Department of Pathophysiology, School of the Basic Medicine, Xuzhou Medical University, Xuzhou 221004, Jiangsu, China.
Institute of Emergency Rescue Medicine, Xuzhou Medical University, Xuzhou 221004, Jiangsu, China.
Int J Mol Sci. 2017 Sep 7;18(9):1922. doi: 10.3390/ijms18091922.
Glial cell line-derived neurotrophic factor (GDNF), a potential therapeutic factor for Parkinson's disease (PD), exerts its biological effects through the Ret receptor tyrosine kinase. The redistribution of Ret into lipid rafts substantially influences Ret signaling, but the mechanisms underlying Ret translocation remain unclear. The purpose of our study was to further explore the signaling mechanisms of GDNF and to determine whether the actin cytoskeleton is involved in the GDNF-induced Ret translocation into lipid rafts. In MN9D dopaminergic neuronal cells, we used density gradient centrifugation and immunofluorescence confocal microscopy to separate and visualize lipid rafts, co-immunoprecipitation to analyze protein-protein interactions, and latrunculin B (Lat B) and jasplakinolide (Jas) to disrupt and enhance the polymerization of the actin cytoskeleton, respectively. The results showed that Ret translocated into lipid rafts and coimmunoprecipitated with actin in response to GDNF treatment. After Lat B or Jas treatment, the Ret-F-actin association induced by GDNF was impaired or enhanced respectively and then the levels of Ret translocated into lipid rafts were correspondingly inhibited or promoted. These data indicate that actin polymerization and cytoskeletal remodeling are integral to GDNF-induced cell signaling in dopaminergic cells and define a new role of the actin cytoskeleton in promoting Ret redistribution into lipid rafts.
胶质细胞源性神经营养因子（GDNF）是一种潜在的治疗帕金森病（PD）的因子，通过 Ret 受体酪氨酸激酶发挥其生物学作用。Ret 重新分布到脂筏中会极大地影响 Ret 信号转导，但 Ret 易位的机制尚不清楚。我们的研究目的是进一步探讨 GDNF 的信号转导机制，并确定肌动蛋白细胞骨架是否参与 GDNF 诱导的 Ret 向脂筏的易位。在 MN9D 多巴胺能神经元细胞中，我们使用密度梯度离心和免疫荧光共聚焦显微镜分离和可视化脂筏，共免疫沉淀分析蛋白-蛋白相互作用，并用 latrunculin B（Lat B）和 jasplakinolide（Jas）分别破坏和增强肌动蛋白细胞骨架的聚合。结果表明，Ret 在 GDNF 处理后向脂筏易位并与肌动蛋白共沉淀。Lat B 或 Jas 处理后，GDNF 诱导的 Ret-F-actin 结合分别受到抑制或增强，随后 GDNF 诱导的 Ret 向脂筏的易位水平相应受到抑制或促进。这些数据表明，肌动蛋白聚合和细胞骨架重塑是 GDNF 诱导多巴胺能细胞信号转导的必要条件，并定义了肌动蛋白细胞骨架在促进 Ret 重新分布到脂筏中的新作用。
