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.
Koivunen
E
E
Cancer Research Center, La Jolla Cancer Research Foundation, California 92037.
Wang
B
B
Ruoslahti
E
E
D000595:Amino Acid Sequence; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D002460:Cell Line; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D017469:Receptors, Fibronectin; D014407:Tumor Cells, Cultured
D005353:Fibronectins; D016023:Integrins; D009842:Oligopeptides; D010456:Peptides, Cyclic; D017469:Receptors, Fibronectin; C047981:arginyl-glycyl-aspartic acid