ATP-dependent 17-Estradiol 17-(-D-Glucuronide) Transport by Multidrug Resistance Protein (MRP)

doi:10.1074/jbc.271.16.9683

ATP-dependent 17-Estradiol 17-(-D-Glucuronide) Transport by Multidrug Resistance Protein (MRP)

INHIBITION BY CHOLESTATIC STEROIDS (*)

From the (1)Cancer Research Laboratories and
(2)Department of Pathology, Queen's University, Kingston, Ontario, K7L 3N6 Canada

** Stauffer Research Professor of Queen's University. To whom correspondence and reprint requests should be addressed:
Cancer Research Laboratories, Rm. 314, Botterell Hall, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Tel.: 613-545-2981; Fax: 613-545-6830.

Abstract

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, LTC, 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, 17β-estradiol 17-(β-D-glucuronide) (E17βG), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (V 1.4 nmol mg min, K 2.5 μM). 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 LTC was competitively inhibited by E17βG (K 22 μM), despite the lack of structural similarity between these two substrates. Competitive inhibition of [³H]E17βG transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [³H]LTC, 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 E17βG transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17β-estradiol 3-(β-D-glucuronide), did not compete for transport of E17βG by MRP, nor did they inhibit photolabeling of the protein with [³H]LTC. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site-specific requirements for glucuronidation of the steroid nucleus.

Footnotes

↵^§ Recipient of an MRCC graduate studentship.
↵^¶ Career Scientist of the Ontario Cancer Foundation.
↵* This work was supported in part by a grant from the Medical Research Council of Canada (MRCC) (to S. P. C. C. and R. G. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MRP

multidrug resistance protein

AMP-PCP

β,-methyleneadenosine 5′-triphosphate

AMP-PNP

adenosine 5′-[β,-imido]triphosphate

ATPS

adenosine 5′-O-(3-thiotriphosphate)

E17αE

17α-ethinyl-17β-estradiol

E3SO17βG

17β-estradiol 3-sulfato-17-(β-D-glucuronide)

E3βG

17β-estradiol 3-(β-D-glucuronide)

E3βG

16α,17β-estriol 3-(β-D-glucuronide)

E16αG

16α,17β-estriol 16-(β-D-glucuronide)

E17βG

17β-estradiol 17-(β-D-glucuronide)

E17βG

16α,17β-estriol 17-(β-D-glucuronide)

glycolithocholate-3-sulfate

3α-hydroxy-5β-cholan-24-oic acid N-[carboxymethyl]amide 3-sulfate

LTC

leukotriene C

mAb

monoclonal antibody

MOAT

multispecific organic anion transporter

PAGE

polyacrylamide gel electrophoresis.
- Received November 30, 1995.
- Revision received January 29, 1996.