Molecular dissection of the α-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10
Molecular dissection of the α-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10
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DOI：
10.1074/jbc.m313626200
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发表时间：
2004-03-19
影响因子：
4.8
通讯作者：
Sekiguchi, K
中科院分区：
文献类型：
--
作者：
Ido, H;Harada, K;Sekiguchi, K
细胞与层粘连蛋白的粘附性相互作用是由整合素和非整合素型受体如α-肌营养不良蛋白聚糖和突触素介导的。层粘连蛋白在其阿尔法链的C末端球状区域与这些受体结合，但这些受体识别的区域尚未被准确定位。在这项研究中，我们试图通过在球状结构域内缺失LG(层粘连蛋白G样)模块的一系列重组层粘连蛋白-10蛋白来定位层粘连蛋白-10(Alpha5β1 Gamma1)与α(3)β(1)和α(6)β(1)整合素和α-肌营养不良聚糖的结合部位。我们发现，LG4-5模块的缺失不会影响层粘连蛋白-10与α(3)β(1)和α(6)β(1)整合素的结合，但完全取消了其与α-营养不良多糖的结合。LG3模块的进一步缺失导致其失去与整合素的结合，这突显了LG3对层粘连蛋白-10结合整合素的重要性。当单独表达为与谷胱甘肽S转移酶或纤维连接蛋白N端70 kDa区域的融合蛋白时，只有LG4能够与α-营养不良聚糖结合，而LG3和任何其他LG模块都不能与整合素结合。LG3和LG4模块的定点突变表明，LG3模块中的Asp-3198参与了层粘连蛋白-10与整合素的结合，而LG4模块与α-肌营养不良蛋白聚糖的结合可能涉及环区的多个碱性氨基酸残基协同参与。
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.