Structural and Functional Analysis of New Type Extracellular Matrix Receptor syndecan-2

小鼠刘易斯肺癌低转移克隆P29的成瘤依赖于富含纤维连接蛋白的基质，而高转移克隆LM 66-H11的生长依赖于基底膜。P29细胞在体外与纤维连接蛋白基质粘附时形成应力纤维，而LM 66-H11细胞则形成皮质作用结构。P29细胞的表型与整合素α5β1和syndrecan-2的表达密切相关，所述整合素α5β1和syndrecan-2具有对COOH末端肝素结合结构域具有特异性亲和力的硫酸乙酰肝素侧链。LM 66-H11细胞表达整合素的水平与P29细胞相同，但syndecan-2的水平明显低于P29细胞。在体外用多配体蛋白聚糖-2的反义寡核苷酸处理P29细胞时，导致其生物合成的选择性抑制，就表型而言，该细胞与LM 66-H11细胞无法区分。P29细胞的表型通过粘附至纤连蛋白重组融合多肽（CH-271）而复制，所述纤连蛋白重组融合多肽（CH-271）包含RGD细胞结合（C-274）和C-末端肝素结合（H271），所述C-末端肝素结合（H271）包含RGD细胞结合（C-274）和C-末端肝素结合（H271）结构域，而LM 66-H11细胞的表型仅由C-274多肽诱导，表明通过整合素α5β1和多配体蛋白聚糖-2的信号传导是多肽所必需的，可以用大量的H-271多肽或多配体蛋白聚糖-2或硫酸乙酰肝素特异性抗体替代。此外，当接种在包含C-274多肽和对多配体蛋白聚糖-2胞外域具有强亲和力的碱性FGF的融合多肽上时，甚至LM 66-H11细胞也形成应力纤维。这些发现表明，聚集的两个受体的肌动蛋白细胞骨架的组织信号的必要性。

Mouse Lewis lung carcinoma-derived low metastatic P29 clone exhibits tumorigenesis dependent on the fibronectin-rich stromal matrix, whereas the growth of highly metastatic LM66-H11 clone depends on the basement membranes. On adhesion to the fibronectin substratum in vitro, P29 cells show stress fiber formation, whereas LM66-H11 cells form cortex action structure. The phenotype of P29 cells is closely correlated to the expression of integrin α5β1 and syndrecan-2 having heparan sulfate side chains with specific affinity to COOH-terminal heparin-binding domain. LM66-H11 cells express the integrin at the same level to that of P29 cells, but syndecan-2 at a significantly lower level. On treatment of P29 cells in vitro with antisense oligonucleotide for syndecan-2, causing selective inhibition of its biosynthesis, the cells turn out to be indistinguishable from LM66-H11 cells as regards the phenotype. The phenotype of P29 cells is reproduced by adhesion to fibronectin recombinant fusion polypeptide (CH-271) comprising RGD cell-binding (C-274) and C-terminal heparin-binding (H271) comprising RGD cell-binding (C-274) and C-terminal heparin-binding (H271) domains whereas the phenotype of LM66-H11 cells in induced only by C-274 polypeptide, indicating that signaling through integrin α5β1 and syndecan-2 is essential for the polypeptide can be replaced by a large amount of H-271 polypeptide, or antibodies specific to syndecan-2 or heparan sulfate. Furthermore, when inoculated on a fusion polypeptide comprising C-274 polypeptide and basic FGF with strong affinity to syndecan-2 ectodomain, even LM66-H11 cells form stress fibers. These findings indicate the necessity of clustering of the two receptors for signaling for the organization of the actin cytoskeletons.