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 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 alpha 2 chain utilizing three different approaches. First, competition assays using mutated recombinant alpha 2 chain defined a 25-amino acid sequence at the C terminus of the long arm as an essential site for assembly with beta 1 and gamma 1 chain. Site-specific mutations and synthetic peptides of this site revealed that both positively charged amino acid residues and the alpha-helical structure within this site were critical. Second, overexpression studies of recombinant alpha 2 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 alpha 2 and gamma 1 chains in the process of assembly. These studies also revealed that the proper 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 initiation of laminin heterotrimer assembly.
Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892.
A
A
Utani
M
M
Nomizu
S
S
Sugiyama
S
S
Miyamoto
P P
PP
Roller
Y
Y
Yamada
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D007797:Laminin / Q000096:biosynthesis* / Q000737:chemistry*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D007797:Laminin; D046911:Macromolecular Substances; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins