title: Identification of domains participating in the substrate specificity and subcellular localization of the multidrug resistance proteins MRP1 and MRP2.
abstract: The human multidrug resistance protein MRP1 and its homolog, MRP2, are both thought to be involved in cancer drug resistance and the transport of a wide variety of organic anions, including the cysteinyl leukotriene C4 (LTC4) (Km = 0.1 and 1 microm). To determine which domain of these proteins is associated with substrate specificity and subcellular localization, we constructed various chimeric MRP1/MRP2 molecules and expressed them in polarized mammalian LLC-PK1 cells. We examined the kinetic properties of each chimeric protein by measuring LTC4 and methotrexate transport in inside-out membrane vesicles, sensitivity to an anticancer agent, etoposide, and subcellular localization by indirect immunofluorescence methods. The following results were determined in these studies: (i) when the NH2-proximal 108 amino acids of MRP2, including transmembrane (TM) helices 1-3, were exchanged with the corresponding region of MRP1, Km(LTC4) values of the chimera decreased approximately 4-fold and Km(methotrexate) values increased approximately 5-fold relative to those of wild-type MRP2 and MRP1, respectively, whereas resistance to etoposide increased approximately 3-fold; (ii) when the NH2-proximal region up to TM9 of MRP2 was exchanged with the corresponding region of MRP1, a further increase in etoposide resistance was observed, and subcellular localization moved from the apical to the lateral membrane; (iii) when two-thirds of MRP2 at the NH2 terminus were exchanged with the corresponding MRP1 region, the chimeric protein transported LTC4 with an efficiency comparable with that achieved by the wild-type MRP1; and (iv) exchange of the COOH-terminal 51 amino acids between MRP1 and MRP2 did not affect the localization of either of the proteins. These results provide a strong framework for further studies aimed at determining the precise domains of MRP1 and MRP2 with affinity for LTC4 and anticancer agents.
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Konno
authors.forename: Takuya
authors.initials: T
authors.lastname: Ebihara
authors.forename: Keiji
authors.initials: K
authors.lastname: Hisaeda
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchiumi
authors.forename: Takanori
authors.initials: T
authors.lastname: Nakamura
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shirakusa
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kuwano
authors.forename: Morimasa
authors.initials: M
authors.lastname: Wada
mesh_terms: D000818:Animals; D001692:Biological Transport; D016764:Cell Polarity; D006801:Humans; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013552:Swine; D014162:Transfection; D019459:Urothelium
chemical_list: D017997:Leukotriene C4; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011993:Recombinant Fusion Proteins; C000715087:ABCC2 protein, human; C421109:multidrug resistance-associated protein 1