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.