Identification of cell adhesive sequences in the N-terminal region of the laminin ?2 chain.

ABSTRACT: The laminin ?2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of ?2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin ?2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin ?1 and anti-integrin ?2?1 antibodies. These results suggest that A2-8 promotes an integrin ?2?1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin ?2?1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin ?2?1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin ?2?1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.

SUBMITTER: Hozumi K 

PROVIDER: S-EPMC3408208 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature