School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4072, Australia.
Cells. 2022 Oct 25;11(21):3358. doi: 10.3390/cells11213358.
Mammalian retromer complex contributes to multiple early endosome-associated trafficking pathways whose origins are dependent on which sorting nexin (SNX) they are complexed with. In an attempt to dissect out the contribution of individual retromer-SNX complexes, we examined the trafficking of EGFR in detail within a series of KO cell line models. We demonstrated that the depletion of retromer subunit Vps35 leads to decreased EGFR protein levels in resting cells with enhanced association of EGFR with lysosomal compartments. Compared to control cells, the addition of EGF to Vps35 KO cells resulted in a reduced rate of EGFR degradation; AKT activation and cell prolferation rates were elevated, while ERK activation remained relatively unchanged. These observations are consistent with a prolonged temporal association of EGFR within early endosomes due to the inefficiency of early endosome-associated protein trafficking pathways or organelle maturation due to retromer absence. We did not fully delineate the discrete contributions from retromer-associated SNXs to the phenotypes observed from retromer Vps35 depletion. While each of the knock-outs of SNX1/2, SNX3, or SNX27 promotes the enhanced association of EGFR with early endosomal compartments, only the decreased EGF-mediated EGFR degradation was observed in SNX1/2 dKO cells, while the enhanced AKT activation was only increased in SNX3 KO or SNX27 KO cells. Despite this, each of the knock-outs showed increased EGF-stimulated cell proliferation rates.
哺乳动物内体再循环复合物（retromer complex）有助于多种早期内体相关的运输途径，其起源取决于与之结合的分选连接蛋白（sorting nexin，SNX）。为了剖析单个内体再循环-SNX 复合物的作用，我们在一系列 KO 细胞系模型中详细研究了 EGFR 的运输。我们发现，内体再循环亚基 Vps35 的耗竭会导致静止细胞中 EGFR 蛋白水平降低，同时 EGFR 与溶酶体区室的结合增强。与对照细胞相比，EGF 的添加会导致 Vps35 KO 细胞中 EGFR 降解速度降低；AKT 激活和细胞增殖率升高，而 ERK 激活保持相对不变。这些观察结果与 EGFR 在早期内体中的时空关联延长一致，这是由于内体相关蛋白运输途径或细胞器成熟的效率降低导致内体再循环缺失。我们没有完全阐明内体再循环相关 SNX 对 Vps35 耗竭后观察到的表型的离散贡献。虽然 SNX1/2、SNX3 或 SNX27 的敲除均促进 EGFR 与早期内体区室的增强结合，但只有在 SNX1/2 dKO 细胞中观察到 EGF 介导的 EGFR 降解减少，而 AKT 激活增强仅在 SNX3 KO 或 SNX27 KO 细胞中增加。尽管如此，每种敲除都显示出 EGF 刺激的细胞增殖率增加。
