Evidence for Two Interacting Ligand Binding Sites in Human Multidrug Resistance Protein 2 (ATP Binding Cassette C2)

Abstract

Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-␤-D-glucuronide (E 2 17␤G), methotrexate, and glutathione-S-dinitrophenol. Complex inhibition and stimulation patterns were obtained, different from those observed with the related transporters MRP1 and MRP3. In contrast to a previous report, we found that the rate of E 2 17␤G transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Halfmaximal transport was obtained at 120 M E 2 17␤G, in contrast to values < 20 M for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E 2 17␤G (halfmaximal transport rates at 65 and 16 M E 2 17␤G, respectively) and shifted the sigmoidal dependence of transport rate on substrate concentration to a more hyperbolic one, without substantially affecting the maximal transport rate. Sulfanitran also stimulated MRP2 activity in cells, i.e. the transport of saquinavir through monolayers of Madin-Darby canine kidney II cells. Some compounds that stimulate E 2 17␤G transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E 2 17␤G. We propose that MRP2 contains two similar but nonidentical ligand binding sites: one site from which substrate is transported and a second site that regulates the affinity of the transport site for the substrate.

Differential Modulation of the Human Liver Conjugate Transporters MRP2 and MRP3 by Bile Acids and Organic Anions

“…However, our data correlate closely to the findings reported by Zelcer et al (39) and may suggest that a relatively low MRP2 expression level in mammalian cell membranes and/or a complex modulation of MRP2 transport (see below) may have masked this phenomenon (for a more detailed discussion, see the accompanying article (39)). …”

“…We thank Prof. Piet Borst for the MRP2 and MRP3 cDNAs. We appreciate that Zelcer et al (39) allowed us to read their paper and critically reviewed our manuscript.…”

The MRP family of drug efflux pumps

“…MRP2 is a lower affinity transporter for conjugates, and MRP2-mediated transport of compounds such as E 2 17bG, an established physiological substrate of the pump (Morikawa et al, 2000), is subject to positive allosteric regulation by bile acids and certain other amphipathic anions (Bodo et al, 2003;Zelcer et al, 2003a). In spite of the similarity in substrate range, the functions of MRP2 in the body are distinct from those of MRP1 as a result of differences in expression pattern and subcellular polarity.…”

Structure and function of ABC transporters: the ATP switch provides flexible control

“…Indeed, it has been shown [80] that two drug molecules can bind to a single protein molecule at the same time. Frequently, one substrate can stimulate transport of the other [58,81,82], possibly because occupancy of two sites more effectively stimulates ATP binding and 'closed NBD dimer' formation. In support of this hypothesis, GSH and drug must both bind to MRP1 to efficiently induce activating conformational changes in the NBDs [28].…”