Characterization of differential protein tethering at the plasma membrane in response to epidermal growth factor signaling.
Characterization of differential protein tethering at the plasma membrane in response to epidermal growth factor signaling.
复制标题
质膜上响应表皮生长因子信号传导的差异蛋白的表征。
DOI：
10.1021/pr201077d
复制
发表时间：
2012
影响因子：
4.4
通讯作者：
Mackeigan,JeffreyP
中科院分区：
文献类型：
--
作者：
Looyenga,BrendanD;Mackeigan,JeffreyP
膜蛋白与皮质肌动蛋白细胞骨架的物理束缚为质膜提供了功能组织，并有助于多种细胞过程，包括细胞信号传导、囊泡运输、内吞作用和迁移。为了发生这些过程，必须根据环境线索动态调节膜蛋白束缚。在这项研究中，我们描述了一种新颖的生化方案，用于分离物理上与肌动蛋白细胞骨架相连的质膜蛋白的补体。我们将此方法与串联液相色谱/质谱 (LC-MS/MS) 结合使用，证明正常人肾 (HK2) 细胞中膜蛋白的细胞骨架束缚受到表皮生长因子 (EGF) 的强烈调节。我们的结果表明，已知参与 EGF 信号传导的几种蛋白质，以及传统上与该途径不相关的其他蛋白质，以动态方式束缚在细胞骨架上。对我们的蛋白质组学调查中的一个目标——受体磷酸酪氨酸磷酸酶 PTPRS 的进一步分析揭示了细胞骨架束缚与 EGF 响应的内体运输之间的相关性。这一发现与之前的研究结果一致，即 PTPRS 参与 EGFR 的脱敏，并提供了一种潜在机制，可以在 EGFR 激活时协调这两种膜蛋白在同一区室中的定位。
Physical tethering of membrane proteins to the cortical actin cytoskeleton provides functional organization to the plasma membrane and contributes to diverse cellular processes including cell signaling, vesicular trafficking, endocytosis, and migration. For these processes to occur, membrane protein tethering must be dynamically regulated in response to environmental cues. In this study, we describe a novel biochemical scheme for isolating the complement of plasma membrane proteins that are physically tethered to the actin cytoskeleton. We utilized this method in combination with tandem liquid chromatography/mass spectrometry (LC–MS/MS) to demonstrate that cytoskeletal tethering of membrane proteins is acutely regulated by epidermal growth factor (EGF) in normal human kidney (HK2) cells. Our results indicate that several proteins known to be involved in EGF signaling, as well as other proteins not traditionally associated with this pathway, are tethered to the cytoskeleton in dynamic fashion. Further analysis of one hit from our proteomic survey, the receptor phosphotyrosine phosphatase PTPRS, revealed a correlation between cytoskeletal tethering and endosomal trafficking in response to EGF. This finding parallels previous indications that PTPRS is involved in the desensitization of EGFR and provides a potential mechanism to coordinate localization of these two membrane proteins in the same compartment upon EGFR activation.