ABCC1 p.Glu535Asp
| Predicted by SNAP2: | A: D (71%), C: D (71%), D: D (75%), F: D (85%), G: D (75%), H: D (80%), I: D (85%), K: D (91%), L: D (80%), M: D (80%), N: D (75%), P: D (91%), Q: D (75%), R: D (91%), S: D (80%), T: D (80%), V: D (75%), W: D (91%), Y: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: 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, 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.Glu535Asp 21177244:47:1000
status: NEW141 Expression and Transport Activity of "Same Charge" Mutants of Lys513 , Lys516 , Glu521 , and Glu535 -To determine whether it was the charge or another property of the amino acid at positions 513, 516, 521, and 535 that was critical for efficient plasma membrane expression of MRP1, same charge mutants (K513R, K516R, E521D, and E535D) were created and again expressed in HEK cells.
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ABCC1 p.Glu535Asp 21177244:141:328
status: NEW142 At 37 °C, levels of the K513R mutant were comparable with wild-type MRP1 levels, whereas levels of the K516R, E521D, and E535D mutants were somewhat reduced (20-40%) (Fig. 4A).
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ABCC1 p.Glu535Asp 21177244:142:126
status: NEW148 A and B, representative immunoblots of whole cell lysates (WCL) (10 g of protein/lane) (A) and membrane vesicles (1 g of protein/lane) (B) prepared from HEK293T cells transfected with wild-type (WT-MRP1) and mutant (K513R, K516R, E521D, and E535D) MRP1 cDNA expression vectors. Untransfected cells were used as a negative control. MRP1 levels were detected with mAb QCRL-1, and the relative protein expression levels, estimated by densitometry, are shown below the blots.
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ABCC1 p.Glu535Asp 21177244:148:257
status: NEW