ABCMdb

ABCC1 p.Trp361Ala

Predicted by SNAP2:	A: D (71%), C: D (66%), D: D (85%), E: D (80%), F: D (63%), G: D (75%), H: D (80%), I: D (66%), K: D (80%), L: D (75%), M: D (66%), N: D (75%), P: D (91%), Q: D (75%), R: D (80%), S: D (71%), T: D (71%), V: D (66%), Y: D (53%),
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). Login to comment
108 All four mutants generated (W361A, W445A, W459A, and W553A) were expressed at levels 60-90% those of wild-type MRP1, indicating that none of the mutations had a major effect on the expression levels of the protein. Login to comment
110 After 1 min, ATP-dependent [3 H]LTC4 uptake by the W445A and W553A MRP1 mutants was reduced by ϳ75 and 50%, respectively, whereas uptake by the W361A and W459A mutants was comparable with wild-type MRP1 (Fig. 3B). Login to comment
113 In contrast, after 1 min, [3 H]E217betaG uptake by the W361A, W445A, and W553A MRP1 mutants was ϳ50, 25, and 10%, respectively, of wild-type MRP1 (Fig. 3D). Login to comment
119 Similarly, GSH-stimulated E13SO4 uptake levels by the W445A and W553A mutants were just 30 and Ͻ10% of wild-type MRP1 levels, respectively, whereas uptake by the W361A mutant was similar to wild-type MRP1, and uptake by W459A MRP1 was reduced by just 25% (Fig. 4B). Login to comment
121 A, ATP-dependent uptake of [3 H]LTC4 was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector pcDNA3.1(-) (E) and vectors containing wild-type MRP1 (f) and MSD2 Trp-Ala mutant MRP1 cDNAs (W361A, Œ; W445A, ; W459A, ࡗ; and W553A, q). Login to comment
124 C, the time course of ATP-dependent uptake of [3 H]E217betaG by wild-type MRP1 and MSD2 mutants W361A, W445A, W459A, and W553A was measured as described for A. D, relative levels of [3 H]E217betaG uptake at 1 min are shown and were determined from the time course shown in C as described for B. Login to comment
127 MTX uptake by the W361A mutant was also reduced (ϳ40%), whereas uptake by the W459A mutant was similar to that of wild-type MRP1. Login to comment
128 Thus, substitution of either Trp445 or Trp553 by an Ala residue resulted in a general loss of MRP1 organic anion transport activity, whereas Ala substitution of Trp361 and Trp459 had a much more moderate and selective effect on the substrate specificity of the protein. Login to comment
153 Relative uptake levels of 3 H-labeled organic anions by membrane vesicles enriched for wild-type MRP1 (solid bar) and W361A, W445A, W459A, and W553A mutant MRP1 proteins (shaded bars) were determined as described under "Experimental Procedures." Login to comment
179 Thus, the W361A mutant exhibited a 25-50% decrease in E217betaG, GSH, and MTX transport activity compared with wild-type MRP1, but LTC4 and E13SO4 transport activities were not affected. Login to comment

[hide] Structural and functional properties of human mult... Curr Med Chem. 2011;18(3):439-81. He SM, Li R, Kanwar JR, Zhou SF
Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1).
Curr Med Chem. 2011;18(3):439-81., [PMID:21143116]

Abstract [show]
Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.

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Sentences [show]
No. Sentence Comment
781 Ala substitution of Trp361 in TM7 and Trp459 in TM9 only resulted in more moderate changes in trasnport activity. Login to comment