ABCMdb

ABCC1 p.Trp1198Ala

Predicted by SNAP2:	A: D (66%), C: D (59%), D: D (85%), E: D (80%), F: D (63%), G: D (80%), H: D (80%), I: D (59%), K: D (80%), L: D (63%), M: D (53%), N: D (80%), P: D (91%), Q: D (71%), R: D (75%), S: D (75%), T: D (80%), V: D (63%), Y: D (66%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, Y: D,

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[hide] Multiple membrane-associated tryptophan residues c... J Biol Chem. 2002 Dec 20;277(51):49495-503. Epub 2002 Oct 17. Koike K, Oleschuk CJ, Haimeur A, Olsen SL, Deeley RG, Cole SP
Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1.
J Biol Chem. 2002 Dec 20;277(51):49495-503. Epub 2002 Oct 17., 2002-12-20 [PMID:12388549]

Abstract [show]
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.

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48 Tryptophan substitutions were generated in the pGEM-3Z and pBluescriptSK(ϩ) plasmids above according to the manufacturer`s instructions with the following mutagenic primers (substituted nucleotides are underlined): MSD1 Trp mutants W40A (5Ј-G GTC CTC GTG GCC GTG CCT TG-3Ј, W47A (5Ј-GT TTT TAC CTC GCC GCC TGT TTC CCC-3Ј), W82A (5Ј-C TTG GGA TTT TTG CTG GCG ATC GTC TGC TGG GC-3Ј), W86A (5Ј-G CTG TGG ATC GTC TGC GCT GCA GAC CTC TTC TAC TC-3Ј), W94A (5Ј-C CTC TTC TAC TCT TTC GCG GAA AGA AGT CGG GGC-3Ј), W142A (5Ј-GGG ATC ATG CTC ACT TTC GCA CTG GTA GCC CTA ATG TG-3Ј), W142F (5Ј-G CTC ACT TTT TTC CTG GTA GCC C-3Ј); MSD2 Trp mutants W361A (5Ј-C ACG AAG GCC CCA GAT GCG CAG GGC TAC TTC TAC-3Ј), W445A (5Ј-G TAC ATT AAC ATG ATC GCG TCA GCC CCC CTG CAA G-3Ј), W445F (5Ј-CG TAC ATT AAC ATG ATC TTC TCA GCC CCC CTG CAA GTC-3Ј), W445Y (5Ј-CC ACG TAC ATT AAC ATG ATC TAC TCA GCG CCC CTG CAA GTC-3Ј), W459A (5Ј-GCT CTC TAC CTC CTG GCG CTG AAT CTG GGC CC-3Ј), W553A (5Ј-G GGC ACC TTC ACC GCG GTC TGC ACG CCC-3Ј), W553F (5Ј-G GGC ACC TTC ACC TTC GTC TGC ACG CCC-3Ј), W553Y (5Ј-GCC CTG GGC ACC TTC ACA TAT GTC TGC ACG CCC-3Ј; and MSD3 Trp mutants W1198A (5Ј-C GTG GCC AAC AGG GCG CTG GCC GTG CGG C-3Ј), W1198F (5Ј-GTG GCC AAC AGG TTC CTG GCC GTG CGG C-3Ј), W1198Y (5Ј-GTG GCC AAC AGG TAC CTG GCC GTG CGG C-3Ј). Login to comment
50 Construction of MRP1-GFP Fusion Proteins-Constructs encoding GFP fusion proteins of selected MRP1 Trp mutations were generated by exchanging the 1.3-kb ClaI/AflII fragment of a pcDNA3.1(-)-MRP1-GFP construct with the comparable fragments containing the W445A, W553A, and W1198A mutations generated above and designated pcDNA3.1-W445A/MRP1-GFP, pcDNA3.1-W553A/MRP1-GFP, and pcDNA3.1-W1198A/MRP1-GFP, respectively (39). Login to comment
132 When expressed in HEK cells, W1198A-MRP1 was found to be expressed at levels that were ϳ35% (range 30-40%) of those of wild-type MRP1, suggesting that MRP1 stability may be affected by this mutation. Login to comment
133 Time courses of [3 H]LTC4 uptake showed that transport of this GSH-conjugated substrate by the W1198A mutant was ϳ65% of that by wild-type MRP1 after correcting for differences in MRP1 expression levels (Fig. 5A). Login to comment
134 In contrast, ATP-dependent [3 H]E217betaG uptake by the W1198A mutant was not detectable (Fig. 5B). Login to comment
144 Transport activity of the W1198A MRP1 mutant was recovered to an even greater extent by Phe and Tyr substitutions. Login to comment
148 In contrast to the W445A and W1198A mutants, more conservative substitutions of the Ala-substituted Trp553 mutant W553A were much less effective in restoring MRP1 transport activity. Login to comment
171 C-E, relative uptake levels of 3 H-labeled organic anions by membrane vesicles enriched for wild-type MRP1 (solid bar) and Trp1198 3 Ala substituted MRP1 (W1198A; shaded bars) were determined as described under "Experimental Procedures." Login to comment
189 Ala substitution of Trp1198 in MSD3 also resulted in a broad and profound decrease in MRP1 transport activity except for FIG. 6. ATP-dependent organic anion transport activity of wild-type and mutant MRP1 containing conservative Phe and Tyr substitutions of Trp1198 in TM16 of MSD3. A, immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (W445A, W445F, and W445Y; W553A, W553F and W553Y; W1198A, W1198F, and W1198Y) MRP1 cDNAs. MRP1 proteins were detected with monoclonal antibody QCRL-1, and relative levels of expression shown under the blot were estimated by densitometry. Login to comment
191 Ala-substituted mutants (W445A, W553A, and W1198A; shaded bars) were included for comparison. Login to comment
194 The Ala-substituted Trp1246 mutant, like W553A and W1198A, also showed significantly reduced transport of multiple MRP1 substrates with the exception of LTC4 (39). Login to comment
196 However, the specific environment of the two Trp residues appears to differ, since Tyr and Phe substitutions restore the transport activity of the W1198A but not W1246A mutant (39). Login to comment
224 HEK293T cells were transfected with pcDNA3.1(-)MRP1K-GFP (WT-MRP1-GFP) (A), pcDNA3.1-W445A/MRP1-GFP (B), pcDNA3.1-W553A/MRP1-GFP (C), and pcDNA3.1-W1198A/MRP1-GFP (D), and 48 h later, cells were processed for confocal fluorescence microscopy. Login to comment

[hide] Mutational analysis of polar amino acid residues w... Drug Metab Dispos. 2006 Apr;34(4):539-46. Epub 2006 Jan 13. Zhang DW, Nunoya K, Vasa M, Gu HM, Cole SP, Deeley RG
Mutational analysis of polar amino acid residues within predicted transmembrane helices 10 and 16 of multidrug resistance protein 1 (ABCC1): effect on substrate specificity.
Drug Metab Dispos. 2006 Apr;34(4):539-46. Epub 2006 Jan 13., [PMID:16415113]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) has a total of 17 transmembrane (TM) helices arranged in three membrane-spanning domains, MSD0, MSD1, and MSD2, with a 5 + 6 + 6 TM configuration. Photolabeling studies indicate that TMs 10 and 11 in MSD1 and 16 and 17 in MSD2 contribute to the substrate binding pocket of the protein. Previous mutational analyses of charged and polar amino acids in predicted TM helices 11, 16, and 17 support this suggestion. Mutation of Trp(553) in TM10 also affects substrate specificity. To extend this analysis, we mutated six additional polar residues within TM10 and the remaining uncharacterized polar residue in TM16, Asn(1208). Although mutation of Asn(1208) was without effect, two of six mutations in TM10, T550A and T556A, modulated the drug resistance profile of MRP1 without affecting transport of leukotriene C4, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), and glutathione. Mutation T550A increased vincristine resistance but decreased doxorubicin resistance, whereas mutation T556A decreased resistance to etoposide (VP-16) and doxorubicin. Although conservative mutation of Tyr(568) in TM10 to Phe or Trp had no apparent effect on substrate specificity, substitution with Ala decreased the affinity of MRP1 for E(2)17betaG without affecting drug resistance or the transport of other substrates tested. These analyses confirm that several amino acids in TM10 selectively alter the substrate specificity of MRP1, suggesting that they interact directly with certain substrates. The location of these and other functionally important residues in TM helices 11, 16, and 17 is discussed in the context of an energy-minimized model of the membrane-spanning domains of MRP1.

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No. Sentence Comment
165 Mutation of Trp1198 to Ala also dramatically decreased overall transport activity (Koike et al., 2002). Login to comment