Synthetic RGD-containing alpha-helical coiled coil peptides promote integrin-dependent cell adhesion.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively.     

Constrained cell recognition peptides engineered into streptavidin.

Streptavidin is widely used as an adaptor molecule in diagnostics, separations, and laboratory assay applications. We have here engineered cell adhesive peptides into the three-dimensional scaffolding of streptavidin to convert streptavidin into a functional protein. The mutations did not alter refolding or tetramer assembly and the slow biotin dissociation rate of wild-type streptavidin was retained. The peptide targets were hexapeptide sequences derived from osteopontin and fibronectin that contain the RGD cell adhesion sequence. Cell binding assays directly demonstrated that rat aortic endothelial cells and human melanoma cells adhered to surfaces coated with either of the two RGD streptavidin mutants in a dose-dependent fashion. Wild-type streptavidin displayed no significant cell binding activity. Inhibition studies with soluble RGD peptides confirmed that the cell adhesion was RGD mediated. Further inhibition studies with antibodies directed against alphavbeta3 demonstrated that the RGD-streptavidin interaction was primarily mediated by this integrin with melanoma cells. These results demonstrate that peptide recognition sequences can be engineered into accessible surface regions of streptavidin without disrupting biotin binding properties. This approach to introducing secondary functional activities into streptavidin may improve streptavidin's utility in existing applications or provide new technology opportunities.     

Enhanced cell adhesion and mature intracellular structure promoted by squaramide-based RGD mimics on bioinert surfaces

Highly selective molecular binding and the subsequent dynamic protein assemblies control the adhesion of mammalian cells. Molecules that inhibit cell adhesion have the therapeutic potential for a wide range of diseases. Here, we report an efficient synthesis (2–4 steps) of a class of squaramide molecules that mimics the natural tripeptide ligand Arg-Gly-Asp (RGD) that mediates mammalian cell adhesion through binding with membrane protein integrin. In solution, this class of squaramides exhibits a higher potency at inhibiting mammalian cell adhesion than RGD tripeptides. When immobilized on a bio-inert background formed by self-assembled monolayers of alkanethiols on gold films, squaramide ligands mediate vastly different intracellular structures than RGD ligands. Immunostaining revealed that the focal adhesions are smaller, but with a larger quantity, for cells adhered on squaramides than that on RGD ligands. Furthermore, the actin filaments are also more fibrous and well distributed for cell adhesion mediated by squaramide than that by RGD ligands. Quantification reveal that squaramide ligands mediate about 1.5 times more total focal adhesion (measured by the summation of the area of all focal adhesions) than that by natural RGD ligands. This result suggests that cell adhesion inhibitors, while blocking the attachment of cells to surfaces, may induce more focal adhesion proteins. Finally, this work demonstrates that immobilizing new ligands on bioinert surfaces provide a powerful tool to study mammalian cell adhesion.     

Interleukin 32 (IL-32) contains a typical α-helix bundle structure that resembles focal adhesion targeting region of focal adhesion kinase-1

IL-32 can be expressed in several isoforms. The amino acid sequences of the major IL-32 isoforms were used to predict the secondary and tertiary protein structure by I-TASSER software. The secondary protein structure revealed coils and α-helixes, but no β sheets. Furthermore, IL-32 contains an RGD motif, which potentially activates procaspase-3 intracellular and or binds to integrins. Mutation of the RGD motif did not result in inhibition of the IL-32β- or IL-32γ-induced cytotoxicity mediated through caspase-3. Although IL-32α interacted with the extracellular part of αVβ3 and αVβ6 integrins, only the αVβ3 binding was inhibited by small RGD peptides. Additionally, IL-32β was able to bind to αVβ3 integrins, whereas this binding was not inhibited by small RGD peptides. In addition to the IL-32/integrin interactions, we observed that IL-32 is also able to interact with intracellular proteins that are involved in integrin and focal adhesion signaling. Modeling of IL-32 revealed a distinct α-helix protein resembling the focal adhesion targeting region of focal adhesion kinase (FAK). Inhibition of FAK resulted in modulation of the IL-32β- or IL-32γ-induced cytotoxicity. Interestingly, IL-32α binds to paxillin without the RGD motif being involved. Finally, FAK inhibited IL-32α/paxillin binding, whereas FAK also could interact with IL-32α, demonstrating that IL-32 is a member of the focal adhesion protein complex. This study demonstrates for the first time that IL-32 binds to the extracellular domain of integrins and to intracellular proteins like paxillin and FAK, suggesting a dual role for IL-32 in integrin signaling.     

The relative importance of topography and RGD ligand density for endothelial cell adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×10(2)-6×10(11) RGD/mm(2). We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×10(5) RGD/mm(2) on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×10(8) RGD/mm(2) irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry.     

Chimeric peptides of statherin and osteopontin that bind hydroxyapatite and mediate cell adhesion

Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moalpha(v) melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moalpha(v) subpopulation demonstrated that the alpha(v)beta(3) integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.     

Integrin alpha3beta1 (CD 49c/29) is a cellular receptor for Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) entry into the target cells

Human herpesvirus-8 (HHV-8) is implicated in the pathogenesis of Kaposi's sarcoma. HHV-8 envelope glycoprotein B possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, antibodies against RGD-dependent alpha3 and beta1 integrins, and by soluble alpha3beta1 integrin. Expression of human alpha3 integrin increased the infectivity of virus for Chinese hamster ovary cells. Anti-gB antibodies immunoprecipitated the virus-alpha3 and -beta1 complexes, and virus binding studies suggest a role for alpha3beta1 in HHV-8 entry. Further, HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK). These findings implicate a role for alpha3beta1 integrin and the associated signaling pathways in HHV-8 entry into the target cells.     

Adhesion of Cultured Human Kidney Mesangial Cells to Native Entactin: Role of Integrin Receptors

Entactin is an extracellular matrix glycoprotein which binds to laminin and is found in most renal basement membranes and in the glomerular mesangial matrix. In the present study, we have characterized specific integrin receptors on cultured human mesangial cells (CHMC) responsible for adhesion to native entactin. The integrin receptors α2,β1, α3,β1, α5,β1, αv,β3, αv,β5, and α6 complexed with either β1 or β4 could be immune precipitated from detergent extracts of metabolically labeled CHMC. Adhesion assays with inhibitory anti integrin monoclonal antibodies (mab) demonstrated that CHMC use both αv,β3 and a β1-containing integrin to bind surfaces coated with native entactin. Optimal binding of CHMC to native entactin required the participation of cations. Using wild type and mutant recombinant entactin fragments, the binding site for the αv,β3 receptor was localized to the RGD sequence on the rod or E domain of entactin. CHMC adhesion to mutant full length recombinant entactin ligands lacking the E domain RGD sequence confirmed the presence of ligand binding site(s) for β1 integrin receptor(s). Differences in CHMC binding characteristics to recombinant and full length entactin compared to native bovine basement membrane entactin were observed. This suggests that tertiary molecular structure may contribute to entactin ligand binding properties. Primary amino acid residue sequences and tertiary structure of entactin may play roles in forming functional cell attachment sites in native basement membrane entactin.     

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.     

Human herpesvirus 8 envelope glycoprotein B mediates cell adhesion via its RGD sequence

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposi's sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes alpha3beta1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBdeltaTM) and a gBdeltaTM mutant (gBdeltaTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBdeltaTM, gBdeltaTM-RGA, and deltaTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBdeltaTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBdeltaTM-RGA and DeltaTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBdeltaTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBdeltaTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBdeltaTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBdeltaTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBdeltaTM protein to the target cells but not by gBdeltaTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBdeltaTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposi's sarcoma pathogenesis.     

Galectin-8 functions as a matricellular modulator of cell adhesion

The interaction of cells with the extracellular matrix regulates cell adhesion and motility. Here we demonstrate that different cell types adhere and spread when cultured in serum-free medium on immobilized galectin-8, a mammalian beta-galactoside-binding protein. At maximal doses, galectin-8 is equipotent to fibronectin in promoting cell adhesion and spreading. Cell adhesion to immobilized galectin-8 is mediated by sugar-protein interactions with integrins, and galectin-8 triggers integrin-mediated signaling cascades including Tyr phosphorylation of focal adhesion kinase and paxillin. Cell adhesion is potentiated in the presence of Mn(2+), whereas it is interrupted in the presence of soluble galectin-8, integrin beta(1) inhibitory antibodies, EDTA, or thiodigalactoside but not by RGD peptides. Furthermore, cells readily adhere onto immobilized monoclonal galectin-8 antibodies, which are equipotent to integrin antibodies in promoting cell adhesion. Cell adhesion to immobilized galectin-8 is partially inhibited by serum proteins, suggesting that complex formation between immobilized galectin-8 and serum components generates a matrix that is less supportive of cell adhesion. Accordingly, cell motility on immobilized galectin-8 readily takes place in the presence of serum. Truncation of the C-terminal half of galectin-8, including one of its two carbohydrate recognition domains, largely abolishes its ability to modulate cell adhesion, indicating that both carbohydrate recognition domains are required to maintain a functional form of galectin-8. Collectively, our findings implicate galectin-8 as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of cell surface integrin receptors. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Because of its dual effects on the adhesive properties of the cells and its association with fibronectin, galectin-8 might be considered a novel type of matricellular protein.     

Dual functionality of the anti-beta1 integrin antibody, 12G10, exemplifies agonistic signalling from the ligand binding pocket of integrin adhesion receptors

Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin alpha4beta1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via alpha5beta1 stimulated all of these processes. In contrast, soluble ligand binding to either alpha4beta1 or alpha5beta1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric alpha5 integrin containing the beta-propeller domain from the ligand binding pocket of alpha4 exhibited the same signaling properties as the full-length alpha4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular beta-propeller domain of the associating alpha-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the alpha cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.     

Neurite outgrowth,RGD-dependent,and RGD-independent adhesion of identified molluscan motoneurons on selected substrates

The extracellular matrix (ECM) provides structural support to cells and tissues and is involved in the regulation of various essential physiological processes, including neurite outgrowth. Most of the adhesive interactions between cells and ECM proteins are mediated by integrins. Integrins typically recognize short linear amino acid sequences in ECM proteins, one of the most common being Arginine-Glycine-Aspartate (RGD). The present study investigated neurite outgrowth and adhesion of identified molluscan neurons on a selection of substrates in vitro. Involvement of RGD binding sites in adhesion to the different substrates was investigated using soluble synthetic RGD peptides. The cells adhered to native (i.e., nondenatured) laminin and type IV collagen, but not to native plasma fibronectin. Denaturation of fibronectin dramatically enhanced cell adhesion. Only the adhesion to denatured fibronectin was inhibited by RGD peptides, indicating that denaturation uncovers a RGD binding site in the protein. Laminin as well as denatured fibronectin, but not type IV collagen, induced neurite outgrowth from a percentage of the RPA neurons. These results demonstrate that molluscan neurons can attach to various substrates using both RGD-dependent and RGD-independent adhesion mechanisms. This suggests that at least two different cell adhesion receptors, possibly belonging to the integrin family, are expressed in these neurons. Moreover, the results show that vertebrate ECM proteins can induce outgrowth from these neurons, suggesting that the mechanisms involved in adhesion as well as outgrowth promoting are evolutionarily well conserved. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 37–52, 1998     

Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands

Integrin-mediated adhesion is regulated by multiple features of the adhesive surface, including its chemical composition, topography, and physical properties. In this study we investigated integrin lateral clustering, as a mechanism to control integrin functions, by characterizing the effect of nanoscale variations in the spacing between adhesive RGD ligands on cell spreading, migration, and focal adhesion dynamics. For this purpose, we used nanopatterned surfaces, containing RGD-biofunctionalized gold dots, surrounded by passivated gaps. By varying the spacing between the dots, we modulated the clustering of the associated integrins. We show that cell-surface attachment is not sensitive to pattern density, whereas the formation of stable focal adhesions and persistent spreading is. Thus cells plated on a 108-nm-spaced pattern exhibit delayed spreading with repeated protrusion-retraction cycles compared to cells growing on a 58-nm pattern. Cell motility on these surfaces is erratic and nonpersistent, leaving thin membrane tethers bound to the RGD pattern. Dynamic molecular profiling indicated that the adhesion sites formed with the 108-nm pattern undergo rapid turnover and contain reduced levels of zyxin. These findings indicate that a critical RGD density is essential for the establishment of mature and stable integrin adhesions, which, in turn, induce efficient cell spreading and formation of focal adhesions.     

The inhibitory receptor PIR-B negatively regulates neutrophil and macrophage integrin signaling

The Ig-like receptor family member, PIR-B, has been shown to play an inhibitory role in receptor signaling within B cells, mast cells, and dendritic cells. As it has been implicated in integrin-mediated responses, we investigated the effect of loss of the PIR-B protein on integrin-mediated signaling in primary murine myeloid cells. The pir-b-/- neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when plated on surfaces coated with either extracellular matrix proteins or cellular adhesion molecules in the presence or absence of the soluble inflammatory agonist TNF-alpha. The pir-b-/- and wild-type cells responded equivalently when stimulated with TNF-alpha in suspension, indicating that the hyperresponsive phenotype of the pir-b-/- cells during adhesion was due to enhanced integrin signaling. Both wild-type and pir-b-/- neutrophils expressed similar levels of integrin subunits. Primary bone marrow-derived macrophages from pir-b-/- mice were also hyperadhesive and spread more rapidly than wild-type cells following plating on surfaces that cross-linked cellular beta2 integrins. Biochemical analysis of macrophages from pir-b-/- mice revealed enhanced phosphorylation and activation of proteins involved in integrin signaling. These observations point to a nonredundant role for PIR-B in the regulation of leukocyte integrin signaling.     

Alpha(v)beta3 and alpha(v)beta5 integrins bind both the proximal RGD site and non-RGD motifs within noncollagenous (NC1) domain of the alpha3 chain of type IV collagen: implication for the mechanism of endothelia cell adhesion

The NC1 domains of human type IV collagen, in particular alpha3NC1, are inhibitors of angiogenesis and tumor growth (Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J., Sado, Y., Ninomiya, Y., Sarras, M. P., Jr., Hudson, B. G., and Brooks, P. C. (2000) J. Biol. Chem. 275, 8051-8061). The recombinant alpha3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-alpha3NC1. Others, using synthetic peptides, have concluded that this RGD site is nonfunctional in cell adhesion, and therefore, the anti-angiogenic activity is attributed exclusively to alpha(v)beta(3) integrin interactions with non-RGD motifs of the RGD-alpha3NC1 domain (Maeshima, Y., Colorado, P. C., and Kalluri, R. (2000) J. Biol. Chem. 275, 23745-23750). This nonfunctionality is surprising given that RGD is a binding site for alpha(v)beta(3) integrin in several proteins. In the present study, we used the alpha3NC1 domain with or without the RGD site, expressed in HEK 293 cells for native conformation, as an alternative approach to synthetic peptides to assess the functionality of the RGD site and non-RGD motifs. Our results demonstrate a predominant role of the RGD site for endothelial adhesion and for binding of alpha(v)beta(3) and alpha(v)beta(5) integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the alpha(v)beta(3) integrin-binding sites of the alpha3NC1 domain, are 10-fold less potent in competing for integrin binding than the native protein, indicating the importance of additional structural and/or conformational features of the alpha3NC1 domain for integrin binding. Therefore, the RGD site, in addition to non-RGD motifs, may contribute to the mechanisms of endothelial cell adhesion in the human vasculature and the anti-angiogenic activity of the RGD-alpha3NC1 domain.     

An RGD Motif Present in Cadherin 17 Induces Integrin Activation and Tumor Growth

Little is known about the mechanism of integrin activation by cadherin 17 (CDH17). Here we observed the presence of a tri-peptide motif, RGD, in domain 6 of the human CDH17 sequence and other cadherins such as cadherin 5 and cadherin 6. The use of CDH17 RAD mutants demonstrated a considerable decrease of proliferation and adhesion in RKO and KM12SM colon cancer cells. Furthermore, RGD peptides inhibited the adhesion of both cell lines to recombinant CDH17 domain 6. The RGD motif added exogenously to the cells provoked a change in β1 integrin to an active, high-affinity conformation and an increase in focal adhesion kinase and ERK1/2 activation. In vivo experiments with Swiss nude mice demonstrated that cancer cells expressing the CDH17 RAD mutant showed a considerable delay in tumor growth and liver homing. CDH17 RGD effects were also active in pancreatic cancer cells. Our results suggest that α2β1 integrin interacts with two different ligands, collagen IV and CDH17, using two different binding sites. In summary, the RGD binding motif constitutes a switch for integrin pathway activation and shows a novel capacity of CDH17 as an integrin ligand. This motif could be targeted to avoid metastatic dissemination in tumors overexpressing CDH17 and other RGD-containing cadherins.     

(Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion

We have prepared protein-peptide conjugates composed of bovine serum albumin (BSA) derivatized with short peptides containing the Arg-Gly-Asp (RGD) sequence derived from the adhesion site of fibronectin. The RGD-BSA conjugates were used to coat tissue culture plastic surfaces which then served as substrata in cell adhesion experiments. Our results indicate that the efficiency of adhesion to RGD-BSA-coated surfaces is highly dependent on the valency of the (RGD)n-BSA conjugates. For example, on surfaces with approximately equal amounts of RGD ligand, CHO cells adhered virtually 100% to the (RGD)n-BSA (n = 20.8) conjugate and not at all to the (RGD)n-BSA (n = 3.5) conjugate. Adhesion on (RGD)n-BSA-coated substrata and on fibronectin- or vitronectin-coated substrata was also examined in terms of the relationship between cell adhesion and the intermolecular distances of adsorbed proteins. It was observed that for substrata coated with relatively compact, symmetric molecules, such as RGD-BSA or vitronectin, adhesion dropped off sharply as intermolecular distances increased; by contrast, for fibronectin, a large asymmetric molecule, adhesion declined more gradually as intermolecular distances increased. Finally, we have examined the role of different cell-surface receptors in the process of adhesion to RGD-BSA substrata. Interestingly, competition and blocking experiments with antibodies and with soluble competing proteins suggest that it is the vitronectin receptor rather than the fibronectin receptor which mediates adhesion to RGD-BSA.     

Extracellular RGD-binding proteins modulate cell adhesion.

Cell/fibronectin adhesion in extracellular matrices is partly mediated by integrin receptor recognition of RGD domains in fibronectin. Since blood contains significant levels of soluble fibronectin we have now investigated the occurrence of extracellular RGD-binding proteins. Attachment assays indicate that extracellular RGD-binding proteins prevent cell adhesion, suggesting their potential as novel secreted modulators of blood-borne cell adhesive interactions. These extracellular RGD-binding proteins also showed electrophoretic changes with reducing agents, suggestive of intrachain disulphide bonds, like those found in RGD-binding integrins. However, they differed from the latter in their electrophoretic profile, which was greatly dependent on the presence of protease inhibitors. Plasma from tumor-bearing mice showed a greater proportion of fast-migrating RGD-binding species under reducing condition compared to similarly treated normal plasma, suggesting that tumor development is associated with a partial degradation of extracellular RGD-binding proteins.     

Cell adhesion to tenascin-X mapping of cell adhesion sites and identification of integrin receptors.

Adhesive properties of tenascin-X (TN-X) were investigated using TN-X purified from bovine skin and recombinant proteins encompassing the RGD sequence located within the tenth fibronectin type-III domain, and the fibrinogen-like domain. Osteosarcoma (MG63) and bladder carcinoma cells (ECV304) cells were shown to adhere to purified TN-X, but did not spread and did not assemble actin stress fibers. Both cell types adhered to recombinant proteins harboring the contiguous fibronectin type-III domains 9 and 10 (FNX 9-10) but not to the FNX 10 domain alone. This adhesion to FNX 9-10 was shown to be mediated by alphavbeta3 integrin, was inhibited by RGD peptides and was strongly reduced in proteins mutated within the RGD site. As antibodies against alphavbeta3 integrin had no effects on cell adhesion to purified TN-X, we suggest that the RGD sequence is masked in intact TN-X. Cell attachment to the recombinant TN-X fibrinogen domain (FbgX) and to purified TN-X was greater for MG63 than for ECV304 cells. A beta1-containing integrin was shown to be involved in MG63 cell attachment to FbgX and to purified TN-X. Although the existence of other cell interaction sites is likely in this huge molecule, these similar patterns of adhesion and inhibition suggest that the fibrinogen domain might be a dominant site in the whole molecule.