Laminin-111 is a large trimeric basement membrane glycoprotein with many active sites. In particular, four peptides active in tumor malignancy studies have been identified in laminin-111 using a systematic peptide screening method followed by various assays. Two of the peptides (IKVAV and AG73) are found on the α1 chain, one (YIGSR) of the β1 chain and one (C16) on the γ1 chain. The four peptides have distinct activities and receptors. Since three of the peptides (IKVAV, AG73 and C16) strongly promote tumor growth, this may explain the potent effects laminin-111 has on malignant cells. The peptide, YIGSR, decreases tumor growth and experimental metastasis via a 32/67 kD receptor while IKVAV increases tumor growth, angiogenesis and protease activity via integrin receptors. AG73 increases tumor growth and metastases via syndecan receptors. C16 increases tumor growth and angiogenesis via integrins. Identification of such sites on laminin-111 will have use in defining strategies to develop therapeutics for ca...

Laminin-1, an extracellular matrix molecule, promotes neurite outgrowth through the interaction of integrin and actin. Monosialoganglioside GM1 in the lipid rafts associates with and activates the NGF receptor TrkA, and enhances neurite outgrowth. However, the role of GM1 in laminin-1-induced neurite outgrowth was still unclear. Here, we describe that laminin-1 binds to GM1 through a carbohydrate moiety and a specific conformation of GM1, induces focal formation of large clusters of GM1, and enhances the relocation of TrkA in the membrane of dorsal root ganglion (DRG) and PC12 cells. We found that laminin-1-mediated clustering of GM1 causes the translocation and enrichment of β1 integrin in lipid rafts – where TrkA colocalizes with β1 integrin – and the activation of Lyn, Akt and MAPK to promote the outgrowth of neurites. Our results suggest that the binding of laminin-1 to GM1 facilitates the formation of a focal microdomain in the membrane, and enhances signal transduction that promotes neurite outgrowth by linking NGF-TrkA signaling with the laminin-integrin signaling pathways.

We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin β1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7.

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.