A SPECIFIC SEQUENCE OF THE LAMININ ALPHA-2 CHAIN CRITICAL FOR THE INITIATION OF HETEROTRIMER ASSEMBLY

Triple-stranded laminin molecules assemble via an alpha-helical coiled -coil structure spanning approximately 600 amino acid residues of each chain. We reported that the C termini of the beta 1 and gamma 1 chain s direct the specific dimer and trimer assembly (Utani, A., Nomizu, M. , Timpl, R., Roller, P. P., and Yamada, Y. (1994) J. Biol. Chem. 269, 19167-19175). In this study, we focused on the mechanism of trimer for mation of the alpha 2 chain utilizing three different approaches. Firs t, competition assays using mutated recombinant alpha 2 chain defined a 25-amino acid sequence at the C terminus of the long arm as an essen tial site for assembly with beta 1 and gamma 1 chain. Site-specific mu tations and synthetic peptides of this site revealed that both positiv ely charged amino acid residues and the alpha-helical structure within this site were critical. Second, overexpression studies of recombinan t alpha 2 chain long arm confirmed that the C-terminal end was critica l for the trimer assembly within NIH 3T3 cells. Third, circular dichro ism spectroscopic examination of the complexes reconstituted in vitro revealed dynamic conformational changes of the alpha 2 and gamma 1 cha ins in the process of assembly. These studies also revealed that the p roper folding of the extreme C terminus of alpha 2 chain was critical for the stability of trimer. From these data, it is concluded that the C terminus of alpha 2 chain long arm is required for the effective in itiation of laminin heterotrimer assembly.