ABCC1 p.Arg1138Glu
| Predicted by SNAP2: | A: D (95%), C: D (80%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (91%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%), |
| 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, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Functional importance of three basic residues clus... J Biol Chem. 2006 Jan 6;281(1):43-50. Epub 2005 Oct 17. Conseil G, Deeley RG, Cole SP
Functional importance of three basic residues clustered at the cytosolic interface of transmembrane helix 15 in the multidrug and organic anion transporter MRP1 (ABCC1).
J Biol Chem. 2006 Jan 6;281(1):43-50. Epub 2005 Oct 17., 2006-01-06 [PMID:16230346]
Abstract [show]
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.
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No. Sentence Comment
47 Substitutions of charged residues were generated in the pGEM-3Z- XmaI/MRP1 plasmid according to the manufacturer`s instructions with the following mutagenic primers (Integrated DNA Technologies, Inc., Coralville, IA), which also introduced an additional silent restriction site (substituted nucleotides are underlined): R1138E, 5Ј-CGT GGC TTC CTC CGA GCA ACT GAA GCG CCT CGA G-3Ј; R1138K, 5Ј-CGT GGC TTC CTC CAA GCA GCT TAA GCG CCT CGA G-3Ј; K1141E, 5Ј-CGG CAG CTG GAG CGC CTG GAG TCG GTC AGC-3Ј; K1141R, 5Ј-CCC GGC AGC TGC GGC GCC TCG AGT CGG-3Ј; R1142E, 5Ј-CCC GGC AGC TGA AGG AGC TCG AGT CGG TCA GCC G-3Ј;R1142K, 5Ј-CCC GGC AGC TGA AGA AGC TTG AGT CGG TCA GCC G-3Ј.
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ABCC1 p.Arg1138Glu 16230346:47:320
status: NEW118 A, membrane vesicles (1 g of protein) prepared from HEK293T cells transfected with wild-type (WT-MRP1), and mutant (R1138E/K, K1141E/R, R1142E/K) MRP1 cDNAs were immunoblotted, and MRP1 was detected with monoclonal antibody QCRL-1.
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ABCC1 p.Arg1138Glu 16230346:118:124
status: NEW122 B, HEK293T cells were transfected with expression vectors encoding WT-MRP1 and MRP1 mutants R1138E, R1138K, K1141E, K1141R, R1142E, and R1142K, and 48 h later, cells were probed with monoclonal antibody QCRL-3 and processed for confocal fluorescence microscopy using Alexa Fluor 488 conjugated secondary antibody.
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ABCC1 p.Arg1138Glu 16230346:122:92
status: NEW135 Finally, the effects of the mutations on MTX transport were relatively minor and ranged from no decrease to a maximum decrease of 35% (R1138E) when compared with wild-type MRP1 (Fig. 3D).
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ABCC1 p.Arg1138Glu 16230346:135:135
status: NEW136 Photolabeling of Arg1138 , Lys1141 , and Arg1142 Mutants of MRP1 with [3 H]LTC4-To ascertain whether the decrease in LTC4 uptake by the R1138E/K, K1141E, and R1142E/K mutants was related to a decrease in substrate binding, membrane vesicles enriched for wild-type or mutant MRP1s were photolabeled with [3 H]LTC4.
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ABCC1 p.Arg1138Glu 16230346:136:136
status: NEW137 As shown in Fig. 4, LTC4 labeling of R1138E and K1141E was decreased by 50% or more, after correcting for differences in protein expression levels, consistent with their decreased LTC4 transport activity.
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ABCC1 p.Arg1138Glu 16230346:137:37
status: NEW139 Therefore, reduced substrate binding appeared to explain, at least in part, the loss of LTC4 transport for the R1138E and K1141E mutants but not the R1138K, R1142E, and R1142K mutants.
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ABCC1 p.Arg1138Glu 16230346:139:111
status: NEW149 ATP-dependent uptake of 3 H-labeled organic anions was measured in membrane vesicles prepared from HEK293T cells transfected with wild-type MRP1 (WT-MRP1) and Arg1138 , His1141 and Arg1142 mutant MRP1 (R1138E/K, K1141E/R, R1141E/K) cDNAs (shown in Fig. 2).
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ABCC1 p.Arg1138Glu 16230346:149:202
status: NEW163 In contrast, when the catalytic activity of MRP1 was measured at 37 °C by the orthovanadate-induced trapping of 8-azido-[␣32 P]ADP in the post-hydrolysis state (Fig. 6B), five of the six mutants (R1138E/K, R1142E/K, and K1141E) showed substantially reduced nucleotide trapping (ϳ40-60% that of wild-type MRP1).
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ABCC1 p.Arg1138Glu 16230346:163:208
status: NEW200 The reduction in GSH and LTC4 transport appeared largely attributable to a substantial decrease in substrate binding in the case of R1138E but not R1138K.
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ABCC1 p.Arg1138Glu 16230346:200:132
status: NEW[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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No. Sentence Comment
739 In contarst, the mutations showed minor to moderate effect on MTX transport, with a maximum decrease for Arg1138Glu).
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ABCC1 p.Arg1138Glu 21143116:739:105
status: NEW744 The Arg1138Glu and Lys1141Glu mutants, but not the Arg1138Lys, Arg1142Glu, and Arg1142Lys mutants, showed a >50% decrease in binding to [3 H]LTC4.
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ABCC1 p.Arg1138Glu 21143116:744:4
status: NEW