RESUMO
Laminins, major components of basement membrane, consist of three different subunits, α, ß, and γ chains, and so far, five α, three ß, and three γ chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin α chain sequences (α1-α5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the ß1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin ß2 and ß3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin ß1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin ß1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin ß2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin ß3 chain, 182-193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2-105, and B3-19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with α3, α6, and ß1 integrins, and B34 and B3-19 interact with ß1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2-64 (LPRAMDYDLLLRW, mouse laminin ß2 chain, 618-630), the Glu(8) residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide has potential for use as a molecular probe for integrins.