Rab4 interacts with the human P-glycoprotein and modulates its surface expression in multidrug resistant K562 cells
Rab4 interacts with the human P-glycoprotein and modulates its surface expression in multidrug resistant K562 cells
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DOI：
10.1002/ijc.25310
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发表时间：
2011-01-01
影响因子：
6.4
通讯作者：
Ferrer-Montiel, Antonio
中科院分区：
文献类型：
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作者：
Ferrandiz-Huertas, Clotilde;Fernandez-Carvajal, Asia;Ferrer-Montiel, Antonio
p -糖蛋白（P-gp）是一种质膜糖蛋白，已被认为是肿瘤多药耐药（MDR）的主要原因。我们利用P-gp的c端结构域进行了酵母2杂交筛选，发现了与P-gp复合物的2个参与囊泡运输的小gtpase Rab4和Rab14。在K562ADR细胞中，瞬时或稳定过表达GFP-Rab4，而不表达Rab14，可降低细胞表面P-gp的存在。结果，GTPase的表达通过增加胞内daunomycin （DNM）的积累，降低了K562ADR细胞的MDR表型。这种效应被组成型活性的Rab4Q72L突变体模仿，但不被显性阴性的Rab4S27N突变体模仿。Rab4调节胞外P-gp从胞内区室转运到质膜，这种调节需要两种蛋白和GTPase活性的相互作用。值得注意的是，与敏感的亲本细胞系相比，K562ADR细胞表现出Rab4水平的显著降低，但其他Rab gtpase的水平没有下降，这表明这些细胞中MDR表型的发展涉及P-gp的上调和伴随的调节其表面表达的蛋白的下调。通过RNA干扰降低K562ADR内源性Rab4水平，增强细胞表面P-gp的表达，减少DNM的摄取。因此，这些发现证实了调节P-gp在癌细胞中的时空分布可能是缓解MDR表型的有效治疗策略，并将信号传递给Rab4作为潜在靶点。
P-glycoprotein (P-gp) is a plasma membrane glycoprotein that has been signaled as a primary cause of multidrug resistance (MDR) in tumors. We performed a yeast 2-hybrid screen using the C-terminal domain of P-gp and identified 2 small GTPases involved in vesicular trafficking, Rab4 and Rab14, which complex with P-gp. The overexpression of GFP-Rab4, either transiently or stably, but not of Rab14, in K562ADR cells decreased the presence of P-gp in the cell surface. As a result, expression of this GTPase reduced the MDR phenotype of K562ADR cells, by augmenting the intracellular accumulation of daunomycin (DNM). This effect was mimicked by the constitutively active Rab4Q72L mutant, but not by the dominant negative Rab4S27N mutant. Rab4 regulated excocytotic P-gp trafficking to the plasma membrane from intracellular compartments, and this modulation required the interaction of both proteins and the GTPase activity. Noteworthy, K562ADR cells exhibited a significant reduction of Rab4 levels, but not of other Rab GTPases, as compared with the sensitive parental cell line, suggesting that the development of the MDR phenotype in these cells involves upregulation of P-gp and a concomitant downregulation of proteins that regulate its surface expression. Attenuation of endogenous Rab4 levels in K562ADR by RNA interference enhanced the expression of P-gp in the cell surface, and reduced the uptake of DNM. Accordingly, these findings substantiate the notion that modulation of the temporal and spatial distribution of P-gp in cancer cells may be a valid therapeutic strategy to alleviate the MDR phenotype, and signal to Rab4 as a potential target.