ATP-dependent 17 beta-estradiol 17-(beta-D-glucuronide) transport by multidrug resistance protein (MRP). Inhibition by cholestatic steroids.
In addition to its ability to confer resistance to a range of natural product type chemotherapeutic agents, multidrug resistance protein (MRP) has been shown to transport the cysteinyl leukotriene, LTC4, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential physiological substrates also includes cholestatic glucuronidated steroids. ATP dependent, osmotically sensitive transport of the naturally occurring conjugated estrogen, 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (Vmax 1.4 nmol mg-1 min-1, K(m) 2.5 micron). The involvement of MRP was confirmed by demonstrating that transport was completely inhibited by a monoclonal antibody specific for an intracellular conformational epitope of the protein. MRP-mediated transport of LTC4, was competitively inhibited by E(2)17 beta G (K(i(app)) 22 micron), despite the lack of structural similarity between these two substrates. Competitive inhibition of [3H]E(2)17 beta G transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [3H]LTC4, demonstrating that the cholestatic steroid and leukotriene conjugates compete either for the same or possibly overlapping sites on the protein. Consistent with the presence of overlapping but non-identical sites, studies using chemotherapeutic drugs to inhibit MRP-mediated E(2)17 beta G transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC4 transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17 beta-estradiol 3-(beta-D-glucuronide), did not compete for transport of E(2)17 beta G by MRP, nor did they inhibit photolabeling of the protein with [3H]LTC4. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site-specific requirements for glucuronidation of the steroid nucleus.
Loe
D W
DW
Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada.
Almquist
K C
KC
Cole
S P
SP
Deeley
R G
RG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001647:Bile Acids and Salts / Q000494:pharmacology*; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D018288:Carcinoma, Small Cell; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000939:Epitopes; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D005965:Glucuronates / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D008175:Lung Neoplasms; D051379:Mice; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D001647:Bile Acids and Salts; D000939:Epitopes; D005965:Glucuronates; D011994:Recombinant Proteins; C025483:estradiol-17 beta-glucuronide; D017997:Leukotriene C4; D004958:Estradiol; D000255:Adenosine Triphosphate