Identification of cell-binding sites on the Laminin alpha 5 N-terminal domain by site-directed mutagenesis.
The newly discovered laminin alpha(5) chain is a multidomain, extracellular matrix protein implicated in various biological functions such as the development of blood vessels and nerves. The N-terminal globular domain of the laminin alpha chains has an important role for biological activities through interactions with cell surface receptors. In this study, we identified residues that are critical for cell binding within the laminin alpha(5) N-terminal globular domain VI (approximately 270 residues) using site-directed mutagenesis and synthetic peptides. A recombinant protein of domain VI and the first four epidermal growth factor-like repeats of domain V, generated in a mammalian expression system, was highly active for HT-1080 cell binding, while a recombinant protein consisting of only the epidermal growth factor-like repeats showed no cell binding. By competition analysis with synthetic peptides for cell binding, we identified two sequences: S2, (123)GQVFHVAYVLIKF(135) and S6, (225)RDFTKATNIRLRFLR(239), within domain VI that inhibited cell binding to domain VI. Alanine substitution mutagenesis indicated that four residues (Tyr(130), Arg(225), Lys(229), and Arg(239)) within these two sequences are crucial for cell binding. Real-time heparin-binding kinetics of the domain VI mutants analyzed by surface plasmon resonance indicated that Arg(239) of S6 was critical for both heparin and cell binding. In addition, cell binding to domain VI was inhibited by heparin/heparan sulfate, which suggests an overlap of cell and heparin-binding sites. Furthermore, inhibition studies using integrin subunit monoclonal antibodies showed that integrin alpha(3)beta(1) was a major receptor for domain VI binding. Our results provide evidence that two sites spaced about 90 residues apart within the laminin alpha(5) chain N-terminal globular domain VI are critical for cell surface receptor binding.
Nielsen
P K
PK
Molecular Biology Section, Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370, USA.
Yamada
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D007797:Laminin / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding
D007797:Laminin; C097043:laminin alpha5