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

J Cell Biol. 1995 Sep;130(5):1189-96. doi: 10.1083/jcb.130.5.1189.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Bacteriophages / genetics
  • Binding Sites / genetics
  • Binding, Competitive / physiology
  • Cell Adhesion / genetics
  • Fibronectins / metabolism
  • Gene Library
  • Humans
  • Immunoblotting
  • Integrins / antagonists & inhibitors
  • Integrins / metabolism*
  • Integrins / ultrastructure
  • Mice
  • Mice, Inbred Strains
  • Molecular Sequence Data
  • Oligopeptides / metabolism*
  • Peptide Fragments / genetics
  • Peptide Fragments / isolation & purification*
  • Peptide Fragments / metabolism*
  • Peptides / immunology
  • Sepharose

Substances

  • Fibronectins
  • Integrins
  • Oligopeptides
  • Peptide Fragments
  • Peptides
  • arginyl-glycyl-aspartic acid
  • Sepharose