From the Department of Biochemistry, Yong Loo Lin School of Medicine, National University Health System, 1E Kent Ridge Road, Singapore 119228 and.
J Biol Chem. 2014 May 2;289(18):12375-89. doi: 10.1074/jbc.M114.548321. Epub 2014 Mar 18.
Rab31 is a member of the Rab5 subfamily of Rab GTPases. Although localized largely to the trans-Golgi network, it shares common guanine nucleotide exchange factors and effectors with other Rab5 subfamily members that have been implicated in endocytic membrane traffic. We investigated whether Rab31 also has a role in the trafficking of the ligand-bound EGF receptor (EGFR) internalized through receptor-mediated endocytosis. We found that loss of Rab31 inhibits, but overexpression enhances, EGFR trafficking to the late endosomes and that the effect of Rab31 silencing could be specifically rescued by overexpression of a silencing-resistant form of Rab31. Rab31 was found to interact with the EGFR by coimmunoprecipitation and affinity pulldown analyses, and the primarily trans-Golgi network-localized Rab31 has increased colocalization with the EGFR in A431 cells 30 min after pulsing with EGF. A glycerol gradient sedimentation assay suggested that Rab31 is sequestered into a high molecular weight complex after stimulation with EGF, as was early endosome antigen 1 (EEA1), a factor responsible for endosomal tethering and fusion events. We found that loss of EEA1 reduced the interaction between Rab31 and the EGFR and abrogated the effect of Rab31 overexpression on the trafficking of the EGFR. Likewise, loss of GAPex5, a Rab31 guanine nucleotide exchange factor that has a role in ubiquitination and degradation of the EGFR, reduced the interaction of Rab31 with the EGFR and its effect on EGFR trafficking. Taken together, our results suggest that Rab31 is an important regulator of endocytic trafficking of the EGFR and functions in an EGFR trafficking complex that includes EEA1 and GAPex5.
Rab31 是 Rab5 亚家族的一员 GTPases。虽然它主要定位于反式高尔基体网络，但它与其他 Rab5 亚家族成员共享常见的鸟嘌呤核苷酸交换因子和效应物，这些成员已被牵连到内吞膜运输中。我们研究了 Rab31 是否也在配体结合的 EGF 受体 (EGFR) 的运输中发挥作用，该受体通过受体介导的内吞作用内化。我们发现，Rab31 的缺失抑制，但过表达增强，EGFR 向晚期内体的运输，并且 Rab31 沉默的影响可以通过过表达沉默抗性形式的 Rab31 特异性挽救。通过共免疫沉淀和亲和下拉分析发现 Rab31 与 EGFR 相互作用，并且在 A431 细胞中用 EGF 脉冲 30 分钟后，主要位于反式高尔基体网络中的 Rab31 与 EGFR 的共定位增加。甘油梯度沉降测定表明，Rab31 在受到 EGF 刺激后被隔离到高分子量复合物中，就像早期内体抗原 1（EEA1）一样，EEA1 是负责内体连接和融合事件的因子。我们发现，EEA1 的缺失减少了 Rab31 与 EGFR 的相互作用，并消除了 Rab31 过表达对 EGFR 运输的影响。同样，Rab31 鸟嘌呤核苷酸交换因子 GAPex5 的缺失，该因子在 EGFR 的泛素化和降解中起作用，减少了 Rab31 与 EGFR 的相互作用及其对 EGFR 运输的影响。总之，我们的结果表明，Rab31 是 EGFR 内吞运输的重要调节剂，并且在包括 EEA1 和 GAPex5 的 EGFR 运输复合物中发挥作用。
