The small GTPases Rab5 and RalA regulate intracellular traffic of P-glycoprotein

Abstract

P-glycoprotein (P-gp) is a plasma membrane glycoprotein that can cause multidrug resistance (MDR) of cancer cells by acting as an ATP-dependent drug efflux pump. The regulatory effects of the small GTPases Rab5 and RalA on the intracellular trafficking of P-gp were investigated in HeLa cells. As expected, overexpressed enhanced green fluorescent protein (EGFP)-tagged P-gp (P-gp-EGFP) is mainly localised to the plasma membrane. However, upon cotransfection of either dominant negative Rab5 (Rab5-S34N) or constitutively active RalA (RalA-G23V) the intracellular P-gp-EGFP levels increased approximately 9 and 13 fold, respectively, compared to control P-gp-EGFP cells. These results suggest that Rab5 and RalA regulate P-gp trafficking between the plasma membrane and an intracellular compartment. In contrast, coexpression of constitutively active Rab5 (Rab5-Q79L) or dominant negative RalA (RalA-S28N) had no effect on the localisation of P-gp-EGFP. Furthermore, the intracellular accumulation of daunorubicin, a substrate for P-gp, increased significantly with an increased intracellular localisation of P-gp-EGFP. These results imply that it may be possible to overcome MDR by controlling the plasma membrane localisation of P-gp.

Intracellular trafficking of P-glycoprotein

“…Although mitochondria localization of P-gp was reported (Munteanu et al, 2006), co-localization of P-gp-EGFP or wild-type P-gp with mitochondrial marker were not observed (Fu et al, 2007, Paterson and Gottesman, 2007). The sites of P-gp synthesis (ER), modification (Golgi), trafficking/recycling (endosomes) and degradation (lysosome, co-localized with LAMP1/2) are indicated in Figure 2.…”

“…Similarly, increased intracellular wild type P-gp was also observed in multidrug resistant MCF-7/Adr cells transfected with Rab5-S34N, suggesting that Rab5 regulates P-gp traffic from the endosome compartment to the plasma membrane in nonpolarized cells (Fu et al, 2007). However, studies in LS174T cells, showed that overexpression of Rab5 resulted in removal of P-gp from the plasma membrane into intracellular compartments, suggesting that Rab5 regulates P-gp endosytosis in these cells (Kim et al, 1997).…”

“…Co-expression of P-gp-EGFP and constitutively active RalA-G23V but not dominant-negative RalA-S28N in HeLa cells resulted in intracellular accumulation of P-gp-EGFP in approximately 72 % of cells (Fu et al, 2007). In addition, wild type P-gp was redistributed from the plasma membrane into an intracellular compartment in multidrug resistant MCF-7/Adr cells transfected with RalA-G23V (Fu et al, 2007), suggesting that RalA could either increased rate of endocytosis or inhibition of recycling of P-gp-EGFP.…”

Intracellular Trafficking of MDR Transporters and Relevance of SNPs

“…In simple non-polarized cells, internalized membrane proteins are first delivered to Rab family of small GTP-ases (Rab, Ral, Rac), known to regulate vesicle traffic from a specific organelle or along a specific pathway [64] to endosome-endosome tethering and fusion [65][66][67][68]. Recently it has been shown that over-expression of Rab5 and RalA GTPase accelerate intracellular trafficking of P-gp, and consequently intracellular levels of daunorubicin, a substrate of P-gp, clearly demonstrating that these small GTPases are involved in P-gp plasma membrane localization, and endocytosis [69]. Furthermore, they showed that increased intracellular P-gp was found in early endosomes, according to Kim et al [15] who previously demonstrated that endocytic/recycling traffic of P-gp is involved in steady-state distribution of transporters and this consequently influences drug resistance of cancer cells.…”

Targeting of Multidrug‐Resistant Human Ovarian Carcinoma Cells With Anti‐P‐Glycoprotein Antibody Conjugates

“…[25–27,54,63–65] Images of conjugate 11 [P-(AP-FITC)-mAb] incubated with A2780/AD cells does exhibit limited internalization within the cells, and based on trafficking studies of Pgp in the literature, it is within early endosomes or lysosomes, and not within recycling endosomes. [6,64] If alteration of Pgp trafficking had occurred, a more intense fluorescence near the perinuclear region, as found for conjugate 10 [P-(AP-FITC)], would be evident. [20,40] Localization of HPMA copolymer conjugates was studied using FITC labeled conjugates, rather than DOX, as alterations in DOX fluorescence occur due to DNA intercalation, effects of the cell medium, whether the cells are sensitive or resistant to anti-cancer agents and the presence of DOX metabolites.…”