Structural Determinants of Substrate Specificity Differences between Human Multidrug Resistance Protein (MRP) 1 (ABCC1) and MRP3 (ABCC3)

Multidrug resistance proteins (MRPs) are members of the C branch of the ATP-binding cassette transporter superfamily. Human MRP1 transports a wide range of natural product drugs and structurally diverse conjugated and unconjugated organic anions. Its closest relative is MRP3. Despite their structural similarity, the homologs differ substantially in their substrate specificity. It is noteworthy that MRP1 transports glutathione (GSH) and GSH conjugates and displays GSH-stimulated transport of a number of unconjugated and conjugated compounds. In contrast, MRP3 does not transport GSH and is a poor transporter of GSH conjugates. However, both proteins transport glucuronide conjugates, such as 17 beta-estradiol 17-(beta-D-glucuronide). We have constructed a series of MRP1/MRP3 hybrids and used them to identify a region of MRP1 that is critical for binding and transport of GSH conjugates such as leukotriene C-4 (LTC4). Substitution of this region encompassing transmembrane helices 8 and 9 and portions of cytoplasmic loops 4 and 5 of MRP1 with the equivalent region of MRP3 eliminated LTC4 transport. Transport of other substrates was either unaffected or enhanced. We identified three residues in this region: Tyr(440), Ile(441), and Met(443), mutation of which differentially affected transport. It is noteworthy that substitution of Tyr(440) with Phe, as found in MRP3, reduced LTC4 and GSH-stimulated estrone-3-sulfate transport without affecting transport of other substrates tested. The mutation increased the K-m for LTC4 5-fold and substantially reduced photolabeling of MRP1 by both [H-3] LTC4 and the GSH derivative, azidophenacyl-[S-35] GSH. These results suggest that Tyr(440) makes a major contribution to recognition of GSH and the GSH moiety of conjugates such as LTC4.