SELECTION OF PEPTIDES BINDING TO THE ALPHA-5-BETA-1 INTEGRIN FROM PHAGE DISPLAY LIBRARY

The alpha5beta1 integrin binds fibronectin through the integrin recogn ition sequence Arg-Gly-Asp (RGD). We have used a 6-amino acid peptide library expressed on filamentous phage to identify peptide ligands for alpha5beta1. We found that this integrin selectively binds RGD-contai ning peptides from the library. Of the 32 different sequences obtained , 28 had the RGD motif, 3 contained sequences related to RGD, and only 1 had a clearly different sequence. One of the RGD-containing phage e ncoded a potentially cyclic insert CRGDCL. The cyclic peptide GACRGDC LGA (where * denotes cysteines forming a disulfide bond) was 10-fold more efficient than any of the linear RGD-containing hexapeptides in i nhibiting the binding of RGD-expressing phage to alpha5beta1 or the at tachment of alpha5beta1-expressing cells to fibronectin. This peptide also inhibited cell attachment mediated by the alpha(v)beta1, alpha(v) beta3, and alpha(v)beta5, integrins with about 10-fold higher efficien cy than linear GRGDSP. One peptide containing an RGD-related sequence, NGRAHA, was also found to inhibit phage attachment and cell adhesion, especially adhesion mediated by the alpha(v)beta5 integrin. These res ults indicate that novel and high affinity ligands for integrins can b e isolated from a random peptide library.