Laminin α1 Chain LG4 Module Promotes Cell Attachment through Syndecans and Cell Spreading through Integrin α2β1

The laminin α1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin α1 contains major binding sites for heparin, sulfatide, and α-dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of lamininα1 LG4 for binding to syndecan and integrin α2β1, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and α-dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and α-dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin α2 and β1 but not by anti-integrin α1 and α6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin α2β1. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or antiintegrin α2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin α1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.