Molecular dissection of the alpha-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10.
The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as alpha-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their alpha chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (alpha5beta1gamma1) for alpha(3)beta(1) and alpha(6)beta(1) integrins and alpha-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4-5 modules did not compromise the binding of laminin-10 to alpha(3)beta(1) and alpha(6)beta(1) integrins but completely abrogated its binding to alpha-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to alpha-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the alpha-dystroglycan binding by the LG4 module.
Ido
Hiroyuki
H
Division of Protein Chemistry, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
Harada
Kenji
K
Futaki
Sugiko
S
Hayashi
Yoshitaka
Y
Nishiuchi
Ryoko
R
Natsuka
Yuko
Y
Li
Shaoliang
S
Wada
Yoshinao
Y
Combs
Ariana C
AC
Ervasti
James M
JM
Sekiguchi
Kiyotoshi
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D017931:DNA Primers; D049030:Dystroglycans; D006493:Heparin / Q000378:metabolism; D006801:Humans; D039201:Integrin alpha3beta1 / Q000378:metabolism*; D039121:Integrin alpha6beta1 / Q000378:metabolism*; D007797:Laminin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D003598:Cytoskeletal Proteins; C483081:DAG1 protein, human; D017931:DNA Primers; D039201:Integrin alpha3beta1; D039121:Integrin alpha6beta1; D007797:Laminin; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C119013:laminin 10; D049030:Dystroglycans; D006493:Heparin