Study on the function of laminin by producing transgenic mice and fruit flies

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Laminins are heterotrimers with alpha, beta, and gamma chains held together by a triple-stranded alpha-helical coiled-coil structure. This structure is highly conserved among laminins from mammalians and invertebrates. Various isoforms of laminin chains have been found and this diversity raised the question how laminin chains are selected for the heterotrimer assembly. We here studied in vivo process of the heterotrimer assembly using four experimental systems. First, we expressed mouse laminin alpha1, beta1 or gamma1 sequence covering various region of the long arm in monkey COS1 cells and found that a common mechanism is shared by laminin chains of different animal origins. We also found that the sequences in E8 domain at C-terminal end of the long arm are crucial for the assembly. Second, we expressed mouse laminin beta1 covering either C-terminal end or whole of the long arm in mouse embryonal carcinoma F9 cells. Both fragments were disulfide-bonded to endogenous gamma1 and formed trimers with alpha1, which was actively secreted into the medium. However, beta1 sequence covering C-terminal end could not support the disulfide-bonding of alpha1 at N-terminus of the long arm. Third, we developed chain specific antibodies directed against Drosophila laminin alpha, beta and gamma and found that the disulfide-bonding between Drosophila beta and gamma is essential for alpha to from alphabetagamma heterotrimer. Fourth, we found that laminin-8 with chain composition of alpha4beta1gamma1 is the only isoform expressed in 3T3-L1 adipocytes. Northern blot showed the levels of alpha4, beta1 and gamma1 mRNAs increase 2.5-fold during the adipose conversion of the cells.