Identification of Cell Binding Sequences in Mouse Laminin γ1 Chain by Systematic Peptide Screening (original) (raw)

1997, Journal of Biological Chemistry

Laminin-1, a major component of basement membranes, consists of three different chains designated ␣1, ␤1, and ␥1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin ␥1 chain, using systematic screening of 165 overlapping synthetic peptides covering the entire chain. We identified 12 cell binding sequences using HT-1080 human fibrosarcoma and B16-F10 mouse melanoma cells in two independent assays employing peptide-conjugated Sepharose beads and peptide-coated dishes. Four peptides (C-16, C-28, C-64, and C-68) located on the globular domains of the ␥1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-␣ 2 ␤ 1 integrin antibodies. Cell attachment to C-16 and C-64 was partially inhibited by EDTA but was not inhibited by anti-integrin antibodies. EDTA and anti-integrin antibodies did not affect cell attachment to C-28. The four peptides were tested in adhesion and differentiation assays with endothelial, neuronal, and human salivary gland cells. C-16 was the most active for all of the cells, whereas the other three peptides showed cell type specificity in their activities. The active core sequences of C-16, C-28, C-64, and C-68 are YVRL, IRVTLN, TTVKYIFR, and SIKIRGTY, respectively. These sequences are highly conserved among the different species and in the laminin ␥2 chain. These results suggest that the specific sequences on the laminin ␥1 chain are biologically active and interact with distinct cell surface receptors. Laminin-1, a major component of the basement membrane matrix, has multiple biological activities including promotion of cell adhesion, spreading, growth, neurite outgrowth, tumor metastasis, and collagenase IV secretion (1-4). There are at least 11 isoforms of laminin, each consisting of three different chains (5). The most extensively characterized laminin, laminin-1 (M r ϭ 900,000) from the mouse Engelbreth-Holm-Swarm tumor consists of ␣1, ␤1, and ␥1 chains, which assemble into a triple-stranded cross-like structure with a coiled-coil domain at the long arm (6). Several active sequences on laminin-1 have been identified using proteolytic fragments, recombinant proteins and syn