title: Rab4 interacts with the human P-glycoprotein and modulates its surface expression in multidrug resistant K562 cells.
abstract: 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.
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Av de la Universidad s/n, 03202 Elche, Alicante, Spain.
authors.forename: Clotilde
authors.initials: C
authors.lastname: Ferrándiz-Huertas
authors.forename: Asia
authors.initials: A
authors.lastname: Fernández-Carvajal
authors.forename: Antonio
authors.initials: A
authors.lastname: Ferrer-Montiel
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000903:Antibiotics, Antineoplastic / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003630:Daunorubicin / Q000378:metabolism / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D020014:K562 Cells; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011485:Protein Binding; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D049452:Green Fluorescent Proteins; C553411:Rab14 protein, human; D020691:rab GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D003630:Daunorubicin