Detachment of cells from culture substrate by soluble fibronectin peptides

The synthetic cell attachment-promoting peptides from fibronectin (Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.)., 309:30-33) were found to detach cultured cells from the substratum when added to the culture in a soluble form. Peptides ranging in length from tetrapeptide to heptapeptide and containing the active L-arginyl-glycyl-L-aspartic acid (Arg-Gly-Asp) sequence had the detaching activity, whereas a series of different peptides with chemically similar structures had no detectable effect on any of the test cells. The Arg-Gly-Asp-containing peptides caused detachment of various cell lines of different species and histogenetic origin. Studies with defined substrates showed that the active peptides could inhibit the attachment of cells to vitronectin in addition to fibronectin, indicating that vitronectin is recognized by cells through a similar mechanism as fibronectin. The peptides did not inhibit the attachment of cells to collagen. However, cells cultured on collagen-coated plastic for 24-36 h, as well as cells with demonstrable type I or type VI collagen in their matrix, were susceptible to the detaching effect of the peptides. These results indicate that the recognition mechanism(s) by which cells bind to fibronectinand vitronectin plays a major role in the substratum attachment of cells and that collagens may not be directly involved in cell-substratum adhesion. Since vitronectin is abundant in serum, it is probably an important component in mediating the attachment of cultured cells. The independence of the effects of the peptide on the presence of serum and the susceptibility of many different cell types to detachment by the peptide show that the peptides perturb an attachment mechanism that is intrinsic to the cells and fundamentally significant to their adhesion.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin.

cDNA clones for vitronectin, a cell adhesion-promoting plasma and tissue protein, were isolated from a lambda gt11 library containing cDNA inserts made from human liver mRNA. The library was screened with anti-vitronectin antibodies and the positive clones were further identified with synthetic oligonucleotide probes deduced from the partial amino acid sequence of vitronectin. Nucleotide sequence analysis showed that the largest insert was 1545 bp long and contained the whole sequence corresponding to plasma vitronectin. It showed that vitronectin contains the entire 44-amino acid somatomedin B peptide at its NH2 terminus and, near its COOH terminus, a 34-amino acid glycosaminoglycan binding site in which half of the amino acids are basic residues. Three potential carbohydrate attachment sites are present in the sequence. An Arg-Gly-Asp sequence, which has previously been shown to be the cell attachment site in fibronectin, was found in vitronectin immediately after the NH2-terminal somatomedin B sequence. No other homologies with fibronectin were found. The Arg-Gly-Asp sequence appears to constitute the cell attachment site of vitronectin, since it is in the region where we have previously localized the cell attachment site, its presence correlate with cell attachment activity among the insert-coded polypeptides, and because previous results have shown that synthetic peptides containing the Arg-Gly-Asp sequence inhibit the cell attachment function of vitronectin. The discovery of an Arg-Gly-Asp cell attachment site in a protein with a known cell attachment function emphasizes the general importance of this sequence in cell recognition.     

A cell surface receptor complex for collagen type I recognizes the Arg- Gly-Asp sequence

To isolate collagen-binding cell surface proteins, detergent extracts of surface-iodinated MG-63 human osteosarcoma cells were chromatographed on affinity matrices of either type I collagen-Sepharose or Sepharose carrying a collagen-like triple-helical peptide. The peptide was designed to be triple helical and to contain the sequence Arg-Gly-Asp, which has been implicated as the cell attachment site of fibronectin, vitronectin, fibrinogen, and von Willebrand factor, and is also present in type I collagen. Three radioactive polypeptides having apparent molecular masses of 250 kD, 70 kD, and 30 kD were distinguishable in that they showed affinity toward the collagen and collagen-like peptide affinity columns, and could be specifically eluted from these columns with a solution of an Arg-Gly-Asp-containing peptide, Gly-Arg-Gly-Asp-Thr-Pro. These collagen-binding polypeptides associated with phosphatidylcholine liposomes, and the resulting liposomes bound specifically to type I collagen or the collagen-like peptide but not to fibronectin or vitronectin or heat-denatured collagen. The binding of these liposomes to type I collagen could be inhibited with the peptide Gly-Arg-Gly-Asp-Thr-Pro and with EDTA, but not with a variant peptide Gly-Arg-Gly-Glu-Ser-Pro. We conclude from these data that these three polypeptides are membrane molecules that behave as a cell surface receptor (or receptor complex) for type I collagen by interacting with it through the Arg-Gly-Asp tripeptide adhesion signal. The lack of binding to denatured collagen suggests that the conformation of the Arg-Gly-Asp sequence is important in the recognition of collagen by the receptor complex.     

Human osteosarcoma cells resistant to detachment by an Arg-Gly-Asp- containing peptide overproduce the fibronectin receptor

MG-63 human osteosarcoma cells were selected for attachment and growth in the presence of increasing concentrations of a synthetic peptide containing the cell attachment-promoting Arg-Gly-Asp sequence derived from the cell-binding region of fibronectin. Cells capable of attachment and growth in 5-mM concentrations of a peptide having the sequence Gly-Arg-Gly-Asp-Ser-Pro overproduce the cell surface receptor for fibronectin. In contrast, these cells show no differences in the numbers of vitronectin receptor they express as compared with the parental MG-63 cells. In agreement with the resistance of the selected cells to detachment by the peptide, 25-fold more Arg-Gly-Asp-containing peptide is required to prevent the attachment of these cells to fibronectin-coated surfaces than is needed to inhibit the attachment of MG-63 cells to the same substrate. However, similar concentrations of this peptide inhibit attachment of both cell lines to vitronectin-coated surfaces. The increase in fibronectin receptor is due to an increase in the levels of mRNA encoding the fibronectin receptor. Because of the nature of the selection process, we reasoned that this increase might be due to amplification of the fibronectin receptor gene, but no increase in gene copy number was detected by Southern blot analysis. The peptide-resistant cells display a very different morphology from that of the MG-63 cells, one that has a greater resemblance to that of osteocytes. The resistant cells also grow much more slowly than the MG-63 cells. The increased fibronectin receptor and altered morphology and growth properties were stable for at least 3 mo in the absence of peptide. The enhanced expression of the fibronectin receptor on the resistant cells indicates that cells are capable of altering the amount of fibronectin receptor on their surface in response to environmental factors and that this may in turn affect the phenotypic properties of the cell.     

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.     

The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation- dependent manner. Binding was inhibited by soluble vitronectin, by RGD- containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins.     

Effects of modifications of the RGD sequence and its context on recognition by the fibronectin receptor

The receptor for fibronectin is a member of the integrin superfamily of cell surface adhesion receptors, many of which recognize the sequence RGD in their ligands. We have developed sensitive enzyme-linked and radioreceptor assays to examine the ligand specificity of the fibronectin receptor. The fibronectin receptor bound only to fibronectin of the various Arg-Gly-Asp (RGD)-containing proteins tested. The smallest amount of receptor detectable in the assay was about 10 ng. Mn2+ enhanced the binding of the receptor to fibronectin 3-10-fold as compared to Ca2+ and Mg2+. Scatchard analysis of the saturation plot from the radioreceptor assay gave a dissociation constant (Kd) of 3 x 10(-8) M for the binding of fibronectin receptor to fibronectin in the presence of Mn2+. Inhibition experiments showed that the affinities of the ligands for the receptor decreased in the order of fibronectin approximately 110-kDa fibronectin fragment greater than GRGDSP peptide greater than 11.5-kDa fragment. Peptides not containing an RGD were several hundred to several thousand-fold less inhibitory than GRGDSP. These included the closely related peptides GRADSP and GRGESP, as well as three peptides containing the reverse sequence DGR. A peptide from the fibrinogen gamma-chain, KQAGDV, which had about 0.5% of the inhibitory activity of the standard GRGDSP peptide, was the most active peptide not containing an RGD. These results document the exquisite specificity of the fibronectin receptor for the RGD sequence.     

Von Willebrand factor promotes endothelial cell adhesion via an Arg-Gly- Asp-dependent mechanism

Von Willebrand factor (vWF) is a constitutive and specific component of endothelial cell (EC) matrix. In this paper we show that, in vitro, vWF can induce EC adhesion and promote organization of microfilaments and adhesion plaques. In contrast, human vascular smooth muscle cells and MG63 osteosarcoma cells did not adhere and spread on vWF. Using antibodies to the beta chains of fibronectin (beta 1) and vitronectin (beta 3) receptors it was found that ECs adherent to vWF show clustering of both receptors. The beta 1 receptor antibodies are arranged along stress fibers at sites of extracellular matrix contact while the beta 3 receptor antibodies were sharply confined at adhesion plaques. ECs release and organize endogenous fibronectin early during adhesion to vWF. Upon blocking protein synthesis and secretion, ECs can equally adhere and spread on vWF but, while the beta 3 receptors are regularly organized, the beta 1 receptors remain diffuse. This suggests that the organization of the beta 1 receptors depend on the release of fibronectin and/or other matrix proteins operated by the same cell. Antibodies to the beta 3 receptors fully block EC adhesion to vWF and detach ECs seeded on this substratum. In contrast, antibodies to the beta 1 receptors are poorly active. Overall these results fit with an accessory role of beta 1 receptors and indicate a leading role for the beta 3 receptors in EC interaction with vWF. To identify the EC binding domain on vWF we used monoclonal antibodies produced against a peptide representing the residues Glu1737-Ser1750 of the mature vWF and thought to be important in mediating its binding to the platelet receptor glycoprotein IIb-IIIa. We found that the antibody that recognizes the residues 1,744-1,746, containing the Arg-Gly-Asp sequence, completely inhibit EC adhesion to vWF whereas a second antibody recognizing the adjacent residues 1,740-1,742 (Arg-Gly-Asp-free) is inactive. Both antibodies do not interfere with EC adhesion to vitronectin. This defines the molecular domain on vWF that is specifically recognized by ECs and reaffirms the direct role of the Arg-Gly-Asp sequence as the integrin receptor recognition site also in the vWF molecule.     

An Arg-Gly-Asp-directed receptor on the surface of human melanoma cells exists in an divalent cation-dependent functional complex with the disialoganglioside GD2

The disialogangliosides GD2 and GD3 play a major role in the ability of human melanoma cells to attach to Arg-Gly-Asp-containing substrates such as fibronectin and vitronectin, since pretreatment of these cells with monoclonal antibodies to the oligosaccharide of GD2 and GD3 can inhibit their attachment and spreading on such adhesive proteins. This report demonstrates that human melanoma cells (M21) synthesize and express a glycoprotein receptor that shares antigenic epitopes with the vitronectin receptor on human fibroblasts and is capable of specifically recognizing the Gly-Arg-Gly-Asp-Ser-Pro sequence. In the presence of calcium, GD2, the major ganglioside of M21 cells, colocalized with this receptor on the surface of human melanoma cells and their focal adhesion plaques as demonstrated by double-label transmission immunoelectron microscopy and indirect immunofluorescence. Biochemical evidence is presented indicating that the vitronectin receptor on M21 human melanoma cells contains associated calcium and GD2. This ganglioside copurified with the glycoprotein receptor for vitronectin on affinity columns containing either an Arg-Gly-Asp-containing peptide, concanavalin A, or lentil lectin. This major Arg-Gly-Asp-directed receptor on M21 cells could be metabolically labeled with 45Ca2+. Chelation of this ion with EDTA caused the dissociation of GD2 from the receptor and rendered the remaining glycoprotein incapable of binding to an Arg-Gly-Asp-containing peptide. Reconstitution experiments demonstrated a requirement for calcium, and not magnesium, for receptor binding to Arg-Gly-Asp and indicated that addition of ganglioside can enhance this interaction.     

Effects of inhibitors of integrin binding on cellular outgrowth from bovine inner cell masses in vitro

Summary Bovine inner cell masses (ICM) cultured on fibronectin give rise to extensive cellular outgrowths containing endoderm. Peptides with the Glu-Ile-Leu-Asp-Val (EILDV) and Arg-Gly-Asp (RGD) sequences inhibit cell migration on fibronectin by binding to the fibronectin-recognition site in several integrins. To identify integrins involved in endodermal cell outgrowth on fibronectin and vitronectin, the effects of the EILDV and RGD peptides were evaluated in vitro. In experiment 1, ICM were cultured on fibronectin in medium containing 0.5 or 1.0 mg/ml EILDV or RGD (or both). Compared with 0 mg/ml, 0.5 mg/ml EILDV suppressed (P<0.10) outgrowth area overall, and 1.0 mg/ml EILDV reduced (P<0.05) outgrowth area after 72 h of culture. Compared with 0 mg/ml, 0.5 and 1.0 mg/ml RGD reduced (P<0.05) outgrowth area after 72 h of culture. Plasminogen activator activity in conditioned medium increased (P<0.05) in 0.5 mg/ml RGD but decreased (P<0.10) in 1.0 mg/ml RGD compared with 0 mg/ml RGD. In experiment 2, bovine ICM were cultured on vitronectin in medium containing 0.5 or 1.0 mg/ml RGD. Neither concentration of RGD (P>0.10) affected the extent of cellular outgrowth on vitronectin. Bovine endodermal cell migration on fibronectin can be modulated by the RGD and EILDV peptides. Despite inhibition, neither peptide completely prevented outgrowth on fibronectin. In contrast, cellular outgrowth on vitronectin was unaffected by RGD. The persistence of cellular outgrowth on fibronectin and the absence of inhibition by RGD for ICM cultured on vitronectin suggests that bovine endodermal cells can use alternative cellular adhesion systems, such as nonintegrin receptors, during outgrowth.     

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

The complement SC5b-9 complex mediates cell adhesion through a vitronectin receptor.

Adhesion of cells to the terminal complement complex of C5b through C9 containing the serum S-protein (SC5b-9) was investigated using a microtiter plate attachment assay with L8 myoblast indicator cells. The skeletal muscle-derived L8 myoblasts bound and spread on substratum coated with SC5b-9, and with the vitronectin/S-protein component of SC5b-9. The myoblasts did not adhere to substratum coated with collagen, laminin, or fibronectin. The cell attachment was blocked by antibody to vitronectin/S-protein, whereas antibody to the other components C5, C6, C7, C8, or C9 had minimal effect. The cells were not bound to free vitronectin because attachment activity was removed by adsorption with an anti-C6 antibody column. The L8 cell attachment was dependent on divalent cations, was blocked by synthetic peptides containing the amino acid sequence Arg-Gly-Asp, and was inhibited by antivitronectin receptor antibody. These results indicate that cells adhere to the SC5b-9 complex through interaction of the vitronectin component with an integrin vitronectin receptor. Cell attachment to terminal C complexes could be used for leukocyte adherence and migration during inflammation, and also for attachment of tissue cells during regeneration after disease or traumatic injury.     

Regulation of the fibronectin receptor affinity by divalent cations

The cell surface receptor for fibronectin is a heterodimeric membrane protein that recognizes an Arg-Gly-Asp sequence in fibronectin and that requires cations such as Mg2+ or Ca2+ for binding to fibronectin. The divalent cation requirements of this receptor were analyzed by measuring attachment of receptor liposomes to ligand-coated surfaces in the presence of different cations. The most striking effect observed was produced by Mn2+, which increased the binding of the receptor liposomes to fibronectin 2-3-fold over their binding in buffers containing Ca2+ and Mg2+. The binding activities of two related adhesion receptors, the vitronectin receptor and platelet GP IIb-IIIa, were supported but not enhanced by Mn2+. Two observations suggest that Mn2+ can compete with Ca2+ for the same cation-binding sites of the receptor. First, Mn2+ could still enhance fibronectin receptor binding activity even in the presence of 10-fold higher concentrations of Ca2+ or Mg2+. Second, Mn2+ inhibited the binding of radioactive Ca2+ to the alpha subunit of the receptor. The increased fibronectin receptor activity in the presence of Mn2+ appeared to be due to an increase in the affinity of the receptor for the Arg-Gly-Asp sequence because a 110-kDa cell attachment fragment and a synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited liposome binding more effectively in the presence of Mn2+ than in the presence of Ca2+/Mg2+. The affinity for the peptide was affected more than the affinity for the fragment, indicating that Mn2+ also induces a change in receptor specificity. Increased receptor binding in the presence of Mn2+ was also apparent in affinity chromatography of the fibronectin receptor on the 110-kDa fibronectin fragment; Mn2+ improved the yield of the receptor 4-fold. Mn2+ similarly increased the number of receptor-fibronectin complexes in preparations analyzed by electron microscopy. These results show that exogenous influences can modulate the affinity and specificity with which the fibronectin receptor binds to its ligands.     

Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin

We have used a rat neural cell line, B65, to investigate the relative contributions of gangliosides and glycoprotein receptors in adhesion to fibronectin. Monoclonal antibodies against two neuroectoderm-associated gangliosides, D1.1 and GD3, inhibit the rate of B65 attachment to fibronectin, suggesting that these gangliosides are involved in the adhesion process. Adhesion to fibronectin is not affected by a third monoclonal antibody against a separate, unidentified cell-surface component of B65 cells. Furthermore, B65 cells lacking D1.1 adhere to fibronectin at a slower rate than B65 cells that express D1.1. The involvement of glycoprotein receptors in adhesion is demonstrated by the ability of antibodies against human fibronectin receptor to inhibit B65 attachment to fibronectin. In addition, adhesion is blocked by a hexapeptide containing the Arg-Gly-Asp fibronectin sequence which is necessary for binding to the receptor. Trypsin treatment of B65 cells in the absence of divalent cations results in proteolysis of the fibronectin receptor with an accompanying loss of ability of the cells to attach to fibronectin. D1.1 and GD3 expression is not affected by this trypsinization, indicating that the gangliosides alone are incapable of mediating attachment. The glycoprotein receptors must be primarily responsible for adhesion to fibronectin with the gangliosides playing a secondary role as enhancers or modulators.     

Differential properties of attachment of human fibroblasts to various extracellular matrix proteins

Human diploid fibroblasts (TIG-3) were shown to attach and spread onto substrata coated with collagen, fibronectin, laminin and vitronectin. The cell attachment to these proteins required divalent cations. Mg2+ stimulated the cell attachment to all the proteins, while Ca2+ alone was not effective for the attachment to collagen and laminin. A mild trypsin treatment had prevented cells from attaching to the laminin, while it had no effect on the attachment to the other proteins. The fibronectin fragment, which retained cell binding activity, inhibited the cells from attaching and spreading onto fibronectin, but it did not cause any inhibition on the other proteins. The synthetic peptide GRGDSP inhibited the cells from attaching and spreading onto fibronectin and vitronectin, while it did not cause any inhibition on collagen and laminin. In attempts to isolate distinct receptors for these proteins, we were able to purify proteins very similar to the fibronectin and vitronectin receptors of human placenta. Based on the differential properties of the attachment of TIG-3 cells to these proteins and biochemical data, we indicate that human diploid fibroblasts have distinctive binding sites (receptors) for collagen, fibronectin, laminin and vitronectin.     

Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets

Synthetic peptides corresponding to the carboxyl terminus of the fibrinogen gamma chain inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to platelets, yet the active decapeptide sequence has only been found in fibrinogen to date. In contrast, all three proteins contain Arg-Gly-Asp sequences, and peptides containing Arg-Gly-Asp are potent inhibitors of their binding to activated platelets. We have analyzed the relationship between these peptide sets by direct binding assays. H12 (gamma 400-411) inhibited the binding of an Arg-Gly-Asp-containing peptide to platelets with similar dose response to inhibition of fibronectin binding. We have previously reported that GPIIb-IIIa binds to immobilized Arg-Gly-Asp peptides and can be eluted by Arg-Gly-Asp-containing peptides in solution. Both H12 and L10 (gamma 402-411) completely eluted GPIIb-IIIa bound to immobilized Arg-Gly-Asp peptides. Conversely, when GPIIb-IIIa was bound to immobilized L10, either L10 or an Arg-Gly-Asp peptide could elute it. Peptide specificity was established by the failure of Gly-Arg-Gly-Glu-Ser-Pro or acetylated L10 to elute GPIIb-IIIa from the immobilized peptides. These results indicate that the two peptide sets interact with the same receptor which contains GPIIb-IIIa.     

Evidence for the presence of an integrin cell adhesion receptor on the oolemma of unfertilized human oocytes.

The Arg-Gly-Asp peptide (RGD), contained in several extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen, is a tripeptide that plays a role as a recognition sequence in many cell-to-cell and cell-to-matrix adhesion mechanisms, through its interaction with several receptors of the integrin family. We previously described the ability of the oolemma of hamster oocytes to bind GRGDTP coupled to the surface of activated immunobeads and demonstrated that RGD-containing oligopeptides inhibit the adhesion of human and hamster spermatozoa to zona-free hamster oocytes and their subsequent penetration. In the present experiments, we show, utilizing immunobeads coated with an RGD-containing peptide (PepTiteTM 2000), that the oolemma of unfertilized human eggs is also able to recognize this adhesion sequence. The binding of PepTiteTM 2000-coated immunobeads to the oolemma was inhibited by the oligopeptide GRGDTP as well as by fibronectin and laminin. When immunobeads were prepared with a PepTiteTM concentration of 10 micrograms/ml, GRGDTP 150 micrograms/ml, laminin 80 micrograms/ml, and fibronectin 60 micrograms/ml inhibited bead rosetting on the egg surface. These data suggest that a specific binding moiety for RGD is present on the human egg surface. The binding of fibronectin to the oolemma was also demonstrated by the rosetting of immunobeads coupled with antifibronectin antibody to human oocytes after their exposure to 1 mg/ml free fibronectin. Such binding of fibronectin to the oolemma could be inhibited by coincubation with a monoclonal antibody directed against the cell adhesion fragment of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (_Vβ₃/β₅) and fibronectin (₅β₁) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.     

Fluorescence-activated cell-sorting analysis of fibronectin peptides binding to Trypanosoma cruzi trypomastigotes

The binding of synthetic peptides modeled from the sequence of the cell attachment site of fibronectin to T. cruzi trypomastigote surface receptors was investigated by fluorescence-activated cell-sorting analysis using fluorescein-labeled peptides. Peptides with the sequence Arg-Gly-Asp-Ser bound to the parasite surface. A low percentage of fresh parasites recently liberated from infected fibroblasts had the capacity to bind the peptide. In contrast, these parasites showed a time-dependent several-fold increase in their ability to bind the Arg-Gly-Asp-Ser-containing peptides during extracellular incubation. From these observations, it appears that the expression of surface receptors on a particular, mature stage of the parasite parallels its ability to adhere to and infect host cells.