ABCC1 p.Lys1181Ala
| Predicted by SNAP2: | A: N (72%), C: N (66%), D: D (53%), E: N (61%), F: D (53%), G: N (61%), H: N (82%), I: N (82%), L: N (87%), M: N (57%), N: N (82%), P: N (61%), Q: N (82%), R: N (97%), S: N (72%), T: N (87%), V: N (57%), W: D (66%), Y: N (57%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: N, S: D, T: D, V: D, W: D, Y: D, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Multiple roles of charged amino acids in cytoplasm... Mol Pharmacol. 2009 Feb;75(2):397-406. Epub 2008 Nov 17. Conseil G, Rothnie AJ, Deeley RG, Cole SP
Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1).
Mol Pharmacol. 2009 Feb;75(2):397-406. Epub 2008 Nov 17., [PMID:19015228]
Abstract [show]
Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu(1169), Glu(1170), and Glu(1172) were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, (1169)AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and (1169)AAQA were accompanied by changes in orthovanadate-induced trapping of [alpha-(32)P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.
Comments [show]
None has been submitted yet.
No. Sentence Comment
6 The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, 1169 AAQA).
X
ABCC1 p.Lys1181Ala 19015228:6:236
status: NEW8 The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and 1169 AAQA were accompanied by changes in orthovanadate-induced trapping of [␣-32 P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP.
X
ABCC1 p.Lys1181Ala 19015228:8:100
status: NEW59 Mutations were first 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 restriction site (substituted nucleotides are underlined): E1144A (5Ј-G AAG CGC CTC GCG TCG GTC AGC-3Ј); R1166A (5Ј C AGC GTC ATT GCA GCA TTC GAG GAG CAG-3Ј); R1166K (5Ј C AGC GTC ATT AAG GCC TTC GAG G-3Ј); 1169 EEQE-1169 AAQA (5Ј-C ATT CGA GCC TTC GCG GCA CAG GCA CGC TTC ATC C-3Ј); R1173A (5Ј-C GAG GAG CAG GAG GCA TTC ATC CAC CAG AG-3Ј); D1179A (5Ј-C CAC CAG AGT GCC CTT AAG GTG GAC G-3Ј), K1181A (5Ј-G AGT GAC CTG GCA GTC GAC GAG AAC C-3Ј); D1183A (5Ј-CTG AAG GTG GCC GAG AAC CAG-3Ј); D1183E (5Ј- CTG AAG GTG GAA GAG AAC CAG-3Ј); and E1184A (5Ј-GT GAC CTG AAG GTA GAC GCG AAC CAG AAG GCC-3Ј).
X
ABCC1 p.Lys1181Ala 19015228:59:700
status: NEW156 On the other hand, the K1181A mutant exhibited a global reduction (ϳ50%) in transport of all tested organic anion substrates.
X
ABCC1 p.Lys1181Ala 19015228:156:23
status: NEW160 Because the single E1144A and K1181A mutants and the 1169 AAQA triple mutant exhibited the most substantial changes in transport activity, their ATP binding and hydrolysis properties were examined.
X
ABCC1 p.Lys1181Ala 19015228:160:30
status: NEW164 Thus, trapping of 8-azido-[␣-32 P]ADP by the K1181A mutant was 40% lower than for wild-type MRP1.
X
ABCC1 p.Lys1181Ala 19015228:164:52
status: NEW177 [3 H]LTC4 Photolabeling of K1181A Mutant MRP1 Protein.
X
ABCC1 p.Lys1181Ala 19015228:177:27
status: NEW178 Because the K1181A mutant showed the most significant decrease (45%) in LTC4 transport activity of the seven single Ala mutants, it was of interest to determine whether binding of this substrate was affected by this mutation.
X
ABCC1 p.Lys1181Ala 19015228:178:12
status: NEW179 However, as shown in Fig. 7, no differences in [3 H]LTC4 photolabeling of the K1181A mutant and wild-type MRP1 in their nucleotide-free states were detected after taking in account relative levels of MRP1 expression (Fig. 4).
X
ABCC1 p.Lys1181Ala 19015228:179:78
status: NEW180 Because vanadate-induced trapping of azidoADP by the K1181A mutant was significantly reduced compared with wild-type MRP1 (Fig. 5, B and C), [3 H]LTC4 photolabeling experiments were also carried out using MRP1-enriched membranes in the ATP-bound and "ADP-trapped" (low affinity) states.
X
ABCC1 p.Lys1181Ala 19015228:180:53
status: NEW181 Under both conditions, low levels of [3 H]LTC4 photolabeling were observed for the K1181A mutant (Fig. 7).
X
ABCC1 p.Lys1181Ala 19015228:181:83
status: NEW185 Shown is an immunoblot of membrane vesicles (1 g of protein) prepared from HEK 293T cells transfected with cDNA expression vectors encoding wild-type, E1144A, 1169 AAQA, R1173A, D1179A, K1181A, and E1184A MRP1 mutants.
X
ABCC1 p.Lys1181Ala 19015228:185:194
status: NEW192 The remaining five single mutants were either poorly expressed (R1166A, D1183A) (Fig. 2) or exhibited significantly reduced transport levels of one or more organic anions (E1144A, D1179A, K1181A) (Table 1).
X
ABCC1 p.Lys1181Ala 19015228:192:188
status: NEW212 Percentage Wild-Type MRP1 Uptake Activity E1144A 1169 AAQA R1173A D1179A K1181A E1184A % LTC4 90 Ϯ 5 70 Ϯ 5 120 Ϯ 5 65 Ϯ 5 55 Ϯ 5 85 Ϯ 5 E217bG 55 Ϯ 5 80 Ϯ 0 130 Ϯ 10 90 Ϯ 5 65 Ϯ 5 95 Ϯ 15 E13SO4 45 Ϯ 5 45 Ϯ 5 125 Ϯ 0 65 Ϯ 0 50 Ϯ 5 90 Ϯ 10 MTX 55 Ϯ 5 105 Ϯ 5 N.D. N.D. 45 Ϯ 5 N.D. GSH 80 Ϯ 5 95 Ϯ 10 N.D. N.D. 30 Ϯ 5 N.D. N.D., not determined.
X
ABCC1 p.Lys1181Ala 19015228:212:73
status: NEW219 8-Azido-[32 P]ATP photolabeling and vanadate-dependent 8-azido- [32 P]ADP trapping of CL7 mutants E1144A, 1169 AAQA and K1181A of MRP1.
X
ABCC1 p.Lys1181Ala 19015228:219:120
status: NEW235 [3 H]LTC4 photolabeling of wild-type and K1181A mutant MRP1 in the presence of ATP and trapped ADP.
X
ABCC1 p.Lys1181Ala 19015228:235:41
status: NEW236 Wild-type (WT-MRP1) and K1181A mutant membrane vesicles (50 g of protein) were incubated in transport buffer containing 5 mM MgCl2 for 20 min in the absence (-) or presence (ϩ) of ATP (1 mM) and sodium orthovanadate (Vi) (1 mM), alone or in combination, and then incubated with [3 H]LTC4 (200 nM, 120 nCi) for a further 30 min followed by UV cross-linking, SDS-PAGE, and fluorography.
X
ABCC1 p.Lys1181Ala 19015228:236:24
status: NEW244 Unlike the R1166A and D1183A mutants, the second group of functionally altered single Ala-substituted mutants (E1144A, D1179A, K1181A) and the 1169 AAQA triple mutant were all expressed at levels comparable with or greater than that of wild-type MRP1 (Fig. 4).
X
ABCC1 p.Lys1181Ala 19015228:244:127
status: NEW255 The altered transport activity and nucleotide interactions of the E1144A, K1181A, and 1169 AAQA mutants may thus result from perturbation of this signaling (Table 1, Fig. 5).
X
ABCC1 p.Lys1181Ala 19015228:255:74
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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
726 These include Cys43 in TM1 [171], Thr73 in CL1 [366], Trp222 in L0 [279], Trp223 in L0 [279], Arg230 in L0 [279], Trp261 in L0 [271, 302, 338], Lys267 in L0 [271, 302, 338], Lys319 in TM6 [339], Tyr324 in TM6 [301], Lys332 in TM6 [166, 339-341], His335 in TM6, Asp336 in TM6 [339], Lys347 in TM6 [339], Lys396 in TM7 [339], Arg433 in TM8 [363], Asp436 in TM8 [339], Trp445 in TM8, Trp459 in TM8, Pro478 in TM9, Thr550 in TM10 [343], Trp553 in TM10 [344], Thr556 in TM10 [343], Pro557 in TM10 [345], Tyr568 in TM10 [343], Arg593 in TM11 [339], Phe594 in TM11 [300], Asn597 in TM11, Ser604 in TM11, Ser605 in TM11, Trp653 in NBD1 [351], Lys684 in Walker A motif of NBD1 [350, 364], Ser685 in Walker A motif of NBD1 [353], Arg723 in NBD1 [366], Gly771 in the ABC signature (C motif) of NBD1 [364], Asp792 in Walker B motif of NBD1 [361], Asp793 in Walker B motif of NBD1 [353], Ala989 in TM12 [367], Pro1120 in TM13, Pro1121 in TM13, Arg1058 at TM13/CL6 interface [366], Glu1089 in TM14, Lys1092 in TM14, Ser1097 in TM14, Asn1100 in TM14, Arg1138 in TM15 [354], Lys1141 in CL7 [354], Arg1142 in CL7 [354], Glu1144Ala in CL7 [355], Pro1150 in CL7 [345, 347, 348], Arg1166Ala in CL7 [355], Asp1179Ala in CL7 [355], Lys1181Ala in CL7 [355], Asp1183 in CL7 [355], Tyr1189 in CL7 [368], Tyr1190 in CL7 [368], Arg1197 in TM16 [357], Trp1198 in TM16 [344], Arg1202 in TM16 [357], Glu1204 in TM16 [357], Tyr1236 in TM17, Thr1241 in TM17, Thr1242 in TM17, Tyr1243 in TM17, Asn1245 in TM17, Trp1246 in TM17 [166, 339-341], Arg1249 in TM17 [342], Tyr1302 in NBD2 [351], Lys1333 in Walker A motif of NBD2 [350], Gly1433 in the ABC signature motif of NBD2 [352, 364], Glu1455 in Walker B motif of NBD2 [322], and His1486 in NBD2 [349].
X
ABCC1 p.Lys1181Ala 21143116:726:1210
status: NEW813 The single mutants Glu1144Ala, Asp1179Ala, and Lys1181Ala) and the triple mutant 1169AAQA showed only moderate and substrate-selective changes in transport activity [355].
X
ABCC1 p.Lys1181Ala 21143116:813:47
status: NEW