop 2.     

A Triad of Lys12, Lys41, Arg78 Spatial Domain,a Novel Identified Heparin Binding Site on Tat Protein,Facilitates Tat-Driven Cell Adhesion

Tat protein, released by HIV-infected cells, has a battery of important biological effects leading to distinct AIDS-associated pathologies. Cell surface heparan sulfate protoglycans (HSPGs) have been accepted as endogenous Tat receptors, and the Tat basic domain has been identified as the heparin binding site. However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat–heparin interactions suggest that the basic domain is not the sole Tat heparin binding site. In the current study, an approach integrating computational modeling, mutagenesis, biophysical and cell-based assays was used to elucidate a novel, high affinity heparin-binding site: a Lys12, Lys41, Arg78 (KKR) spatial domain. This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization. The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events.