Department of Life Sciences, Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea.
Cell Signal. 2012 Aug;24(8):1522-30. doi: 10.1016/j.cellsig.2012.03.020. Epub 2012 Apr 4.
Syndecan-4, a transmembrane heparan sulfate proteoglycan, plays a critical role in cell adhesion. Both the transmembrane and cytoplasmic domains of syndecan-4 are known to contribute to its functions, but the regulatory mechanisms underlying the functional interplay between the two domains were previously unclear. Here, we examined the functional relationship between these two domains. Fluorescence resonance energy transfer (FRET)-based assays showed that syndecan-4 expression enhanced RhoA activation. Furthermore, rat embryonic fibroblasts (REFs) plated on fibronectin fragments lacking the heparin-binding domain that interacts with syndecan-4 showed much lower RhoA activation than that in cells plated on full-length fibronectin, indicating that RhoA is involved in syndecan-4-mediated cell adhesion signaling. Syndecan-4 mutants defective in transmembrane domain-induced oligomerization and syndecan-4 phosphorylation-mimicking cytoplasmic domain mutants showed decreases in RhoA activation and RhoA-related functions, such as adhesion, spreading and focal adhesion formation, and subsequent increase in cell migration, but the inhibitory effect was much higher in cells expressing the transmembrane domain mutants. The cytoplasmic domain mutants (but not the transmembrane domain mutants) retained the capacity to form SDS-resistant dimers, and the cytoplasmic mutants showed less inhibition of syndecan-4-mediated protein kinase C activation compared to the transmembrane domain mutants. Finally, cytoplasmic domain activation failed to overcome the inhibition conferred by mutation of the transmembrane domain. Taken together, these data suggest that the transmembrane domain plays a major role in regulating syndecan-4 functions, and further show that a domain hierarchy exists in the regulation of syndecan-4.
硫酸乙酰肝素蛋白聚糖 4（Syndecan-4）是一种跨膜蛋白聚糖，在细胞黏附中起着关键作用。Syndecan-4 的跨膜和胞质结构域都被认为对其功能有贡献，但这两个结构域之间功能相互作用的调控机制以前并不清楚。在这里，我们研究了这两个结构域之间的功能关系。荧光共振能量转移（FRET）测定表明 Syndecan-4 的表达增强了 RhoA 的激活。此外，在缺乏与 Syndecan-4 相互作用的肝素结合域的纤维连接蛋白片段上接种的大鼠胚胎成纤维细胞（REFs）比在全长纤维连接蛋白上接种的细胞显示出低得多的 RhoA 激活，表明 RhoA 参与 Syndecan-4 介导的细胞黏附信号转导。在跨膜结构域诱导寡聚化和 Syndecan-4 磷酸化模拟胞质结构域突变体中缺陷的 Syndecan-4 突变体显示 RhoA 激活和 RhoA 相关功能（如黏附、铺展和焦点形成）降低，随后细胞迁移增加，但在表达跨膜结构域突变体的细胞中抑制作用更高。胞质结构域突变体（而不是跨膜结构域突变体）保留了形成 SDS 抗性二聚体的能力，并且胞质突变体显示出比跨膜结构域突变体更小的抑制 Syndecan-4 介导的蛋白激酶 C 激活的作用。最后，胞质结构域的激活未能克服跨膜结构域突变赋予的抑制作用。总之，这些数据表明跨膜结构域在调节 Syndecan-4 功能中起着主要作用，并进一步表明在 Syndecan-4 调节中存在结构域层次结构。
