ABCC1 p.Trp142Ala
| Predicted by SNAP2: | A: D (71%), C: D (71%), D: D (91%), E: D (85%), F: D (63%), G: D (85%), H: D (85%), I: D (75%), K: D (91%), L: D (75%), M: D (66%), N: D (85%), P: D (91%), Q: D (85%), R: D (91%), S: D (80%), T: D (80%), V: D (75%), Y: D (59%), |
| 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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No. Sentence Comment
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).
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ABCC1 p.Trp142Ala 12388549:48:571
status: NEW95 In contrast, levels of the W142A MRP1 mutant were ϳ35% (range 30-40%) of wild-type MRP1 (Fig. 2A).
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ABCC1 p.Trp142Ala 12388549:95:27
status: NEW96 Reduced expression levels of the W142A-MRP1 mutant were observed in multiple different transfection assays using several independently derived cDNA clones.
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ABCC1 p.Trp142Ala 12388549:96:33
status: NEW99 A, immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild type (WT-MRP1), and mutant (W40A, W47A, W82A, W86A, W94A, and W142A) MRP1 cDNAs. MRP1 proteins were detected with monoclonal antibody QCRL-1, and relative levels of expression shown under the blot were estimated by densitometry.
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ABCC1 p.Trp142Ala 12388549:99:175
status: NEW169 Despite this unusually high frequency (4.5%), we found that none of the six singly Ala-substituted Trp mutants in the TM segments of this region showed significantly reduced E217betaG or LTC4 transport activity, and only one of them, W142A, was expressed at significantly lower levels than wild-type MRP1.
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ABCC1 p.Trp142Ala 12388549:169:234
status: NEW