A specific sequence of the laminin α2 chain critical for the initiation of heterotrimer assembly

Abstract

Triple-stranded laminin molecules assemble via an α-helical coiled-coil structure spanning approximately 600 amino acid residues of each chain. We reported that the C termini of the β1 and γ1 chains 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 formation of the α2 chain utilizing three different approaches. First, competition assays using mutated recombinant α2 chain defined a 25-amino acid sequence at the C terminus of the long arm as an essential site for assembly with β1 and γ1 chain. Site-specific mutations and synthetic peptides of this site revealed that both positively charged amino acid residues and the α-helical structure within this site were critical. Second, overexpression studies of recombinant α2 chain long arm confirmed that the C-terminal end was critical for the trimer assembly within NIH 3T3 cells. Third, circular dichroism spectroscopic examination of the complexes reconstituted in vitro revealed dynamic conformational changes of the α2 and γ1 chains in the process of assembly. These studies also revealed that the proper folding of the extreme C terminus of α2 chain was critical for the stability of trimer. From these data, it is concluded that the C terminus of α2 chain long arm is required for the effective initiation of laminin heterotrimer assembly.