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1.
Koike K Conseil G Leslie EM Deeley RG Cole SP 《The Journal of biological chemistry》2004,279(13):12325-12336
Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette transporter that confers resistance to drugs and mediates the transport of organic anions. MRP1 has a core structure of two membrane spanning domains (MSDs) each followed by a nucleotide binding domain. This core structure is preceded by a third MSD with five transmembrane (TM) helices, whereas MSD2 and MSD3 each contain six TM helices. We investigated the consequences of Ala substitution of 18 Pro residues in both the non-membrane and TM regions of MSD2 and MSD3 on MRP1 expression and organic anion transport function. All MRP1-Pro mutants except P1113A were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. In addition, five mutants containing substitutions of Pro residues in or proximal to the TM helices of MSD2 (TM6-Pro(343), TM8-Pro(448), TM10-Pro(557), and TM11-Pro(595)) and MSD3 (TM14-Pro(1088)) exhibited significantly reduced transport of five organic anion substrates. In contrast, mutation of Pro(1150) in the cytoplasmic loop (CL7) linking TM15 to TM16 caused a substantial increase in 17beta-estradiol-17-beta-(D-glucuronide) and methotrexate transport, whereas transport of other organic anions was reduced or unchanged. Significant substrate-specific changes in the ATP dependence of transport and binding by the P1150A mutant were also observed. Our findings demonstrate the importance of TM6, TM8, TM10, TM11, and TM14 in MRP1 transport function and suggest that CL7 may play a differential role in coupling the activity of the nucleotide binding domains to the translocation of different substrates across the membrane. 相似文献
2.
Situ D Haimeur A Conseil G Sparks KE Zhang D Deeley RG Cole SP 《The Journal of biological chemistry》2004,279(37):38871-38880
The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding. 相似文献
3.
Koike K Oleschuk CJ Haimeur A Olsen SL Deeley RG Cole SP 《The Journal of biological chemistry》2002,277(51):49495-49503
The multidrug resistance protein, MRP1, is a clinically important ATP-binding cassette transporter in which the three membrane-spanning domains (MSDs), which contain up to 17 transmembrane (TM) helices, and two nucleotide binding domains (NBDs) are configured MSD1-MSD2-NBD1-MSD3-NBD2. In tumor cells, MRP1 confers resistance to a broad spectrum of drugs, but in normal cells, it functions as a primary active transporter of organic anions such as leukotriene C(4) and 17beta-estradiol 17beta-(D-glucuronide). We have previously shown that mutation of TM17-Trp(1246) eliminates 17beta-estradiol 17beta-(D-glucuronide) transport and drug resistance conferred by MRP1 while leaving leukotriene C(4) transport intact. By mutating the 11 remaining Trp residues that are in predicted TM segments of MRP1, we have now determined that five of them are also major determinants of MRP1 function. Ala substitution of three of these residues, Trp(445) (TM8), Trp(553) (TM10), and Trp(1198) (TM16), eliminated or substantially reduced transport levels of five organic anion substrates of MRP1. In contrast, Ala substitutions of Trp(361) (TM7) and Trp(459) (TM9) caused a more moderate and substrate-selective reduction in MRP1 function. More conservative substitutions (Tyr and Phe) of the Trp(445), Trp(553), and Trp(1198) mutants resulted in substrate selective retention of transport in some cases (Trp(445) and Trp(1198)) but not others (Trp(553)). Our findings suggest that the bulky polar aromatic indole side chain of each of these five Trp residues contributes significantly to the transport activity and substrate specificity of MRP1. 相似文献
4.
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 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta 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 beta 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 beta G and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E(2)17beta 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. 相似文献
5.
The polytopic 5-domain multidrug resistance protein 1 (MRP1/ABCC1) extrudes a variety of drugs and organic anions across the plasma membrane. Four charged residues in the fifth cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 are critical for stable expression of MRP1 at the plasma membrane. Thus Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) all cause misfolding of MRP1 and target the protein for proteasome-mediated degradation. Of four chemical chaperones tested, 4-phenylbutyric acid (4-PBA) was the most effective at restoring expression of MRP1 mutants K513A, K516A, E521A, and E535A. However, although 4-PBA treatment of K513A resulted in wild-type protein levels (and activity), the same treatment had little or no effect on the expression of K516A. On the other hand, 4-PBA treatment allowed both E521A and E535A to exit the endoplasmic reticulum and be stably expressed at the plasma membrane. However, the 4-PBA-rescued E535A mutant exhibited decreased transport activity associated with reduced substrate affinity and conformational changes in both halves of the transporter. By contrast, E521A exhibited reduced transport activity associated with alterations in the mutant interactions with ATP as well as a distinct conformational change in the COOH-proximal half of MRP1. These findings illustrate the critical and complex role of CL5 for stable expression of MRP1 at the plasma membrane and more specifically show the differential importance of Glu(521) and Glu(535) in interdomain interactions required for proper folding and assembly of MRP1 into a fully transport competent native structure. 相似文献
6.
The multidrug resistance protein, MRP1 (ABCC1), is an ATP-binding cassette transporter that confers resistance to chemotherapeutic agents. MRP1 also mediates transport of organic anions such as leukotriene C(4) (LTC(4)), 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), estrone 3-sulfate, methotrexate (MTX), and GSH. We replaced three charged amino acids, Lys(332), His(335), and Asp(336), predicted to be in the sixth transmembrane (TM6) helix of MRP1 with neutral and oppositely charged amino acids and determined the effect on substrate specificity and transport activity. All mutants were expressed in transfected human embryonic kidney cells at levels comparable with wild-type MRP1, and confocal microscopy showed that they were correctly routed to the plasma membrane. Vesicular transport studies revealed that the MRP1-Lys(332) mutants had lost the ability to transport LTC(4), and GSH transport was reduced; whereas E(2)17betaG, estrone 3-sulfate, and MTX transport were unaffected. E(2)17betaG transport was not inhibited by LTC(4) and could not be photolabeled with [(3)H]LTC(4), indicating that the MRP1-Lys(332) mutants no longer bound this substrate. Substitutions of MRP1-His(335) also selectively diminished LTC(4) transport and photolabeling but to a lesser extent. Kinetic analyses showed that V(max) (LTC(4)) of these mutants was decreased but K(m) was unchanged. In contrast to the selective loss of LTC(4) transport in the Lys(332) and His(335) mutants, the MRP1-Asp(336) mutants no longer transported LTC(4), E(2)17betaG, estrone 3-sulfate, or GSH, and transport of MTX was reduced by >50%. Lys(332), His(335), and Asp(336) of TM6 are predicted to be in the outer leaflet of the membrane and are all capable of forming intrahelical and interhelical ion pairs and hydrogen bonds. The importance of Lys(332) and His(335) in determining substrate specificity and of Asp(336) in overall transport activity suggests that such interactions are critical for the binding and transport of LTC(4) and other substrates of MRP1. 相似文献
7.
Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein. 相似文献
8.
Human multidrug resistance protein 1 (MRP1) confers resistance to many natural product chemotherapeutic agents and actively transports structurally diverse organic anion conjugates. We previously demonstrated that two hydrogen-bonding amino acid residues in the predicted transmembrane 17 (TM17) of MRP1, Thr(1242) and Trp(1246), were important for drug resistance and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. To determine whether other residues with hydrogen bonding potential within TM17 influence substrate specificity, we replaced Ser(1233), Ser(1235), Ser(1237), Gln(1239), Thr(1241), and Asn(1245) with Ala and Tyr(1236) and Tyr(1243) with Phe. Mutations S1233A, S1235A, S1237A, and Q1239A had no effect on any substrate tested. In contrast, mutations Y1236F and T1241A decreased resistance to vincristine but not to VP-16, doxorubicin, and epirubicin. Mutation Y1243F reduced resistance to all drugs tested by 2-3-fold. Replacement of Asn(1245) with Ala also decreased resistance to VP-16, doxorubicin, and epirubicin but increased resistance to vincristine. This mutation also decreased E(2)17betaG transport approximately 5-fold. Only mutation Y1243F altered the ability of MRP1 to transport both leukotriene 4 and E(2)17betaG. Together with our previous results, these findings suggest that residues with side chain hydrogen bonding potential, clustered in the cytoplasmic half of TM17, participate in the formation of a substrate binding site. 相似文献
9.
The multidrug resistance protein 1 (MRP1) mediates drug and organic anion efflux across the plasma membrane. The 17 transmembrane (TM) helices of MRP1 are linked by extracellular and cytoplasmic (CL) loops of various lengths and two cytoplasmic nucleotide binding domains. In this study, three basic residues clustered at the predicted TM15/CL7 interface were investigated for their role in MRP1 expression and activity. Thus, Arg1138, Lys1141, and Arg1142 were replaced with residues of the same or opposite charge, expressed in human embryonic kidney cells, and the properties of the mutant proteins were assessed. Neither Glu nor Lys substitutions of Arg1138 and Arg1142 affected MRP1 expression; however, all four mutants showed a decrease in organic anion transport with a relatively greater decrease in leukotriene C4 and glutathione transport. These mutations also modulated MRP1 ATPase activity as reflected by a decreased vanadate-induced trapping of 8-azido-[32P]ADP. Mutation of Lys1141 to either Glu or Arg reduced MRP1 expression, and routing to the plasma membrane was impaired. However, only the Glu-substituted Lys1141 mutant showed a decrease in organic anion transport, and this was associated with decreased substrate binding and vanadate-induced trapping of 8-azido-ADP. These studies identified a cluster of basic amino acids likely at the TM15/CL7 interface as a region important for both MRP1 expression and activity and demonstrated that each of the three residues plays a distinct role in the substrate specificity and catalytic activity of the transporter. 相似文献
10.
The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) configured MSD1-MSD2-NBD1-MSD3-NBD2. MRP1 overexpression in tumor cells results in an ATP-dependent efflux of many oncolytic agents and arsenic and antimony oxyanions. MRP1 also transports GSSG and GSH as well as conjugated organic anions, including leukotriene C(4) and 17beta-estradiol 17-(beta-D-glucuronide) and certain xenobiotics in association with GSH. Previous studies have shown that portions of MSD1 and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. In the present study, Cys residues at positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Ala and Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organic anion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of 14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteins were also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organic anions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substituted Cys43 and Cys265 MRP1 mutants exhibited a 2.5-fold decrease and a 3-fold increase in arsenite resistance, respectively; Cys43Ser MRP1 also conferred lower levels of vincristine resistance. These results indicate that certain Cys residues in the NH(2) proximal region of MRP1 can be important for its structure and selected transport activities. 相似文献
11.
Ren XQ Furukawa T Aoki S Sumizawa T Haraguchi M Nakajima Y Ikeda R Kobayashi M Akiyama S 《Biochemistry》2002,41(48):14132-14140
MRP1 is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. Our recent study demonstrated that GSH is required for the labeling of MRP1(932)(-)(1531) with a photoanalogue of agosterol A (AG-A) and suggested that GSH interacts with the L(0) region of MRP1. In this study, we further characterized the GSH-dependent binding site of azido AG-A on MRP1. Coexpression of the N- and C-terminal halves of MRP1 (residues 1-1222, TM1-16, and 1223-1531, TM17, respectively) in Sf21 insect cells reconstituted a functional drug transporter with a K(m) for LTC(4) (97 nM) similar to that of intact MRP1. In membrane vesicles from those cells, GSH-dependent photolabeling of the MRP1 fragment (1-1222) required the coexpression of the C-terminal MRP1 fragment (1223-1531). An MRP1 fragment extending from residue 1 to 1295 however could be photolabeled by azido AG-A in a GSH-dependent manner. These data indicate that amino acids 1223-1295 of MRP1 are required for AG-A binding to MRP1 in a GSH-dependent manner. However, cross-linking of the photolabel to MRP1 occurs at a more upstream site. An arginine residue at position 1249 of MRP1 was shown to be important for the GSH-dependent binding of AG-A to MRP1. Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Furthermore, this mutant attenuated MRP1 function by decreasing the level of LTC(4) substrate transport and impairing resistance to the drug vincristine (VCR). In summary, this study demonstrates that a region of MRP1 (amino acids 1223-1295), which includes TM helix 17, is required for azido AG-A binding to MRP1 in a GSH-dependent manner. A GSH-dependent drug binding site may exist in this region. Furthermore, our findings suggest that the charged amino acid Arg(1249) proximal to the C-terminus of TM helix 17 is indispensable for MRP1-substrate interaction and the function of MRP1. 相似文献
12.
Ito K Oleschuk CJ Westlake C Vasa MZ Deeley RG Cole SP 《The Journal of biological chemistry》2001,276(41):38108-38114
The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX. 相似文献
13.
Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the drug-binding domain of P-gp. MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a K(m) value (25 microM) that was similar to that of verapamil. 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil. Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the binding of verapamil. 相似文献
14.
Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu(1089) of MRP1 as being critical for anthracycline resistance. However, Glu(1089) mutations had no effect on E(2)17betaG transport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala(1239) in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E(2)17betaG transport 3-fold. Any mutation of mrp1 Ala(1239), including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr(1242) for E(2)17betaG transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E(2)17betaG transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C(4) transport approximately 2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C(4) and E(2)17betaG. These results demonstrate the important role of Thr(1242) for E(2)17betaG transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16. 相似文献
15.
The Multidrug Resistance Protein, MRP1 (ABCC1) confers drug resistance and transports organic anions such as leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). Previous studies showed that portions of the first membrane spanning domain (MSD1) and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. We have replaced 12 prolines in MSD1 and CL3 with alanine and determined the effects of these substitutions on MRP1 expression and transport activity. All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A. The expressed mutants also transported LTC(4) and E(2)17betaG, and their K(m) (LTC(4)) values were similar to wild-type MRP1. Expression of the double mutant MRP1-P42/51A was reduced by >80% although it localized to the plasma membrane and transported organic anions. MRP1 expression was also reduced when the first transmembrane helix (amino acids 37-54) was deleted. In contrast, the phenotypes of the multiply substituted CL3 mutants MRP1-P196/205/207/209A and MRP1-P235/255A were comparable to wild-type MRP1. However, Pro(255)-substituted MRP1 mutants showed reduced immunoreactivity with a monoclonal antibody (MAb) whose epitope is located in CL3. We conclude that certain prolines in MSD1 and CL3 play a role in the expression and structure of MRP1. 相似文献
16.
Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1. 相似文献
17.
Campbell JD Koike K Moreau C Sansom MS Deeley RG Cole SP 《The Journal of biological chemistry》2004,279(1):463-468
The human ATP-binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), confers resistance to a broad range of anti-cancer agents and transports a variety of organic anions. At present, essentially no structural data exists for MRP1 that might be used to elucidate its mechanism of transport. Consequently, we have applied a modeling strategy incorporating crystal and indirect structural data from other ABC transporters to construct a model of the transmembrane domains of the core region of MRP1 that includes the amino acid side chains. Three conserved Trp residues and one non-conserved Tyr residue, shown previously to be of functional importance (Koike, K., Oleschuk, C. J., Haimeur, A., Olsen, S. L., Deeley, R. G., and Cole, S. P. C. (2002) J. Biol. Chem. 277, 49495-49503), were found to line the "pore" in our model proximal to the membrane cytosol interface. A fifth aromatic residue (Phe594) was identified that, with the Trp and Tyr residues, completed a ring or "basket" of aromatic amino acids and, accordingly, we postulated that it would also be of functional importance. To test this idea, MRP1-Phe594 mutants were expressed in human embryonic kidney cells, and their properties were examined using membrane vesicles. Substitution of Phe594 with Ala substantially reduced or eliminated the transport of five organic anion substrates by MRP1 and abrogated the binding of leukotriene C4. On the other hand, the conservatively substituted F594W and F594Y mutants remained transport competent, although significant substrate- and substitution-specific changes were observed. These studies provide some structural insight into a possible substrate binding/transport site of MRP1 at the beginning of a putative substrate translocation pathway and demonstrate the usefulness of modeling for directing structure-function analyses of this transporter. 相似文献
18.
The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT. 相似文献
19.
Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3
Oleschuk CJ Deeley RG Cole SP 《American journal of physiology. Gastrointestinal and liver physiology》2003,284(2):G280-G289
Multidrug resistance protein 3 (MRP3) is an ATP-dependent transporter of 17beta-estradiol 17beta(d-glucuronide) (E(2)17betaG), leukotriene C(4) (LTC(4)), methotrexate, and the bile salts taurocholate and glycocholate. In the present study, the role of a highly conserved Trp residue at position 1242 on MRP3 transport function was examined by expressing wild-type MRP3 and Ala-, Cys-, Phe-, Tyr-, and Pro-substituted mutants in human embryonic kidney 293T cells. Four MRP3-Trp(1242) mutants showed significantly increased E(2)17betaG uptake, whereas transport by the Pro mutant was undetectable. Similarly, the Pro mutant did not transport LTC(4). By comparison, LTC(4) transport by the Ala, Cys, Phe, and Tyr mutants was reduced by approximately 35%. The Ala, Cys, Phe, and Tyr mutants all showed greatly reduced methotrexate and leucovorin transport, except the Tyr mutant, which transported leucovorin at levels comparable with wild-type MRP3. In contrast, the MRP3-Trp(1242) substitutions did not significantly affect taurocholate transport or taurocholate and glycocholate inhibition of E(2)17betaG uptake. Thus Trp(1242) substitutions markedly alter the substrate specificity of MRP3 but leave bile salt binding and transport intact. 相似文献
20.
S W Hall M Nagashima L Zhao J Morser L L Leung 《The Journal of biological chemistry》1999,274(36):25510-25516
A collection of 56 purified thrombin mutants, in which 76 charged or polar surface residues on thrombin were mutated to alanine, was used to identify key residues mediating the interactions of thrombin with thrombomodulin (TM), protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI). Comparison of protein C activation in the presence and absence of TM identified 11 residues mediating the thrombin-TM interaction (Lys(21), Gln(24), Arg(62), Lys(65), His(66), Arg(68), Thr(69), Tyr(71), Arg(73), Lys(77), Lys(106)). Three mutants (E25A, D51A, R89A/R93A/E94A) were found to have decreased ability to activate TAFI yet retained normal protein C activation, whereas three other mutants (R178A/R180A/D183A, E229A, R233A) had decreased ability to activate protein C but maintained normal TAFI activation. One mutant (W50A) displayed decreased activation of both substrates. Mapping of these functional residues on thrombin revealed that the 11 residues mediating the thrombin-TM interaction are all located in exosite I. Residues important in TAFI activation are located above the active-site cleft, whereas residues involved in protein C are located below the active-site cleft. In contrast to the extensive overlap of residues mediating TM binding and fibrinogen clotting, these data show that distinct domains in thrombin mediate its interactions with TM, protein C, and TAFI. These studies demonstrate that selective enzymatic properties of thrombin can be dissociated by site-directed mutagenesis. 相似文献