Lee SungKyoung, Shatadal Shalini, Griep Anne E
Invest Ophthalmol Vis Sci. 2016 Feb;57(2):707-18. doi: 10.1167/iovs.15-17727.
We previously showed that Discs large-1 (Dlg-1) regulates lens fiber cell structure and the fibroblast growth factor receptor (Fgfr) signaling pathway, a pathway required for fiber cell differentiation. Herein, we investigated the mechanism through which Dlg-1 regulates Fgfr signaling.
Immunofluorescence was used to measure levels of Fgfr1, Fgfr2, and activated Fgfr signaling intermediates, pErk and pAkt, in control and Dlg-1-deficient lenses that were haplodeficient for Fgfr1 or Fgfr2. Immunoblotting was used to measure levels of N-cadherin, EphA2, β-catenin, and tyrosine-phosphorylated EphA2, Fgfr1, Fgfr2, and Fgfr3 in cytoskeletal-associated and cytosolic fractions of control and Dlg-1-deficient lenses. Complex formation between Dlg-1, N-cadherin, β-catenin, Fgfr1, Fgfr2, Fgfr3, and EphA2 was assessed by coimmunoprecipitation.
Lenses deficient for Dlg-1 and haplodeficient for Fgfr1 or Fgfr2 showed increased levels of Fgfr2 or Fgfr1, respectively. Levels of pErk and pAkt correlated with the level of Fgfr2. N-cadherin was reduced in the cytoskeletal-associated fraction and increased in the cytosolic fraction of Dlg-1-deficient lenses. Dlg-1 complexed with β-catenin, EphA2, Fgfr1, Fgfr2, and Fgfr3. EphA2 complexed with N-cadherin, β-catenin, Fgfr1, Fgfr2, and Fgfr3. Levels of these interactions were altered in Dlg-1-deficient lenses. Loss of Dlg-1 led to changes in Fgfr1, Fgfr2, Fgfr3, and EphA2 levels and to greater changes in the levels of their activation.
Dlg-1 complexes with and regulates the activities of EphA2, Fgfr1, Fgfr2, and Fgfr3. As EphA2 contains a Psd95/Dlg/ZO-1 (PDZ) binding motif, whereas Fgfrs do not, we propose that the PDZ protein, Dlg-1, modulates Fgfr signaling through regulation of EphA2.
我们之前的研究表明，盘状大蛋白1（Dlg-1）调节晶状体纤维细胞结构和成纤维细胞生长因子受体（Fgfr）信号通路，该信号通路是纤维细胞分化所必需的。在此，我们研究了Dlg-1调节Fgfr信号通路的机制。
采用免疫荧光法检测Fgfr1、Fgfr2以及活化的Fgfr信号中间体pErk和pAkt在Fgfr1或Fgfr2单倍体缺乏的对照晶状体和Dlg-1缺陷晶状体中的水平。采用免疫印迹法检测N-钙黏蛋白、EphA2、β-连环蛋白以及酪氨酸磷酸化的EphA2、Fgfr1、Fgfr2和Fgfr3在对照晶状体和Dlg-1缺陷晶状体的细胞骨架相关组分和胞质组分中的水平。通过免疫共沉淀评估Dlg-1、N-钙黏蛋白、β-连环蛋白、Fgfr1、Fgfr2、Fgfr3和EphA2之间的复合物形成。
Dlg-1缺陷且Fgfr1或Fgfr2单倍体缺乏的晶状体分别显示Fgfr2或Fgfr1水平升高。pErk和pAkt水平与Fgfr2水平相关。在Dlg-1缺陷晶状体的细胞骨架相关组分中N-钙黏蛋白减少，在胞质组分中增加。Dlg-1与β-连环蛋白、EphA2、Fgfr1、Fgfr2和Fgfr3形成复合物。EphA2与N-钙黏蛋白、β-连环蛋白、Fgfr1、Fgfr2和Fgfr3形成复合物。在Dlg-1缺陷晶状体中这些相互作用的水平发生改变。Dlg-1的缺失导致Fgfr1、Fgfr2、Fgfr3和EphA2水平的变化以及它们活化水平的更大变化。
Dlg-1与EphA2、Fgfr1、Fgfr2和Fgfr3形成复合物并调节其活性。由于EphA2含有一个Psd95/Dlg/ZO-1（PDZ）结合基序，而Fgfrs没有，我们提出PDZ蛋白Dlg-1通过调节EphA2来调节Fgfr信号通路。
