The mechanism for tumor angiogenesis in hepatocellular carcinoma : focused on the signal transduction pathway by Flt-1

我们确定了四个酪氨酸磷酸化位点的血管内皮生长因子受体-1（Flt-1）。PLC-γ、SHP-2、Grb 2、Crk和Nck以磷酸酪氨酸依赖性方式与Flt-1结合。使用突变体受体表达细胞的分析表明，Tyr 1213和Tyr 1333的磷酸化对于VEGF诱导的内皮细胞生长是必需的。共表达Flt-1和另一种VEGF受体KDR可增强VEGF诱导的信号传导，肝素可通过增强PLC-γ磷酸化来调节VEGF诱导的信号传导，我们还鉴定了一种以磷酸酪氨酸依赖方式与Tyr 1213结合的新分子。分子克隆结果表明，该分子与人Grb 2相关衔接蛋白（Grb 2-related adaptor protein，Grp）具有很高的同源性，表明该分子为小鼠Grp。已显示Grap仅在造血细胞中表达。然而，通过RT-PCR，我们检测的所有类型的内皮细胞都表达Grap，表明该分子在内皮细胞生长中的重要性。体外研究表明，葡萄糖的SH 3结构域结合c-Cb 1和Sos-1。该信号转导通路的下游应进一步研究。

We identified four tyrosine-phosphorylation sites on VEGF receptor-1 (Flt-1). PLC-γ, SHP-2, Grb2, Crk and Nck bind to Flt-1 in a phosphoyrosine-dependent manner. The analysis using mutant receptor-expressing cells indicates that the phosphorylation of Tyr1213 and Tyr1333 is essential for VEGF-induced endothelial cell growth. VEGF-induced signal was enhanced by coexpressing Flt-1 and another VEGF receptor, KDR.Heparin modulates the VEGF-induced signaling by enhancing PLC-γ phosphorylation.We also identified a novel molecule that binds to Tyr1213 in a phosphotyrosine-dependent manner. Molecular cloning revealed this 27 kDa molecule has a high homology with human Grap (Grb2-related adaptor protein), indicating that this molecule was a mouse Grap. Grap has been shown to be expressed exclusively in hematopoietic cells. However, all kinds of endothelial cells we examined expressed Grap by RT-PCR, indicating the importance of this molecule in endothelial cell growth. In vitro studies showed that the SH3 domain of Grap binds c-Cb1 and Sos-1. Down stream of this signal transduction pathway should be further investigated.