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.

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Avian neural crest cell attachment to laminin: involvement of divalent cation dependent and independent integrins

Development ◽

10.1242/dev.113.4.1069 ◽

1991 ◽

Vol 113 (4) ◽

pp. 1069-1084 ◽

Cited By ~ 2

Author(s):

T. Lallier ◽

M. Bronner-Fraser

Keyword(s):

Neural Crest ◽

Cell Attachment ◽

Divalent Cations ◽

Neural Crest Cell ◽

Neural Crest Cells ◽

Cell Interaction ◽

Cell Binding ◽

Cell Adherence ◽

Beta 1 Integrin ◽

Crest Cell

The mechanisms of neural crest cell interaction with laminin were explored using a quantitative cell attachment assay. With increasing substratum concentrations, an increasing percentage of neural crest cells adhere to laminin. Cell adhesion at all substratum concentrations was inhibited by the CSAT antibody, which recognizes the chick beta 1 subunit of integrin, suggesting that beta 1-integrins mediate neural crest cell interactions with laminin. The HNK-1 antibody, which recognizes a carbohydrate epitope, inhibited neural crest cell attachment to laminin at low coating concentrations (greater than 1 microgram ml-1; Low-LM), but not at high coating concentration of laminin (10 micrograms ml-1; High-LM). Attachment to Low-LM occurred in the absence of divalent cations, whereas attachment to High-LM required greater than 0.1 mM Ca2+ or Mn2+. Neural crest cell adherence to the E8 fragment of laminin, derived from its long arm, was similar to that on intact laminin at high and low coating concentrations, suggesting that this fragment contains the neural crest cell binding site(s). The HNK-1 antibody recognizes a protein of 165,000 Mr which is also found in immunoprecipitates using antibodies against the beta 1 subunit of integrin and is likely to be an integrin alpha subunit or an integrin-associated protein. Our results suggest that the HNK-1 epitope on neural crest cells is present on or associated with a novel or differentially glycosylated form of beta 1-integrin, which recognizes laminin in the apparent absence of divalent cations. We conclude that neural crest cells have at least two functionally independent means of attachment to laminin which are revealed at different substratum concentrations and/or conformations of laminin.

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Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain.

Molecular Biology of the Cell ◽

10.1091/mbc.5.5.565 ◽

1994 ◽

Vol 5 (5) ◽

pp. 565-574 ◽

Cited By ~ 145

Author(s):

D R Senger ◽

C A Perruzzi ◽

A Papadopoulos-Sergiou ◽

L Van de Water

Keyword(s):

Cell Surface ◽

Cleavage Site ◽

Cell Attachment ◽

Binding Domain ◽

Adhesion Assay ◽

Cell Binding ◽

Thrombin Cleavage ◽

Close Proximity ◽

Alpha V Beta 3 ◽

Cell Binding Domain

Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were performed with uncleaved and cleaved forms of OPN. For all cell lines examined, thrombin-cleaved OPN promoted markedly greater cell attachment and spreading than uncleaved OPN. Cell attachment and spreading on thrombin-cleaved OPN was inhibited both by the soluble GRGDS peptides and an OPN-specific antibody raised to the GRGDS domain of OPN, thus implicating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest that possibility that thrombin cleavage allows greater accessibility of the GRGDS domain to cell surface receptors. To investigate receptors that recognize uncleaved and thrombin-cleaved OPN, affinity chromatography was performed on placental extracts; the cell surface integrin alpha v beta 3 bound to columns constructed either with native or thrombin-cleaved OPN and was selectively eluted from each with soluble GRGDS peptide and EDTA. Moreover, adhesion assays performed in the presence of alpha v beta 3 blocking monoclonal antibody LM609 identified alpha v beta 3 as a major functional receptor for thrombin-cleaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue injury, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.

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Screening and identification of a novel hepatocellular carcinoma cell binding peptide by using a phage display library

Journal of Huazhong University of Science and Technology [Medical Sciences] ◽

10.1007/s11596-008-0316-1 ◽

2008 ◽

Vol 28 (3) ◽

pp. 299-303 ◽

Cited By ~ 5

Author(s):

Xiaohua Zhu ◽

Hua Wu ◽

Sha Luo ◽

Zhiqun Xianyu ◽

Dan Zhu

Keyword(s):

Hepatocellular Carcinoma ◽

Phage Display ◽

Carcinoma Cell ◽

Phage Display Library ◽

Cell Binding ◽

Binding Peptide ◽

Screening And Identification ◽

Hepatocellular Carcinoma Cell

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Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.

The Journal of Cell Biology ◽

10.1083/jcb.110.6.2145 ◽

1990 ◽

Vol 110 (6) ◽

pp. 2145-2155 ◽

Cited By ~ 254

Author(s):

A Sonnenberg ◽

C J Linders ◽

P W Modderman ◽

C H Damsky ◽

M Aumailley ◽

...

Keyword(s):

Cell Adhesion ◽

Cell Types ◽

Integrin Subunit ◽

Integrin Receptors ◽

Cell Binding ◽

Vitronectin Receptor ◽

Beta 1 Integrin ◽

Integrin Alpha 6 ◽

Alpha V Beta 3 ◽

Integrin Family

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.

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Effects on Rotavirus Cell Binding and Infection of Monomeric and Polymeric Peptides Containing α2β1 and αxβ2 Integrin Ligand Sequences

Journal of Virology ◽

10.1128/jvi.78.21.11786-11797.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 11786-11797 ◽

Cited By ~ 22

Author(s):

Kate L. Graham ◽

Weiguang Zeng ◽

Yoshikazu Takada ◽

David C. Jackson ◽

Barbara S. Coulson

Keyword(s):

Cell Surface ◽

Cell Attachment ◽

Surface Expression ◽

Cell Entry ◽

Cell Surface Expression ◽

Αvβ3 Integrin ◽

Cell Binding ◽

Virus Binding ◽

Genistein Treatment ◽

Integrin Ligand

ABSTRACT Integrin-using rotaviruses bind MA104 cell surface α2β1 integrin via the Asp-Gly-Glu (DGE) sequence in virus spike protein VP4 and interact with αxβ2 integrin during cell entry through outer capsid protein VP7. Infection is inhibited by the α2β1 ligand Asp-Gly-Glu-Ala (DGEA) and the αxβ2 ligand Gly-Pro-Arg-Pro (GPRP), and virus-α2β1 binding is increased by α2β1 activation. In this study, we analyzed the effects of monomers and polymers containing DGEA-, GPRP-, and DGEA-related peptides on rotavirus binding and infection in intestinal (Caco-2) and kidney (MA104) cells and virus binding to recombinant α2β1. Blockade of rotavirus-cell binding and infection by peptides and anti-α2 antibody showed that Caco-2 cell entry is dependent on virus binding to α2β1 and interaction with αxβ2. At up to 0.5 mM, monomeric DGEA and DGAA inhibited binding to α2β1 and infection. At higher concentrations, DGEA and DGAA showed a reduced ability to inhibit virus-cell binding and infection that depended on virus binding to α2β1 but occurred without alteration in cell surface expression of α2, β2, or αvβ3 integrin. This loss of DGEA activity was abolished by genistein treatment and so was dependent on tyrosine kinase signaling. It is proposed that this signaling activated existing cell surface α2β1 to increase virus-cell attachment and entry. Polymeric peptides containing DGEA and GPRP or GPRP only were inhibitory to SA11 infection at approximately 10-fold lower concentrations than peptide monomers. As polymerization can improve peptide inhibition of virus-receptor interactions, this approach could be useful in the development of inhibitors of receptor recognition by other viruses.

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Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin.

The Journal of Cell Biology ◽

10.1083/jcb.113.4.931 ◽

1991 ◽

Vol 113 (4) ◽

pp. 931-941 ◽

Cited By ~ 48

Author(s):

S L Goodman ◽

M Aumailley ◽

H von der Mark

Keyword(s):

Cell Surface ◽

Cell Attachment ◽

Heat Denaturation ◽

Surface Receptors ◽

Murine Tumor ◽

Heat Stable ◽

Multiple Cell ◽

Beta 1 Integrin ◽

Mediate Cell ◽

Alpha V Beta 3

Cell surface molecules that interact with the cross formed by the three short arms of murine tumor laminin were studied using thermal perturbation, antibody and peptide blocking, and affinity chromatography. Several potential receptors for the laminin short arms were revealed that differed from those mediating cell attachment to the E8 (long arm) fragment. Two cell lines, Rugli and L8 attached well to E1-X (short arm) fragments of laminin. This attachment was blocked by antibodies against alpha 1 integrin chains. Other cells were unable to attach strongly to E1-X, but attached to P1. This attachment was unaffected by anti-beta 1 integrin antibodies, but specifically blocked by the peptide GRGDS. By contrast, binding of Rugli cells was RGD independent and blocked by anti-beta 1 integrin antibodies. G7 and C2C12 myoblasts were very sensitive to GRGDS (ID50 approximately 2 micrograms.ml-1) for attachment to P1 which implied that a non-beta 1 series integrin, possibly alpha v beta 3, was involved. On heat denaturation of P1(3) attachment remained sensitive to RGDS and ID50 was unchanged. On heat denaturation of E1-X, attachment remained sensitive to RGDS but the ID50 increased to approximately 200 micrograms.ml-1. Cellular beta 1 integrins were retained on laminin affinity columns. A beta 1 integrin with an approximately 190 kD alpha-chain could be isolated from Rugli cells whose attachment could be blocked by anti-alpha 1 antibodies and not from cells blocked by RGDS peptides. Anti-alpha 1 antibodies blocked Rugli attachment to native laminin, but only when the E8 cell binding sites on laminin were also blocked. Thus, a receptor related to alpha 1 beta 1 integrin can function simultaneously with a receptor for E8. Anti-alpha 1 also blocked attachment to heated laminin, suggesting that the heat-stable attachment activity in laminin involved the E1-X binding site. Thus, at least two putative receptors mediate attachment to the short arms of laminin. One, related to alpha 1 beta 1 integrin, recognizes RGDS-independent sites in E1-X defined by P1 (within domains III, IIIa, IIIb), and one is an RGD-dependent molecule recognizing sites in P1, and is not a beta 1 integrin.

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Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library

Journal of Immunological Methods ◽

10.1016/s0022-1759(00)00340-9 ◽

2001 ◽

Vol 248 (1-2) ◽

pp. 17-30 ◽

Cited By ~ 78

Author(s):

Tara Heitner ◽

Anne Moor ◽

Jennifer L Garrison ◽

Cara Marks ◽

Tayyaba Hasan ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Phage Display ◽

Growth Factor Receptor ◽

Phage Display Library ◽

Cell Binding ◽

Epidermal Growth ◽

Receptor Antibodies ◽

Selection Of

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Beta 1 integrin-dependent and -independent polymerization of fibronectin.

The Journal of Cell Biology ◽

10.1083/jcb.132.1.227 ◽

1996 ◽

Vol 132 (1) ◽

pp. 227-238 ◽

Cited By ~ 201

Author(s):

K Wennerberg ◽

L Lohikangas ◽

D Gullberg ◽

M Pfaff ◽

S Johansson ◽

...

Keyword(s):

Large Cell ◽

Mouse Cell ◽

Integrin Subunit ◽

Cell Binding ◽

Matrix Assembly ◽

Focal Contacts ◽

Fibronectin Matrix ◽

Beta 1 Integrin ◽

Alpha V Beta 3 ◽

Fibronectin Fibrils

The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.

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