Characterization of the Role of Polar Amino Acid Residues within Predicted Transmembrane Helix 17 in Determining the Substrate Specificity of Multidrug Resistance Protein 3 ² Da-Wei Zhang, ‡,§ Hong-Mei Gu, | Monika Vasa, ‡ Mario Muredda, ‡,⊥ Susan P. C. Cole, ‡,§ and Roger G. Deeley* ,‡,§,⊥ DiVision of Cancer Biology and Genetics, Cancer Research Institute, and Departments of Pathology, Biochemistry, and Anatomy and Cell Biology, Queen’s UniVersity, Kingston, Ontario K7L 3N6, Canada ReceiVed March 21, 2003; ReVised Manuscript ReceiVed May 29, 2003 ABSTRACT: Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1. Like MRP1, MRP3 confers resistance to etoposide (VP-16) and actively transports 17-estradiol 17-(- D-glucuronide) (E 2 17G), cysteinyl leukotriene 4 (LTC 4 ), and methotrexate, although with generally lower affinity. Unlike MRP1, MRP3 also transports monovalent bile salts. We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E 2 17G transport. We have now examined the importance of the hydrogen-bonding potential of residues in TM17 of MRP3 on both substrate specificity and overall activity. Mutation S1229A reduced only methotrexate transport. Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E 2 17G and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E 2 17G but increased taurocholate transport without affecting transport of methotrexate. Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. On the basis of the substrates analyzed, residues Ser 1229 , Ser 1231 , Gln 1235 , and Asn 1241 play an important role in determining the specificity of MRP3, while mutation of Tyr 1232 , Ser 1233 , and Thr 1237 affects overall activity. Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix. Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr 1237 , markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined. The frequent occurrence of resistance to a wide variety of structurally and functionally unrelated anticancer drugs is a significant barrier to successful treatment of cancer patients. Experimentally and in some cases clinically, mul- tidrug resistance (MDR) 1 is often associated with overex- pression of ATP-dependent drug-efflux pumps, such as multidrug resistance protein 1 (MRP1), P-glycoprotein (P- gp), and breast cancer resistance protein (BCRP/MXR), all of which belong to the ATP binding cassette (ABC) superfamily of transporters (1-10). MRP1, or ABCC1, is a member of the ABCC branch of the superfamily and can confer resistance to many commonly used, structurally diverse natural product chemotherapeutic agents, including anthracyclines, Vinca alkaloids, and epipodophyllotoxins (11-14). The protein is also a primary active transporter of many glutathione-, glucuronate-, and sulfate-conjugated organic anions (15-19). Since the identification of MRP1, several MRP1-related proteins have been cloned, including MRP2 to -7 and ABCC11 and -12 (20-29). Among the MRP family mem- bers, MRP3 shares the highest degree of structural resem- blance to MRP1 (58% amino acid identity). MRP3, which is composed of 1527 amino acid residues, is predicted to contain a typical ABC transporter core structure, consisting of two membrane-spanning domains (MSDs) and two nucleotide binding domains, plus an additional NH 2 -terminal MSD (MSD1) that is comprised of five transmembrane (TM) helices (24). Thus, the predicted topology of MRP3 is similar to that of MRP1 (30-33), containing a total of 17 TM helices with an extracellular NH 2 terminus. ² This work was supported by a grant from the National Cancer Institute of Canada with funds from the Terry Fox Run. D.-W.Z. was supported in part by a Queen’s University Graduate Award. S.P.C.C. is a Canada Research Chair in Cancer Biology and Senior Scientist of Cancer Care Ontario. R.G.D. is a Stauffer Research Professor of Queen’s University and Vice President of Research Cancer Care Ontario. * To whom correspondence should be addressed at Cancer Research Laboratories, Botterell Hall, Queen’s University, Kingston, Ontario K7L 3N6, Canada. Tel: 613-533-2979. Fax: 613-533-6830. E-mail: deeleyr@post.queensu.ca. ‡ Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University. § Department of Pathology, Queen’s University. | Department of Anatomy and Cell Biology, Queen’s University. ⊥ Department of Biochemistry, Queen’s University. 1 Abbreviations: MDR, multidrug resistance; MRP, multidrug resistance protein; P-gp, P-glycoprotein; BCRP/MXR, breast cancer resistance protein; MSD, membrane-spanning domain; TM, transmem- brane; NBD, nucleotide binding domain; mAb, monoclonal antibody; VP-16, etoposide; E 217, 17-estradiol 17-(-D-glucuronide); LTC4, leukotriene C4; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HEK, human embryonic kidney; MTX, methotrexate; TC, taurocholate. 9989 Biochemistry 2003, 42, 9989-10000 10.1021/bi034462b CCC: $25.00 © 2003 American Chemical Society Published on Web 08/02/2003