ABCC1 p.Arg532Ala
| Predicted by SNAP2: | A: D (80%), C: D (85%), D: D (95%), E: D (91%), F: D (91%), G: D (91%), H: D (85%), I: D (85%), K: D (80%), L: D (80%), M: D (85%), N: D (80%), P: D (91%), Q: D (85%), S: D (80%), T: D (85%), V: D (85%), W: D (95%), Y: D (91%), |
| 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, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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.
Comments [show]
None has been submitted yet.
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.
X
ABCC1 p.Arg532Ala 21177244:47:867
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).
X
ABCC1 p.Arg532Ala 21177244:112:102
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).
X
ABCC1 p.Arg532Ala 21177244:164:152
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.
X
ABCC1 p.Arg532Ala 21177244:166:66
status: NEW168 In contrast, the Km (E217betaG) values for the R501A, E507A, and R532A mutants were increased 3-5-fold (18-25 M).
X
ABCC1 p.Arg532Ala 21177244:168:65
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.
X
ABCC1 p.Arg532Ala 21177244:186:44
status: NEWX
ABCC1 p.Arg532Ala 21177244:186:146
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%.
X
ABCC1 p.Arg532Ala 21177244:187:117
status: NEW199 Levels and organic anion transport activity of MRP1 mutant proteins R501A, K503A, E507A, and R532A.
X
ABCC1 p.Arg532Ala 21177244:199:93
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.
X
ABCC1 p.Arg532Ala 21177244:200:198
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."
X
ABCC1 p.Arg532Ala 21177244:204:81
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).
X
ABCC1 p.Arg532Ala 21177244:207:145
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).
X
ABCC1 p.Arg532Ala 21177244:210:295
status: NEW236 [3 H]LTC4 photolabeling of wild-type and MRP1 mutants R501A, E507A, R532A, and G511I.
X
ABCC1 p.Arg532Ala 21177244:236:68
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.
X
ABCC1 p.Arg532Ala 21177244:253:153
status: NEW