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.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

Amino acids and peptides. Part 39: a bivalent poly(ethylene glycol) hybrid containing an active site (RGD) and its synergistic site (PHSRN) of fibronectin

Fibronectin contains the active sequence Arg-Gly-Asp (RGD), along with its synergic site Pro-His-Ser-Arg-Asn (PHSRN). However, the PHSRN peptide does not show synergic activity when it is mixed with the RGD peptide, indicating that a spatial array between RGD and PHSRN in fibronectin may be necessary for synergic activity. Here, we have used an amino acid type poly(ethylene glycol) derivative (aaPEG) to design a bivalent PEG hybrid of fibronectin active peptides. We prepared the aaPEG hybrid peptides PHSRN-aaPEG, aaPEG-RGD, and PHSRN-aaPEG-RGD, and tested their biological activity. Whereas aaPEG-RGD promoted cell spreading activity, PHSRN-aaPEG had no activity. The PHSRN-aaPEG-RGD hybrid strongly promoted cell spreading compared with aaPEG-RGD. These results suggest that the PHSRN sequence in the PHSRN-aaPEG-RGD molecule synergistically enhances the cell spreading activity of the RGD sequence, and that the bivalent aaPEG hybrid method may be useful for conjugating functionally active peptides.     

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.     

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)     

In vitro and in vivo analyses of the biological activity of RGD peptides towards Ab Bomirski melanoma

The RGD sequence is present in many extracellular matrix proteins and intracellular proteins, including caspases. Synthetic RGD peptides may affect adhesion, migration and tumour metastasis, or directly induce apoptosis. Several RGD peptides were synthesised, and their anti-adhesive and cytotoxic properties were analysed in vitro. The most active peptide (poly RGD) was also tested in vivo to assess its modulatory activity on melanoma growth. Synthetic RGD peptides inhibit the adhesion of Ab melanoma cells to fibronectin. Poly RGD significantly inhibits primary tumour growth. There was no observed cytotoxicity of poly RGD towards Ab cells in a medium with 10% serum; however, under the same conditions, the anti-adhesive effect of poly RGD was still visible. Experiments on Jurkat cells indicated a weak cytotoxicity of poly RGD and a significant cytotoxicity of GRGDNP (the reference cytotoxic peptide), retained only under serum-free conditions. The anti-tumour effect of poly RGD observed in the Ab Bomirski melanoma model is probably due to an anti-adhesive mechanism. The proapoptotic activity of RGD peptides is dependent on the absence of serum.     

Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.     

Triflavin,an Arg-Gly-Asp-containing peptide,inhibits B16-F10 mouse melanoma cell adhesion to matrix proteins via direct binding to tumor cells

Triflavin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, inhibits B16-F10 mouse melanoma cell adhesion to extracellular matrices, e.g., fibronectin, vitronectin, fibrinogen, and collagen type I. In this study, GRGDS inhibits B16-F10 mouse melanoma cell adhesion to immobilized triflavin in a dose-dependent manner. In addition, flow-cytometric analysis and the fluorescence staining method in which FITC-triflavin is utilized as a binding ligand were used. GRGDS inhibits the binding of FITC-triflavin to B16-F10 cells. Additionally, the above results suggest that triflavin directly binds to its receptors expressed on B16-F10 cell surface primarily via its RGD sequence, there-by inhibiting B16-F10 cell adhesion to extracellular matrices.     

RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities

Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with peptide I. Whereas both peptides promoted RGD-independent cell adhesion, peptide CS1 failed to bind heparin, and exogenous peptide CS1 failed to inhibit peptide I-mediated cell adhesion. The results demonstrate a role for distinct heparin-dependent and -independent cell adhesion determinants on the 33-kD fragment, neither of which are related to the RGD-dependent integrin interaction with fibronectin.     

纤维连接蛋白或精-甘-天冬氨酸肽促进兔支气管上皮细胞NO合成

为验证细胞外基质成分纤维连接蛋白（ｆｉｂｒｏｎｅｃｔｉｎ,Ｆｎ）对气道上皮细胞具有调控作用,本文用硝酸还原酶法测定了原代培养的兔支气管上皮细胞（ｂｒｏｎｃｈｉａｌ ｅｐｉｔｈｅｌｉａｌ ｃｅｌｌｓ,ＢＥＣ）的一氧化氮（ｎｉｔｒｉｃ ｏｘｉｄｅ,ＮＯ）释放,并测定了细胞内一氧化氮合酶（ｎｉｔｒｉｃ ｏｘｉｄｅ ｓｙｎｔｈｅｓａｓｅ,ＮＯＳ）活性,观察纤维连接蛋白、精－甘－天冬氨酸肽（Ａｒｇ－Ｇｌｙ－     

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.     

Triflavin, an Arg-Gly-Asp-containing peptide, inhibits human cervical carcinoma (HeLa) cell-substratum adhesion through an RGD-dependent mechanism

Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins, that have been isolated from the venoms of various vipers and shown to be potent inhibitors of platelet aggregation. The interaction of tumor cells with extracellular matrices such as fibronectin, vitronectin, and collagen has been shown to be mediated through a family of cell surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) sequence within each adhesive protein. In this study, we show that triflavin dose-dependently inhibited adhesion of human cervical carcinoma (HeLa) cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, and vitronectin). On the other hand, triflavin exerted a limited inhibitory effect on cell adhesion to laminin and collagen (type I and IV). On a molar basis, triflavin is approximately 800 times more potent than Gly-Arg-Gly-Asp-Ser (GRGDS) at inhibiting cell adhesion. When immobilized on plate, triflavin significantly promoted HeLa cell adhesion, and this attachment was inhibited by GRGDS. Furthermore, FITC-conjugated triflavin bound to cells in a saturable manner and its binding was inhibited by GRGDS. In addition, triflavin did not affect [³H]thymidine uptake of HeLa cells during a 3-day incubation. These results suggest that triflavin probably binds to integrin receptors expressed on HeLa cell surface via its RGD sequence within its molecule, thereby inhibiting the adhesion of extracellular matrices to HeLa cells.     

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin.

The binding of fibronectin (Fn) to several integrins involves the Arg-Gly-Asp (RGD) tripeptide sequence. However, linear synthetic RGD peptides do not completely mimic the cell attachment activity of intact Fn or certain large Fn fragments. This suggests that the integrin-Fn interaction involves a more extended surface of Fn than that provided by the RGD sequence. To test this possibility, three novel monoclonal anti-Fn antibodies that inhibit its binding to a purified integrin, alpha IIb beta 3, were developed. The epitopes of these three antibodies mapped to a region at least 55 residues amino-terminal of the RGD sequence. Further, recombinant fragments of Fn containing these epitopes and lacking the RGD site also inhibited the binding of Fn to purified alpha IIb beta 3. These fragments, which spanned Fn residues 1359-1436, bound to alpha IIb beta 3 in a divalent cation-dependent manner. In addition, this region of Fn bound specifically to alpha IIb beta 3 on thrombin-stimulated but not resting platelets. These results demonstrate the presence of additional sequences in Fn that interact with integrin alpha IIb beta 3 and suggest that multiple sites in Fn are involved in its recognition by this integrin.     

Inhibition of murine melanoma cell-matrix adhesion and experimental metastasis by albolabrin, an RGD-containing peptide isolated from the venom of Trimeresurus albolabris

Albolabrin, a 7.5-kDa cysteine-rich protein isolated from the venom of Trimeresurus albolabris, contains the arginine-glycine-aspartic acid (RGD) cell recognition sequence found in many cell adhesion-promoting extracellular matrix proteins, such as fibronectin and laminin. Albolabrin belongs to a family of RGD-containing peptides, termed disintegrins, recently isolated from the venom of various vipers and discovered to be potent inhibitors of both platelet aggregation and cell-substratum adhesion. Here we report that albolabrin inhibited the attachment of B16-F10 mouse melanoma cells to either fibronectin or laminin absorbed on plastic. When immobilized on plastic, albolabrin promoted B16-F10 melanoma cell attachment; this was inhibited by either RGD-serine (RGDS) or antibodies to integrins, suggesting that albolabrin binds via its RGD amino sequence to integrin receptors expressed on the melanoma cell surface. In an in vivo experimental metastasis system, albolabrin at a concentration of 300-600 nM inhibited C57BL/6 mouse lung colonization by tail vein-injected mouse melanoma cells and was at least 2000 times more active than RGDS in this assay. We propose that albolabrin inhibits tumor cell metastasis by inhibiting integrin-mediated attachment of melanoma cells to RGD-containing components of the extracellular matrix in the mouse lung.     

Plasmodium falciparum-infected erythrocytes bind to the RGD motif of fibronectin via the band 3-related adhesin

Previously it was shown that Plasmodium falciparum-infected erythrocytes bound to thrombospondin by the interaction of the peptidic sequence, HPLQKTY, of the band 3 protein of infected erythrocytes, and the RGD motif of thrombospondin. Here, we show that falciparum-parasitized erythrocytes bind to immobilized fibronectin by the RGD sequence of fibronectin. Involvement of the HPLQKTY region of band 3 in binding was demonstrated by inhibition of adhesion of parasitized erythrocytes to fibronectin by an HPLQKTY-containing peptide and the binding of the HPLQKTY peptide to the RGD sequence of immobilized fibronectin. Since fibronectin occurs on endothelial cells and platelets, this interaction may contribute to the binding of falciparum-infected erythrocytes to such host cells.     

Role of the RGD sequence in parasite adhesion to host cells

For many protozoan parasites, one of the first events in the process of infection is attachment to the surface of host cells. This adhesion phase usually involves ligand-receptor interactions, and has stimulated interest in the biochemical characterization of those host cell and parasite surface components involved. In this article, Ali Ouaissi discusses the strategy employed by pathogens such as Trypanosoma cruzi, Trichomonas, Leishmania and Treponema pallidum, in binding to their host cells' fibronectin receptors. Two systems appear available - to bind to the dimeric cell surface fibronectin through the Arginine-Glycine-Aspartic acid (RGD) sequence that is not occupied by the host cell surface receptors, or to present a surface antigen representing a 'fibronectin-like' molecule containing the RGD sequence directly to the host cell fibronectin receptors.     

Cell adhesion by aqueous extract of human placenta used as wound healer

Aqueous extract of human placenta, used as wound healer, has shown significant cell adhesion property on mouse peritoneal macrophages and P388D1 cultured macrophage cell line. This property was offered primarily by fibronectin type III like peptide present in the extract and is comparable to fibronectin on a molar basis. The peptide induce adhesion of cell through cell surface receptors having K(d) = 2.8 +/- 0.9 x 10(-5) M suggesting weak binding. This is in support of integrins receptors that typically exhibit low affinities. Cell adhesion was partially inhibited by Arg-Gly-Asp (RGD) peptide, anti-beta1 integrin suggesting that integrin beta1 receptors have roles to play in the process.     

Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor for interstitial matrix

The proteoglycan (PG) on the surface of NMuMG mouse mammary epithelial cells consists of at least two functional domains, a membrane- intercalated domain which anchors the PG to the plasma membrane, and a trypsin-releasable ectodomain which bears both heparan and chondroitin sulfate chains. The ectodomain binds cells to collagen types I, III, and V, but not IV, and has been proposed to be a matrix receptor. Because heparin binds to the adhesive glycoproteins fibronectin, an interstitial matrix component, and laminin, a basal lamina component, we asked whether the cell surface PG also binds these molecules. Cells harvested with either trypsin or EDTA bound to fibronectin; binding of trypsin-released cells was inhibited by the peptide GRGDS but not by heparin, whereas binding of EDTA-released cells was inhibited only by a combination of GRDS and heparin, suggesting two distinct cell binding mechanisms. In the presence of GRGDS, the EDTA-released cells bound to fibronectin via the cell surface PG. Binding via the cell surface PG was to the COOH-terminal heparin binding domain of fibronectin. In contrast with the binding to fibronectin, EDTA-released cells did not bind to laminin under identical assay conditions. Liposomes containing the isolated intact cell surface PG mimic the binding of whole cells. These results indicate that the mammary epithelial cells have at least two distinct cell surface receptors for fibronectin: a trypsin- resistant molecule that binds cells to the sequence RGD and a trypsin- labile, heparan sulfate-rich PG that binds cells to the COOH-terminal heparin binding domain. Because the cell surface PG binds cells to the interstitial collagens (types I, III, and V) and to fibronectin, but not to basal lamina collagen (type IV) or laminin, we conclude that the cell surface PG is a receptor on epithelial cells specific for interstitial matrix components.     

Comparative cell response to artificial extracellular matrix proteins containing the RGD and CS5 cell-binding domains

This study addresses endothelial cell adhesion and spreading on a family of artificial extracellular matrix (aECM) proteins designed for application in small-diameter vascular grafts. The aECM proteins contain domains derived from elastin and from fibronectin. aECM 1 contains the RGD sequence from the tenth type III domain of fibronectin; aECM 3 contains the fibronectin CS5 cell-binding domain. Negative control proteins aECM 2 and 4 are scrambled versions of aECM 1 and 3, respectively. Competitive peptide inhibition studies and comparisons of positive and negative control proteins confirm that adhesion of HUVECs to aECM proteins 1 and 3 is sequence specific. When subjected to a normal detachment force of 780 pN, 3-fold more HUVECs remained adherent to aECM 1 than to aECM 3. HUVECs also spread more rapidly on aECM 1 than on aECM 3. These results (i) indicate that cellular responses to aECM proteins can be modulated through choice of cell-binding domain and (ii) recommend the RGD sequence for applications that require rapid endothelial cell spreading and matrix adhesion.     

A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins

Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*- peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD.