Basement membranes are thin sheets of extracellular matrix surrounding most tissues and play a critical role in tissue development, repair, and maintenance.
The aim of this project is to understand the molecular mechanisms underlying the role of the basement membranes in these biological processes. Basement membranes contain a unique set of proteins, such as collagen IV, laminin, perlecan and nidogen/entactin. Laminin is a family of heteromeric glycoproteins specific in basement membranes and has a number of biological activities. Mouse laminin beta3 and gamma2 chains were cloned and their primary structure determined. Their tissue-specific expression was also studied by in situ and Northern hybridization. The diversity of laminin chains has raised the question of how the chains are selected and assembled into intact laminin molecules. The assembly of laminin chains to double- and triple-stranded structures was studied with recombinant proteins and synthetic peptides. We have found that chain selection is controlled by the C-terminal region of the alpha-helical domain of each chain. We found that thermodynamically stable trimer formation requires a minimum sequence of about 50 amino acids, including the core sequence necessary for assembly. Circular dichroism (CD) analysis revealed that beta1 peptide homodimers are more stable than beta1/gamma1 heterodimers. However, a mixture of beta1 and gamma1 peptides preferentially forms beta1/gamma1 heterodimers. The relatively unstable structure of beta1/gamma1 heterodimers can facilitate the formation of stable heterotrimers with the alpha chain. We have screened a series of overlapping peptides that cover most of the domain G of laminin alpha1 chain (amino acids residues 2111-3060) to identified bioactive sites. Five new peptides (AG-10, AG-22, AG-32, AG-56 and AG-73) had cell attachment activity. AG-32 and AG-73 were chemotactic and promoted neurite outgrowth. We have cloned and characterized a new rhoGAP protein, p190B. Our results suggest that signals from extracellular matrix molecules may influence the activity of p190 and rho proteins. We have found that transcription of the collagen IV genes in F9 cells is regulated not only by enhancers and protein factors, but also by the chromatin structure.
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