title: Characterization of binding of leukotriene C4 by human multidrug resistance protein 1: evidence of differential interactions with NH2- and COOH-proximal halves of the protein.
abstract: Multidrug resistance protein 1 (MRP1) is capable of actively transporting a wide range of conjugated and unconjugated organic anions. The protein can also transport additional conjugated and unconjugated compounds in a GSH- or S-methyl GSH-stimulated manner. How MRP1 binds and transports such structurally diverse substrates is not known. We have used [(3)H]leukotriene C(4) (LTC(4)), a high affinity glutathione-conjugated physiological substrate, to photolabel intact MRP1, as well as fragments of the protein expressed in insect cells. These studies revealed that: (i) LTC(4) labels sites in the NH(2)- and COOH-proximal halves of MRP1, (ii) labeling of the NH(2)-half of MRP1 is localized to a region encompassing membrane-spanning domain (MSD) 2 and nucleotide binding domain (NBD) 1, (iii) labeling of this region is dependent on the presence of all or part of the cytoplasmic loop (CL3) linking MSD1 and MSD2, but not on the presence of MSD1, (iv) labeling of the NH(2)-proximal site is preferentially inhibited by S-methyl GSH, (v) labeling of the COOH-proximal half of the protein occurs in a region encompassing transmembrane helices 14-17 and appears not to require NBD2 or the cytoplasmic COOH-terminal region of the protein, (vi) labeling of intact MRP1 by LTC(4) is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC(4) binding to the NH(2)-proximal site, and (vii) the attenuation of LTC(4) binding to the NH(2)-proximal site is a consequence of ATP hydrolysis and trapping of Vi-ADP exclusively at NBD2. These data suggest that MRP1-mediated transport involves a conformational change, driven by ATP hydrolysis at NBD2, that alters the affinity with which LTC(4) binds to one of two sites composed, at least in part, of elements in the NH(2)-proximal half of the protein.
authors.affiliation: Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Y M
authors.initials: YM
authors.lastname: Qian
authors.forename: W
authors.initials: W
authors.lastname: Qiu
authors.forename: M
authors.initials: M
authors.lastname: Gao
authors.forename: C J
authors.initials: CJ
authors.lastname: Westlake
authors.forename: S P
authors.initials: SP
authors.lastname: Cole
authors.forename: R G
authors.initials: RG
authors.lastname: Deeley
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D017997:Leukotriene C4 / Q000737:chemistry* / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation; D019920:Photoaffinity Labels / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D018076:DNA, Complementary; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019920:Photoaffinity Labels; D014357:Trypsin; D014638:Vanadates; C421109:multidrug resistance-associated protein 1