ABCC1 p.Arg501Ala
| Predicted by SNAP2: | A: D (75%), C: D (75%), D: D (91%), E: D (85%), F: D (85%), G: D (85%), H: D (75%), I: D (80%), K: D (71%), L: D (80%), M: D (75%), N: D (80%), P: D (91%), Q: D (71%), S: D (75%), T: D (80%), V: D (80%), W: D (91%), Y: D (85%), |
| 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] Expression and function of human MRP1 (ABCC1) is d... J Biol Chem. 2011 Mar 4;286(9):7202-13. Epub 2010 Dec 20. Iram SH, Cole SP
Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2.
J Biol Chem. 2011 Mar 4;286(9):7202-13. Epub 2010 Dec 20., 2011-03-04 [PMID:21177244]
Abstract [show]
Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.
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No. Sentence Comment
47 To generate point mutations in CL5, plasmid pBluescriptSK(ϩ)BamHI/SpHI-MRP1 containing a 1.9-kb fragment from pcDNA3.1(-)-MRP1k was used as the template with the following mutagenic primers (substituted nucleotides are underlined): R501A, 5Ј-GAG CAA AGA CAA TGC GAT CAA GCT GAT G-3Ј; K503A, 5Ј-GAC AAT CGG ATC GCG CTG ATG AAC G-3Ј; E507A, 5Ј-GCT GAT GAA CGC AAT TCT CAA TGG G-3Ј; G511I, 5Ј-CTC AAT ATT ATC AAA GTG CTA AAG CTT TAT GCC TGG GAG CTG GC-3Ј; K513A, 5Ј-CTC AAT GGG ATC GCA GTG CTA AAG C-3Ј; K513R, 5Ј-CTC AAT GGG ATC AGA GTG CTA AAG C-3Ј; K516A, 5Ј-GAT CAA AGT GCT AGC GCT TTA TGC CTG-3Ј; K516R, 5Ј-GAT CAA AGT GCT AAG ACT TTA TGC CTG-3Ј; E521A, 5Ј-CTT TAT GCC TGG GCG CTG GCA TTC-3Ј; E521D, 5Ј-CTT TAT GCC TGG GAC CTG GCA TTC-3Ј; R532A, 5Ј-GTG CTG GCC ATT GCG CAG GAG GAG CT-3Ј; E535A, 5Ј-CAT CAG GCA GGA GGC CTT GAA GGT GCT GAA G-3Ј; and E535D, 5Ј- CAG GCA GGA GGA TCT GAA GGT GCT GAA G-3Ј.
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ABCC1 p.Arg501Ala 21177244:47:238
status: NEW112 On the other hand, levels of the four remaining CL5 Ala-substituted mutants (R501A, K503A, E507A, and R532A) were comparable with wild-type MRP1 (see Fig. 6A).
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ABCC1 p.Arg501Ala 21177244:112:77
status: NEW163 Effect of Ala substitution of Arg501 , Lys503 , Glu507 , and Arg532 on MRP1 Transport Activity-As mentioned earlier, levels of the Ala-substituted mutants of Arg501 , Lys503 , Glu507 , and Arg532 were comparable with that of wild-type MRP1 (Fig. 6A), and the mutant proteins all routed correctly to the plasma membrane (results not shown).
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ABCC1 p.Arg501Ala 21177244:163:10
status: NEW164 When their transport properties were examined, however, a 50-60% decrease in the [3 H]LTC4 and [3 H]E217betaG transport levels of the R501A, E507A, and R532A mutants was observed (Fig. 6, B and C).
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ABCC1 p.Arg501Ala 21177244:164:134
status: NEW166 The reduced E217betaG transport activity of the R501A, E507A, and R532A mutants was analyzed further by determining the kinetic parameters of uptake; the results of these experiments are summarized in Table 1.
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ABCC1 p.Arg501Ala 21177244:166:48
status: NEW168 In contrast, the Km (E217betaG) values for the R501A, E507A, and R532A mutants were increased 3-5-fold (18-25 M).
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ABCC1 p.Arg501Ala 21177244:168:47
status: NEW186 Photolabeling of MRP1 Mutants R501A, E507A, R532A, and G511I with [3 H]LTC4-To determine whether the decrease in LTC4 uptake by the R501A, E507A, R532A, and G511I mutants was associated with decreased binding of this substrate to MRP1, membrane vesicles enriched for wild-type or mutant MRP1 were photolabeled with [3 H]LTC4.
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ABCC1 p.Arg501Ala 21177244:186:30
status: NEWX
ABCC1 p.Arg501Ala 21177244:186:132
status: NEW187 As shown in Fig. 8A, [3 H]LTC4 labeling of the R501A and E507A mutants was decreased by 50%, whereas labeling of the R532A mutant was decreased by 70%.
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ABCC1 p.Arg501Ala 21177244:187:47
status: NEW199 Levels and organic anion transport activity of MRP1 mutant proteins R501A, K503A, E507A, and R532A.
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ABCC1 p.Arg501Ala 21177244:199:68
status: NEW200 A, shown is a representative immunoblot of membrane vesicles (MV) (1 g of protein/lane) prepared from HEK293T cells transfected with wild-type (WT-MRP1) and mutant (R501A, K503A, E507A, and R532A) cDNA expression vectors. Untransfected cells were used as a negative control. MRP1 was detected with mAb QCRL-1, and the relative protein expression levels were estimated by densitometry and are shown below the blot.
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ABCC1 p.Arg501Ala 21177244:200:173
status: NEW204 TABLE 1 Kinetic parameters of E217betaG uptake by MRP1 mutants R501A, E507A, and R532A Km and Vmax values for E217betaG uptake by membrane vesicles (4 g of protein) prepared from HEK293T cells transfected with wild-type or mutant proteins were determined by measuring ATP-dependent uptake at eight different E217betaG concentrations (0.25-25 M) for 1 min at 37 °C as described under "Experimental Procedures."
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ABCC1 p.Arg501Ala 21177244:204:63
status: NEW207 Transfectant Km Vmax a (Vmax/Km) ؋ 10-3 M pmol/mg/min mg/liter/min WT-MRP1 5.4 1088 0.20 R501A 22.9 1274 0.05 E507A 18.1 1150 0.06 R532A 24.5 1031 0.04 a Vmax values have been corrected for differences in protein expression of the mutants relative to WT-MRP1 (Fig. 7A).
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ABCC1 p.Arg501Ala 21177244:207:103
status: NEW210 Of the charged amino acid mutants, only K503A exhibited properties comparable with those of wild-type MRP1, whereas the seven remaining Ala-substituted mutants were either poorly expressed (K513A, K516A, E521A, and E535A) or exhibited significantly reduced transport activity (R501A, E507A, and R532A).
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ABCC1 p.Arg501Ala 21177244:210:277
status: NEW236 [3 H]LTC4 photolabeling of wild-type and MRP1 mutants R501A, E507A, R532A, and G511I.
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ABCC1 p.Arg501Ala 21177244:236:54
status: NEW253 Nevertheless, although the K503A mutant exhibited properties similar to wild-type MRP1, the LTC4 and E217betaG transport activities of R501A, E507A, and R532A were significantly reduced.
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ABCC1 p.Arg501Ala 21177244:253:135
status: NEW[hide] Differential functional rescue of Lys(513) and Lys... Biochim Biophys Acta. 2014 Mar;1838(3):756-65. doi: 10.1016/j.bbamem.2013.11.002. Epub 2013 Nov 11. Iram SH, Cole SP
Differential functional rescue of Lys(513) and Lys(516) processing mutants of MRP1 (ABCC1) by chemical chaperones reveals different domain-domain interactions of the transporter.
Biochim Biophys Acta. 2014 Mar;1838(3):756-65. doi: 10.1016/j.bbamem.2013.11.002. Epub 2013 Nov 11., [PMID:24231430]
Abstract [show]
Multidrug resistance protein 1 (MRP1) extrudes drugs as well as pharmacologically and physiologically important organic anions across the plasma membrane in an ATP-dependent manner. We previously showed that Ala substitutions of Lys(513) and Lys(516) in the cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 cause misfolding of MRP1, abrogating its expression at the plasma membrane in transfected human embryonic kidney (HEK) cells. Exposure of HEK cells to the chemical chaperones glycerol, DMSO, polyethylene glycol (PEG) and 4-aminobutyric acid (4-PBA) improved levels of K513A to wild-type MRP1 levels but transport activity was only fully restored by 4-PBA or DMSO treatments. Tryptic fragmentation patterns and conformation-dependent antibody immunoreactivity of the transport-deficient PEG- and glycerol-rescued K513A proteins indicated that the second nucleotide binding domain (NBD2) had adopted a more open conformation than in wild-type MRP1. This structural change was accompanied by differences in ATP binding and hydrolysis but no changes in substrate Km. In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by >80% due to reduced substrate Km. Tryptic fragmentation patterns indicated a more open conformation of the third membrane spanning domain of MRP1. Thus, despite their close proximity to one another in CL5, Lys(513) and Lys(516) participate in different interdomain interactions crucial for the proper folding and assembly of MRP1.
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No. Sentence Comment
26 Thus, Ala substitution of Arg501 , Glu507 or Arg532 caused a significant reduction in organic anion transport activity while Ala substitution of Lys513 , Lys516 , Glu521 or Glu535 abrogated MRP1 expression by disrupting the folding and assembly of the transporter, targeting it for degradation by the proteasome [8,9].
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ABCC1 p.Arg501Ala 24231430:26:6
status: NEW