PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
 
J Cell Biol. 1995 September 1; 130(5): 1189–1196.
PMCID: PMC2120548

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

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (1.1M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abrams C, Deng YJ, Steiner B, O'Toole T, Shattil SJ. Determinants of specificity of a baculovirus-expressed antibody Fab fragment that binds selectively to the activated form of integrin alpha IIb beta 3. J Biol Chem. 1994 Jul 22;269(29):18781–18788. [PubMed]
  • Bajt ML, Ginsberg MH, Frelinger AL, 3rd, Berndt MC, Loftus JC. A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site. J Biol Chem. 1992 Feb 25;267(6):3789–3794. [PubMed]
  • Bajt ML, Loftus JC. Mutation of a ligand binding domain of beta 3 integrin. Integral role of oxygenated residues in alpha IIb beta 3 (GPIIb-IIIa) receptor function. J Biol Chem. 1994 Aug 19;269(33):20913–20919. [PubMed]
  • Bowditch RD, Hariharan M, Tominna EF, Smith JW, Yamada KM, Getzoff ED, Ginsberg MH. Identification of a novel integrin binding site in fibronectin. Differential utilization by beta 3 integrins. J Biol Chem. 1994 Apr 8;269(14):10856–10863. [PubMed]
  • Calvete JJ, Rivas G, Schäfer W, McLane MA, Niewiarowski S. Glycoprotein IIb peptide 656-667 mimics the fibrinogen gamma chain 402-411 binding site on platelet integrin GPIIb/IIIa. FEBS Lett. 1993 Nov 29;335(1):132–135. [PubMed]
  • D'Souza SE, Ginsberg MH, Burke TA, Lam SC, Plow EF. Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science. 1988 Oct 7;242(4875):91–93. [PubMed]
  • D'Souza SE, Ginsberg MH, Burke TA, Plow EF. The ligand binding site of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit. J Biol Chem. 1990 Feb 25;265(6):3440–3446. [PubMed]
  • D'Souza SE, Ginsberg MH, Lam SC, Plow EF. Chemical cross-linking of arginyl-glycyl-aspartic acid peptides to an adhesion receptor on platelets. J Biol Chem. 1988 Mar 15;263(8):3943–3951. [PubMed]
  • D'Souza SE, Haas TA, Piotrowicz RS, Byers-Ward V, McGrath DE, Soule HR, Cierniewski C, Plow EF, Smith JW. Ligand and cation binding are dual functions of a discrete segment of the integrin beta 3 subunit: cation displacement is involved in ligand binding. Cell. 1994 Nov 18;79(4):659–667. [PubMed]
  • Dickinson CD, Gay DA, Parello J, Ruoslahti E, Ely KR. Crystals of the cell-binding module of fibronectin obtained from a series of recombinant fragments differing in length. J Mol Biol. 1994 Apr 22;238(1):123–127. [PubMed]
  • Dickinson CD, Veerapandian B, Dai XP, Hamlin RC, Xuong NH, Ruoslahti E, Ely KR. Crystal structure of the tenth type III cell adhesion module of human fibronectin. J Mol Biol. 1994 Mar 4;236(4):1079–1092. [PubMed]
  • Gartner TK, Taylor DB. The amino acid sequence Gly-Ala-Pro-Leu appears to be a fibrinogen binding site in the platelet integrin, glycoprotein IIb. Thromb Res. 1990 Nov 15;60(4):291–309. [PubMed]
  • Ginsberg MH, Loftus JC, Plow EF. Cytoadhesins, integrins, and platelets. Thromb Haemost. 1988 Feb 25;59(1):1–6. [PubMed]
  • Haas TA, Plow EF. Integrin-ligand interactions: a year in review. Curr Opin Cell Biol. 1994 Oct;6(5):656–662. [PubMed]
  • Healy JM, Murayama O, Maeda T, Yoshino K, Sekiguchi K, Kikuchi M. Peptide ligands for integrin alpha v beta 3 selected from random phage display libraries. Biochemistry. 1995 Mar 28;34(12):3948–3955. [PubMed]
  • Hemler ME. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol. 1990;8:365–400. [PubMed]
  • Hibbs ML, Xu H, Stacker SA, Springer TA. Regulation of adhesion of ICAM-1 by the cytoplasmic domain of LFA-1 integrin beta subunit. Science. 1991 Mar 29;251(5001):1611–1613. [PubMed]
  • Humphries MJ, Akiyama SK, Komoriya A, Olden K, Yamada KM. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J Cell Biol. 1986 Dec;103(6 Pt 2):2637–2647. [PMC free article] [PubMed]
  • Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. [PubMed]
  • Juliano RL, Haskill S. Signal transduction from the extracellular matrix. J Cell Biol. 1993 Feb;120(3):577–585. [PMC free article] [PubMed]
  • Koivunen E, Gay DA, Ruoslahti E. Selection of peptides binding to the alpha 5 beta 1 integrin from phage display library. J Biol Chem. 1993 Sep 25;268(27):20205–20210. [PubMed]
  • Koivunen E, Wang B, Dickinson CD, Ruoslahti E. Peptides in cell adhesion research. Methods Enzymol. 1994;245:346–369. [PubMed]
  • Koivunen E, Wang B, Ruoslahti E. Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library. J Cell Biol. 1994 Feb;124(3):373–380. [PMC free article] [PubMed]
  • Koivunen E, Wang B, Ruoslahti E. Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins. Biotechnology (N Y) 1995 Mar;13(3):265–270. [PubMed]
  • Lee JO, Rieu P, Arnaout MA, Liddington R. Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell. 1995 Feb 24;80(4):631–638. [PubMed]
  • Main AL, Harvey TS, Baron M, Boyd J, Campbell ID. The three-dimensional structure of the tenth type III module of fibronectin: an insight into RGD-mediated interactions. Cell. 1992 Nov 13;71(4):671–678. [PubMed]
  • Michishita M, Videm V, Arnaout MA. A novel divalent cation-binding site in the A domain of the beta 2 integrin CR3 (CD11b/CD18) is essential for ligand binding. Cell. 1993 Mar 26;72(6):857–867. [PubMed]
  • Morla A, Zhang Z, Ruoslahti E. Superfibronectin is a functionally distinct form of fibronectin. Nature. 1994 Jan 13;367(6459):193–196. [PubMed]
  • O'Neil KT, Hoess RH, Jackson SA, Ramachandran NS, Mousa SA, DeGrado WF. Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library. Proteins. 1992 Dec;14(4):509–515. [PubMed]
  • O'Toole TE, Katagiri Y, Faull RJ, Peter K, Tamura R, Quaranta V, Loftus JC, Shattil SJ, Ginsberg MH. Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol. 1994 Mar;124(6):1047–1059. [PMC free article] [PubMed]
  • Obara M, Kang MS, Yamada KM. Site-directed mutagenesis of the cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function. Cell. 1988 May 20;53(4):649–657. [PubMed]
  • Ohno S. Active sites of ligands and their receptors are made of common peptides that are also found elsewhere. J Mol Evol. 1995 Jan;40(1):102–106. [PubMed]
  • Pasqualini R, Chamone DF, Brentani RR. Determination of the putative binding site for fibronectin on platelet glycoprotein IIb-IIIa complex through a hydropathic complementarity approach. J Biol Chem. 1989 Aug 25;264(24):14566–14570. [PubMed]
  • Pierschbacher MD, Hayman EG, Ruoslahti E. Location of the cell-attachment site in fibronectin with monoclonal antibodies and proteolytic fragments of the molecule. Cell. 1981 Oct;26(2 Pt 2):259–267. [PubMed]
  • Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 1984 May 3;309(5963):30–33. [PubMed]
  • Plow EF, Srouji AH, Meyer D, Marguerie G, Ginsberg MH. Evidence that three adhesive proteins interact with a common recognition site on activated platelets. J Biol Chem. 1984 May 10;259(9):5388–5391. [PubMed]
  • Pytela R, Pierschbacher MD, Argraves S, Suzuki S, Ruoslahti E. Arginine-glycine-aspartic acid adhesion receptors. Methods Enzymol. 1987;144:475–489. [PubMed]
  • Ruoslahti E. Integrins. J Clin Invest. 1991 Jan;87(1):1–5. [PMC free article] [PubMed]
  • Santoro SA, Lawing WJ., Jr Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins. Cell. 1987 Mar 13;48(5):867–873. [PubMed]
  • Smith GP, Scott JK. Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol. 1993;217:228–257. [PubMed]
  • Smith JW, Cheresh DA. The Arg-Gly-Asp binding domain of the vitronectin receptor. Photoaffinity cross-linking implicates amino acid residues 61-203 of the beta subunit. J Biol Chem. 1988 Dec 15;263(35):18726–18731. [PubMed]
  • Smith JW, Hu D, Satterthwait A, Pinz-Sweeney S, Barbas CF., 3rd Building synthetic antibodies as adhesive ligands for integrins. J Biol Chem. 1994 Dec 30;269(52):32788–32795. [PubMed]
  • Takada Y, Ylänne J, Mandelman D, Puzon W, Ginsberg MH. A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques. J Cell Biol. 1992 Nov;119(4):913–921. [PMC free article] [PubMed]
  • Taub R, Gould RJ, Garsky VM, Ciccarone TM, Hoxie J, Friedman PA, Shattil SJ. A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain in fibrinogen. J Biol Chem. 1989 Jan 5;264(1):259–265. [PubMed]
  • Tomiyama Y, Brojer E, Ruggeri ZM, Shattil SJ, Smiltneck J, Gorski J, Kumar A, Kieber-Emmons T, Kunicki TJ. A molecular model of RGD ligands. Antibody D gene segments that direct specificity for the integrin alpha IIb beta 3. J Biol Chem. 1992 Sep 5;267(25):18085–18092. [PubMed]
  • Vuori K, Ruoslahti E. Association of insulin receptor substrate-1 with integrins. Science. 1994 Dec 2;266(5190):1576–1578. [PubMed]
  • Yatohgo T, Izumi M, Kashiwagi H, Hayashi M. Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct Funct. 1988 Aug;13(4):281–292. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press