title: 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.
authors.affiliation: Cancer Research Center, La Jolla Cancer Research Foundation, California 92037, USA.
authors.forename: R
authors.initials: R
authors.lastname: Pasqualini
authors.forename: E
authors.initials: E
authors.lastname: Koivunen
authors.forename: E
authors.initials: E
authors.lastname: Ruoslahti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000502:physiology; D002448:Cell Adhesion / Q000235:genetics; D005353:Fibronectins / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D016023:Integrins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010455:Peptides / Q000276:immunology; D012685:Sepharose
chemical_list: D005353:Fibronectins; D016023:Integrins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; C047981:arginyl-glycyl-aspartic acid; D012685:Sepharose