Transport of Cyclic Nucleotides and Estradiol 17-β-d-Glucuronide by Multidrug Resistance Protein 4
2001; Elsevier BV; Volume: 276; Issue: 36 Linguagem: Inglês
10.1074/jbc.m104833200
ISSN1083-351X
AutoresZhe‐Sheng Chen, Kun Hee Lee, Gary D. Kruh,
Tópico(s)Hepatitis B Virus Studies
ResumoHuman multidrug resistance protein 4 (MRP4) has recently been determined to confer resistance to the antiviral purine analog 9-(2-phosphonylmethoxyethyl)adenine and methotrexate. However, neither its substrate selectivity nor physiological functions have been determined. Here we report the results of investigations of thein vitro transport properties of MRP4 using membrane vesicles prepared from insect cells infected with MRP4 baculovirus. It is shown that expression of MRP4 is specifically associated with the MgATP-dependent transport of cGMP, cAMP, and estradiol 17-β-d-glucuronide (E217βG). cGMP, cAMP, and E217βG are transported withKm and Vmax values of 9.7 ± 2.3 μm and 2.0 ± 0.3 pmol/mg/min, 44.5 ± 5.8 μm and 4.1 ± 0.4 pmol/mg/min, and 30.3 ± 6.2 μm and 102 ± 16 pmol/mg/min, respectively. Consistent with its ability to transport cyclic nucleotides, it is demonstrated that the MRP4 drug resistance profile extends to 6-mercaptopurine and 6-thioguanine, two anticancer purine analogs that are converted in the cell to nucleotide analogs. On the basis of its capacity to transport cyclic nucleotides and E217βG, it is concluded that MRP4 may influence diverse cellular processes regulated by cAMP and cGMP and that its substrate range is distinct from that of any other characterized MRP family member. Human multidrug resistance protein 4 (MRP4) has recently been determined to confer resistance to the antiviral purine analog 9-(2-phosphonylmethoxyethyl)adenine and methotrexate. However, neither its substrate selectivity nor physiological functions have been determined. Here we report the results of investigations of thein vitro transport properties of MRP4 using membrane vesicles prepared from insect cells infected with MRP4 baculovirus. It is shown that expression of MRP4 is specifically associated with the MgATP-dependent transport of cGMP, cAMP, and estradiol 17-β-d-glucuronide (E217βG). cGMP, cAMP, and E217βG are transported withKm and Vmax values of 9.7 ± 2.3 μm and 2.0 ± 0.3 pmol/mg/min, 44.5 ± 5.8 μm and 4.1 ± 0.4 pmol/mg/min, and 30.3 ± 6.2 μm and 102 ± 16 pmol/mg/min, respectively. Consistent with its ability to transport cyclic nucleotides, it is demonstrated that the MRP4 drug resistance profile extends to 6-mercaptopurine and 6-thioguanine, two anticancer purine analogs that are converted in the cell to nucleotide analogs. On the basis of its capacity to transport cyclic nucleotides and E217βG, it is concluded that MRP4 may influence diverse cellular processes regulated by cAMP and cGMP and that its substrate range is distinct from that of any other characterized MRP family member. multidrug resistance protein (MRP1-MRP5, gene symbols ABCC1–ABCC5) leukotriene C4 S-(2, 4-dinitrophenyl)glutathione estradiol 17-β-d-glucuronide multispecific organic anion transporter (MOAT-B, MOAT-C, and MOAT-D are alternative names for MRP4, MRP5, and MRP3, respectively, and cMOAT is an alternative name for MRP2) 6-mercaptopurine 2-deoxycorfomycin 2-mercaptopurine 6-thioguanine 2-chlorodeoxyadenosine Dulbecco's modified Eagle's medium 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazine methosulfate 9-(2-phosphonylmethoxyethyl)adenine phosphate-buffered saline The multidrug resistance protein (MRP)1 family of ATP-binding cassette transporters first came to light as a result of the identification in drug-resistant cell lines of theMr 190,000 protein product and cDNA of its founding member, MRP1 (1Marquardt D. McCrone S. Center M.S. Cancer Res. 1990; 50: 1426-1430PubMed Google Scholar, 2McGrath T. Latoud C. Arnold S.T. Safa A.R. Felsted R.L. Center M.S. Biochem. Pharmacol. 1989; 38: 3611-3619Crossref PubMed Scopus (151) Google Scholar, 3Cole S.P. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (2988) Google Scholar). Based upon the determination of complete coding sequences and putative topologies, this family is now known to consist of at least seven members (4Hopper E. Belinsky M.G. Zeng H. Tosolini A. Testa J.R. Kruh G.D. Cancer Lett. 2001; 162: 181-191Crossref PubMed Scopus (175) Google Scholar). The substrate selectivities and drug resistance profiles of several of these pumps have been determined. MRP1, MRP2 (cMOAT), and MRP3 (MOAT-D), which confer resistance to certain natural product agents and methotrexate (5Grant C.E. Valdimarsson G. Hipfner D.R. Almquist K.C. Cole S.P. Deeley R.G. Cancer Res. 1994; 54: 357-361PubMed Google Scholar, 6Kruh G.D. Chan A. Myers K. Gaughan K. Miki T. Aaronson S.A. Cancer Res. 1994; 54: 1649-1652PubMed Google Scholar, 7Breuninger L.M. Paul S. Gaughan K. Miki T. Chan A. Aaronson S.A. Kruh G.D. Cancer Res. 1995; 55: 5342-5347PubMed Google Scholar, 8Zaman G.J. Flens M.J. van Leusden M.R. de Haas M. Mulder H.S. Lankelma J. Pinedo H.M. Scheper R.J. Baas F. Broxterman H.J. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8822-8826Crossref PubMed Scopus (697) Google Scholar, 9Cole S.P. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar, 10Cui Y. Konig J. Buchholz J.K. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937PubMed Google Scholar, 11Kawabe T. Chen Z.S. Wada M. Uchiumi T. Ono M. Akiyama S. Kuwano M. FEBS Lett. 1999; 456: 327-331Crossref PubMed Scopus (158) Google Scholar, 12Zeng H. Bain L.J. Belinsky M.G. Kruh G.D. Cancer Res. 1999; 59: 5964-5967PubMed Google Scholar, 13Kool M. van der Linden M. de Haas M. Scheffer G.L. de Vree J.M. Smith A.J. Jansen G. Peters G.J. Ponne N. Scheper R.J. Elferink R.P. Baas F. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6914-6919Crossref PubMed Scopus (588) Google Scholar, 14Hooijberg J.H. Broxterman H.J. Kool M. Assaraf Y.G. Peters G.J. Noordhuis P. Scheper R.J. Borst P. Pinedo H.M. Jansen G. Cancer Res. 1999; 59: 2532-2535PubMed Google Scholar), are the best characterized family members. These three transporters are lipophilic anion pumps whose substrates include glutathioneS-conjugates, such as leukotriene C4(LTC4) and S-(2,4-dinitrophenyl)glutathione (DNP-SG), and glucuronate conjugates such as estradiol 17-β-d-glucuronide (Ε217βG) (10Cui Y. Konig J. Buchholz J.K. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937PubMed Google Scholar, 11Kawabe T. Chen Z.S. Wada M. Uchiumi T. Ono M. Akiyama S. Kuwano M. 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Chem. 1999; 274: 15181-15185Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 22Zeng H. Liu G. Rea P.A. Kruh G.D. Cancer Res. 2000; 60: 4779-4784PubMed Google Scholar). However, whereas MRPs 1–3 have similar substrate ranges, they subserve distinct physiological functions. MRP1 is distinguished from MRP2 and MRP3 by its higher affinity for LTC4, a feature that is reflected in the specific role it plays in mediating immune responses involving cellular export of this cysteinyl leukotriene (23Wijnholds J. Evers R. van Leusden M.R. Mol C.A. Zaman G.J. Mayer U. Beijnen J.H. van der Valk M. Krimpenfort P. Borst P. Nat. Med. 1997; 3: 1275-1279Crossref PubMed Scopus (400) Google Scholar,24Robbiani D.F. Finch R.A. Jager D. Muller W.A. Sartorelli A.C. Randolph G.J. Cell. 2000; 103: 757-768Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). By contrast with MRP1, which is ubiquitously expressed and localized at basolateral surfaces of polarized cells (25Kruh G.D. Gaughan K.T. Godwin A. Chan A. J. Natl. Cancer Inst. 1995; 87: 1256-1258Crossref PubMed Scopus (91) Google Scholar, 26Flens M.J. Zaman G.J. van der Valk P. Izquierdo M.A. Schroeijers A.B. Scheffer G.L. van der Groep P. de Haas M. Meijer C.J. Scheper R.J. Am. J. Pathol. 1996; 148: 1237-1247PubMed Google Scholar, 27Evers R. de Haas M. Sparidans R. Beijnen J. Wielinga P.R. Lankelma J. Borst P. Br. J. Cancer. 2000; 83: 375-383Crossref PubMed Scopus (208) Google Scholar), MRP2 is primarily expressed in the hepatocyte canaliculus where it functions as an apical efflux pump for organic anions such as bilirubin glucuronide and in provision of the biliary fluid constituent glutathione (28Keppler D. Kartenbeck J. Prog. Liver Dis. 1996; 14: 55-67PubMed Google Scholar). MRP3 is also a glutathione and glucuronate conjugate pump but has the additional capability of mediating the transport of monoanionic bile acids (22Zeng H. Liu G. Rea P.A. Kruh G.D. Cancer Res. 2000; 60: 4779-4784PubMed Google Scholar, 29Hirohashi T. Suzuki H. Takikawa H. Sugiyama Y. J. Biol. Chem. 2000; 275: 2905-2910Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). This substrate selectivity, together with its induction at basolateral surfaces of hepatocytes under cholestatic conditions (30Hirohashi T. Suzuki H. Ito K. Ogawa K. Kume K. Shimizu T. Sugiyama Y. Mol. Pharmacol. 1998; 53: 1068-1075PubMed Google Scholar, 31Konig J. Rost D. Cui Y. Keppler D. Hepatology. 1999; 29: 1156-1163Crossref PubMed Scopus (429) Google Scholar, 32Ortiz D.F. Li S. Iyer R. Zhang X. Novikoff P. Arias I.M. Am. J. Physiol. 1999; 276: G1493-G1500PubMed Google Scholar), has led to the notion that it functions as a compensatory backup mechanism to eliminate from hepatocytes potentially toxic compounds that are ordinarily excreted into the bile.Two members of the MRP family, MRP4 (MOAT-B) and MRP5 (MOAT-C), are no more related to each other than they are to MRPs 1–3 in terms of degree of amino acid identity, but they are structurally distinct from the latter proteins in that MRPs 4 and 5 do not possess a third (N-terminal) membrane spanning domain (4Hopper E. Belinsky M.G. Zeng H. Tosolini A. Testa J.R. Kruh G.D. Cancer Lett. 2001; 162: 181-191Crossref PubMed Scopus (175) Google Scholar, 33Lee K. Belinsky M.G. Bell D.W. Testa J.R. Kruh G.D. Cancer Res. 1998; 58: 2741-2747PubMed Google Scholar, 34Belinsky M.G. Bain L.J. Balsara B.B. Testa J.R. Kruh G.D. J. Natl. Cancer Inst. 1998; 90: 1735-1741Crossref PubMed Scopus (162) Google Scholar). The topological dissimilarity of MRP5 is reflected in its distinct drug resistance capabilities and substrate selectivity. By contrast with MRPs 1–3, MRP5 is not known to confer resistance to natural product anticancer agents or methotrexate, but instead it has the facility for conferring resistance to purine analogs (35McAleer M.A. Breen M.A. White N.L. Matthews N. J. Biol. Chem. 1999; 274: 23541-23548Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 36Wijnholds J. Mol C.A. van Deemter L. de Haas M. Scheffer G.L. Baas F. Beijnen J.H. Scheper R.J. Hatse S. De Clercq E. Balzarini J. Borst P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7476-7481Crossref PubMed Scopus (432) Google Scholar). Similarly, membrane vesicle transport assays suggest that glutathione and glucuronate conjugates are not substrates of MRP5. Instead it is able to transport cyclic nucleotides (37Jedlitschky G. Burchell B. Keppler D. J. Biol. Chem. 2000; 275: 30069-30074Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar).The functional characteristics of MRP4, the other MRP family member that lacks an N-terminal membrane spanning domain, have yet to be defined in any detail. The drug resistance capabilities of MRP4 have been assessed to some degree in transfected NIH3T3 cells and in a drug-selected cell line in which MRP4 is overexpressed (38Lee K. Klein-Szanto A.J. Kruh G.D. J. Natl. Cancer Inst. 2000; 92: 1934-1940Crossref PubMed Scopus (209) Google Scholar, 39Schuetz J.D. Connelly M.C. Sun D. Paibir S.G. Flynn P.M. Srinivas R.V. Kumar A. Fridland A. Nat. Med. 1999; 5: 1048-1051Crossref PubMed Scopus (514) Google Scholar). These studies indicate that MRP4 has the facility for conferring resistance to the antimetabolite methotrexate and the antiviral purine analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA). However, almost nothing is known about its in vitro transport properties or physiological functions. In the present report we begin to address these questions by the analysis of MRP4-mediated transport in membrane vesicles prepared from MRP4-enriched insect cells. In so doing it is demonstrated that MRP4, like MRP5, catalyzes the MgATP-energized transport of cGMP and cAMP. However, by contrast with MRP5, MRP4 is also able to transport the glucuronide E217βG and is a higher affinity transporter of cAMP. In addition, it is shown that the resistance profile of MRP4 extends to include anticancer purine analogs. These findings indicate that the substrate range of MRP4 is distinct from all other characterized MRPs and have important implications regarding the cellular physiology of cyclic nucleotides and cellular resistance mechanisms associated with commonly used anticancer purine analogs.DISCUSSIONIn the present study the in vitro transport properties of human MRP4 were investigated to gain insight into its substrate selectivity and potential physiological functions. Cyclic nucleotides were selected as one class of target compounds because MRP4 has been determined previously to confer resistance to the antiviral nucleotide analog PMEA (38Lee K. Klein-Szanto A.J. Kruh G.D. J. Natl. Cancer Inst. 2000; 92: 1934-1940Crossref PubMed Scopus (209) Google Scholar, 39Schuetz J.D. Connelly M.C. Sun D. Paibir S.G. Flynn P.M. Srinivas R.V. Kumar A. Fridland A. Nat. Med. 1999; 5: 1048-1051Crossref PubMed Scopus (514) Google Scholar) and because cyclic nucleotides have been established recently as transport substrates of an MRP family member (MRP5) whose protein topology resembles that of MRP4 (37Jedlitschky G. Burchell B. Keppler D. J. Biol. Chem. 2000; 275: 30069-30074Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). In agreement with these structural and drug resistance features, it was determined that MRP4 can indeed transport cGMP and cAMP. In addition, it is shown that cGMP is a higher affinity substrate of MRP4 than is cAMP, as is also the case for MRP5. However, whereas both transporters are able to mediate transport of cyclic nucleotides there are significant differences in their kinetic parameters. The affinity of MRP4 for cGMP (Km = 9.7 μm) is ∼5-fold lower than that of MRP5 (Km = 2.1 μm) (37Jedlitschky G. Burchell B. Keppler D. J. Biol. Chem. 2000; 275: 30069-30074Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). By contrast, the affinity of MRP4 for cAMP (Km = 44.5 μm) is ∼9-fold higher than that reported for MRP5 (Km = 379 μm). The markedly higher affinity of MRP4 for cAMP may be of considerable significance in view of the involvement of this signaling molecule in diverse regulatory processes.Cellular efflux of cyclic nucleotides has been described in both prokaryotes and eukaryotes (46Brunton L.L. Mayer S.E. J. Biol. Chem. 1979; 254: 9714-9720Abstract Full Text PDF PubMed Google Scholar, 47Brunton L.L. Heasley L.E. Methods Enzymol. 1988; 159: 83-93Crossref PubMed Scopus (27) Google Scholar, 48Goldenbaum P.E. Hall G.A. J. Bacteriol. 1979; 140: 459-467Crossref PubMed Google Scholar, 49Saier Jr., M.H. Feucht B.U. McCaman M.T. J. Biol. Chem. 1975; 250: 7593-7601Abstract Full Text PDF PubMed Google Scholar). Analyses employing a variety of cultured cells and membrane vesicle preparations have established that cyclic nucleotide efflux in mammals is energy-dependent and mediated by amphipathic anion transporters in that it can be blocked by inhibitors of organic anion pumps (46Brunton L.L. Mayer S.E. J. Biol. Chem. 1979; 254: 9714-9720Abstract Full Text PDF PubMed Google Scholar, 47Brunton L.L. Heasley L.E. Methods Enzymol. 1988; 159: 83-93Crossref PubMed Scopus (27) Google Scholar, 50–63). The present study and that by Jedlitschky et al. (37Jedlitschky G. Burchell B. Keppler D. J. Biol. Chem. 2000; 275: 30069-30074Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar) indicate that MRP4 and MRP5, respectively, are components of the previously described membrane efflux systems for these critical signaling molecules (Fig.7). However, while these studies have identified molecular components of the export systems, the precise physiological roles of cyclic nucleotide efflux are not completely understood. A well defined role for cellular export of cyclic nucleotides is best established for the slime mold Dictyostelium discoideum, for which cAMP effluxed by solitary amoebae under low nutrient conditions acts both as a chemoattractant that mediates formation of multicellular aggregates and as a differentiation agent (64Janssens P.M. Van Haastert P.J. Microbiol. Rev. 1987; 51: 396-418Crossref PubMed Google Scholar). In mammals it is thought that efflux of cyclic nucleotides subserves two functions. One proposed function is that it contributes to the modulation of cyclic nucleotide signaling by reducing intracellular levels of these second messengers. In support of this notion is the consistent observation that triggered elevations in intracellular cyclic nucleotide levels are associated with enhanced cellular efflux (52Billiar T.R. Curran R.D. Harbrecht B.G. Stadler J. Williams D.L. Ochoa J.B. Di Silvio M. Simmons R.L. Murray S.A. Am. J. Physiol. 1992; 262: C1077-C1082Crossref PubMed Google Scholar, 53Patel M.J. Wypij D.M. Rose D.A. Rimele T.J. Wiseman J.S. J. Pharmacol. Exp. Ther. 1995; 273: 16-25PubMed Google Scholar, 54Woods M. Houslay M.D. Biochem. Pharmacol. 1991; 41: 385-394Crossref PubMed Scopus (24) Google Scholar, 55Hamet P. Pang S.C. Tremblay J. J. Biol. Chem. 1989; 264: 12364-12369Abstract Full Text PDF PubMed Google Scholar, 57Fehr T.F. Dickinson E.S. Goldman S.J. Slakey L.L. J. Biol. Chem. 1990; 265: 10974-10980Abstract Full Text PDF PubMed Google Scholar). A second proposed function for efflux is in provision of extracellular cAMP involved in intercellular signaling. This idea is consistent with the detection of cAMP in a variety of extracellular fluids (65Ashman D.F. Lipton R. Melicow M.M. Price T.D. Biochem. Biophys. Res. Commun. 1963; 11: 330-334Crossref PubMed Scopus (206) Google Scholar, 66Huang C.L. Ives H.E. Cogan M.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8015-8018Crossref PubMed Scopus (75) Google Scholar, 67Broadus A.E. Kaminsky N.I. Hardman J.G. Sutherland E.W. Liddle G.W. J. Clin. Invest. 1970; 49: 2222-2236Crossref PubMed Scopus (209) Google Scholar) and is also supported by characterizations of cellular activities attributed to extracellular cAMP and presumably mediated by proteins located in the plasma membranes of target cells (see for example Refs. 68Friedlander G. Couette S. Coureau C. Amiel C. J. Clin. Invest. 1992; 90: 848-858Crossref PubMed Google Scholar, 69Kubler D. Pyerin W. Bill O. Hotz A. Sonka J. Kinzel V. J. Biol. Chem. 1989; 264: 14549-14555Abstract Full Text PDF PubMed Google Scholar, 70Wen S.C. Chang C.M. Reitherman R.W. Harding B.W. Endocrinology. 1985; 116: 935-944Crossref PubMed Scopus (12) Google Scholar, 71Sorbera L.A. Morad M. Science. 1991; 253: 1286-1289Crossref PubMed Scopus (41) Google Scholar). It might be expected that MRP4, as a result of its higher affinity for cAMP by comparison with MRP5, plays a more prominent role in modulating intracellular cAMP levels and in efflux of cAMP involved in intercellular signaling. On the other hand MRP5 might be a more potent factor in the modulation of intracellular cGMP levels. Detailed studies concerning the tissue-specific expression patterns of MRP4 and MRP5, which are currently understood primarily at the transcript level (33Lee K. Belinsky M.G. Bell D.W. Testa J.R. Kruh G.D. Cancer Res. 1998; 58: 2741-2747PubMed Google Scholar,34Belinsky M.G. Bain L.J. Balsara B.B. Testa J.R. Kruh G.D. J. Natl. Cancer Inst. 1998; 90: 1735-1741Crossref PubMed Scopus (162) Google Scholar, 38Lee K. Klein-Szanto A.J. Kruh G.D. J. Natl. Cancer Inst. 2000; 92: 1934-1940Crossref PubMed Scopus (209) Google Scholar, 72Kool M. de Haas M. Scheffer G.L. Scheper R.J. van Eijk M.J. Juijn J.A. Baas F. Borst P. Cancer Res. 1997; 57: 3537-3547PubMed Google Scholar), should provide further insights as to which of these pumps are deployed in specific situations.Whereas the substrate selectivity of MRP4 is similar to that of MRP5 with regard to transport of cyclic nucleotides, our experiments indicate that there are also significant differences. By contrast with MRP5 (37Jedlitschky G. Burchell B. Keppler D. J. Biol. Chem. 2000; 275: 30069-30074Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar), MRP4 is able to transport the glucuronide E217βG. In this regard MRP4 is similar to MRPs 1–3, for which this compound is an established substrate. The affinity of E217βG transport by MRP4 (Km = 30.3 μm) is comparable to the Km value we previously reported for human MRP3 (25.6 μm) (22Zeng H. Liu G. Rea P.A. Kruh G.D. Cancer Res. 2000; 60: 4779-4784PubMed Google Scholar). However, both MRP1 (Km = 1.5–2.5 μm) and MRP2 (Km = 7.2 μm) are higher affinity transporters of this substrate (10Cui Y. Konig J. Buchholz J.K. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937PubMed Google Scholar, 15Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994PubMed Google Scholar, 17Loe D.W. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). In addition to the transport of E217βG, the substrate range of MRP4 is distinct from MRP5 with regard to at least one other compound, namely methotrexate. Transport of this anionic antimetabolite was inferred previously from studies demonstrating that MRP4-transfected cells are resistant to and accumulate reduced amounts of this agent (38Lee K. Klein-Szanto A.J. Kruh G.D. J. Natl. Cancer Inst. 2000; 92: 1934-1940Crossref PubMed Scopus (209) Google Scholar). In further support of the notion that methotrexate is an MRP4 substrate, it is demonstrated here that this agent can inhibit MRP4-mediated transport of E217βG. As with E217βG transport, MRP4-mediated transport of methotrexate is similar to MRPs 1–3 which are also able to confer resistance to and transport methotrexate (13Kool M. van der Linden M. de Haas M. Scheffer G.L. de Vree J.M. Smith A.J. Jansen G. Peters G.J. Ponne N. Scheper R.J. Elferink R.P. Baas F. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6914-6919Crossref PubMed Scopus (588) Google Scholar, 14Hooijberg J.H. Broxterman H.J. Kool M. Assaraf Y.G. Peters G.J. Noordhuis P. Scheper R.J. Borst P. Pinedo H.M. Jansen G. Cancer Res. 1999; 59: 2532-2535PubMed Google Scholar, 22Zeng H. Liu G. Rea P.A. Kruh G.D. Cancer Res. 2000; 60: 4779-4784PubMed Google Scholar).In the present study we demonstrate that the drug resistance profile of MRP4 extends beyond the antiviral purine analog PMEA, an acyclic phosphonate, to include the commonly used anticancer purine analog 6-MP. It is further demonstrated that the pump functions to reduce intracellular levels of this agent by an energy-dependent efflux mechanism. However, we have not detected transport of 6-MP in membrane vesicle assays. 2Z.-S. Chen and G. D. Kruh, unpublished observations. It is therefore unlikely that 6-MP or 6-TG, both of which are uncharged purine base analogs, are direct substrates of MRP4. Rather, the facility of MRP4 for transporting cyclic nucleotides and E217βG, both of which are amphipathic anions, suggests that the nucleotide metabolites of 6-MP and 6-TG, which are the toxic forms of these agents, are likely to be the anionic species effluxed by the pump. By contrast with 6-MP and 6-TG, PMEA is an amphipathic anion (45Pisarev V.M. Lee S.H. Connelly M.C. Fridland A. Mol. Pharmacol. 1997; 52: 63-68Crossref PubMed Scopus (40) Google Scholar). Hence, in this case it is likely that either PMEA and/or its di- or triphosphorylated metabolites are direct substrates of MRP4. These notions concerning how MRP4 confers resistance to antiviral and anticancer purine analogs are supported by analyses of species effluxed from MRP4-overexpressing CEMr-1 cells treated with PMEA and MRP5-transduced cells treated with PMEA and 6-MP (13Kool M. van der Linden M. de Haas M. Scheffer G.L. de Vree J.M. Smith A.J. Jansen G. Peters G.J. Ponne N. Scheper R.J. Elferink R.P. Baas F. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6914-6919Crossref PubMed Scopus (588) Google Scholar, 73Robbins B.L. Connelly M.C. Marshall D.R. Srinivas R.V. Fridland A. Mol. Pharmacol. 1995; 47: 391-397PubMed Google Scholar).In view of the fact that 6-MP and methotrexate are significant components of chemotherapeutic regimens used in the treatment of childhood leukemias, the ability of MRP4 to confer resistance to both of these antimetabolites is noteworthy. In this regard MRP4 is unique among characterized MRP family members that confer resistance to either methotrexate (MRPs 1–3) or 6-MP (MRP5) but not to both agents. Whether MRP4 or MRP5 contributes to clinical resistance associated with either of these agents remains to be determined. The multidrug resistance protein (MRP)1 family of ATP-binding cassette transporters first came to light as a result of the identification in drug-resistant cell lines of theMr 190,000 protein product and cDNA of its founding member, MRP1 (1Marquardt D. McCrone S. Center M.S. Cancer Res. 1990; 50: 1426-1430PubMed Google Scholar, 2McGrath T. Latoud C. Arnold S.T. Safa A.R. Felsted R.L. Center M.S. Biochem. 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