ABCC1 p.Arg1222Met
| Predicted by SNAP2: | A: D (85%), C: D (75%), D: D (91%), E: D (91%), F: D (85%), G: D (85%), H: D (85%), I: D (85%), K: N (57%), L: D (85%), M: D (85%), N: D (85%), P: D (91%), Q: D (80%), S: D (71%), T: D (85%), 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: N, 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] A positively charged amino acid proximal to the C-... Biochemistry. 2002 Dec 3;41(48):14132-40. Ren XQ, Furukawa T, Aoki S, Sumizawa T, Haraguchi M, Nakajima Y, Ikeda R, Kobayashi M, Akiyama S
A positively charged amino acid proximal to the C-terminus of TM17 of MRP1 is indispensable for GSH-dependent binding of substrates and for transport of LTC4.
Biochemistry. 2002 Dec 3;41(48):14132-40., 2002-12-03 [PMID:12450376]
Abstract [show]
MRP1 is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. Our recent study demonstrated that GSH is required for the labeling of MRP1(932)(-)(1531) with a photoanalogue of agosterol A (AG-A) and suggested that GSH interacts with the L(0) region of MRP1. In this study, we further characterized the GSH-dependent binding site of azido AG-A on MRP1. Coexpression of the N- and C-terminal halves of MRP1 (residues 1-1222, TM1-16, and 1223-1531, TM17, respectively) in Sf21 insect cells reconstituted a functional drug transporter with a K(m) for LTC(4) (97 nM) similar to that of intact MRP1. In membrane vesicles from those cells, GSH-dependent photolabeling of the MRP1 fragment (1-1222) required the coexpression of the C-terminal MRP1 fragment (1223-1531). An MRP1 fragment extending from residue 1 to 1295 however could be photolabeled by azido AG-A in a GSH-dependent manner. These data indicate that amino acids 1223-1295 of MRP1 are required for AG-A binding to MRP1 in a GSH-dependent manner. However, cross-linking of the photolabel to MRP1 occurs at a more upstream site. An arginine residue at position 1249 of MRP1 was shown to be important for the GSH-dependent binding of AG-A to MRP1. Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Furthermore, this mutant attenuated MRP1 function by decreasing the level of LTC(4) substrate transport and impairing resistance to the drug vincristine (VCR). In summary, this study demonstrates that a region of MRP1 (amino acids 1223-1295), which includes TM helix 17, is required for azido AG-A binding to MRP1 in a GSH-dependent manner. A GSH-dependent drug binding site may exist in this region. Furthermore, our findings suggest that the charged amino acid Arg(1249) proximal to the C-terminus of TM helix 17 is indispensable for MRP1-substrate interaction and the function of MRP1.
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No. Sentence Comment
72 MRP1 constructs encoding R1222M and R1249A were generated in a PCR using the forward primers 5'-CATG- CACAGCCTCAGTGCTG-3' and 5'-GCGATGTCATCT- GAAATGGAAACC-3', respectively (bold denotes mismatched bases encoding the mutations).
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ABCC1 p.Arg1222Met 12450376:72:25
status: NEW73 An EcoRV site (underlined) was created by silent mutation (bold) in the R1222M construct using the reverse primer 5'-GATATC- ACCGCAAACAGGGCAGC-3'.
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ABCC1 p.Arg1222Met 12450376:73:72
status: NEW78 The EcoRV-KpnI fragment from the construct encoding R1222M was cloned into the multiple cloning site II of the pFastBac DUAL plasmid between the SmaI and KpnI sites.
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ABCC1 p.Arg1222Met 12450376:78:52
status: NEW169 Arginine at position 1222 in the azido AG-A-photolabeled MRP11-1222 fragment was replaced with Met, and the functions of MRP1 R1222M were compared with those of MRP1 R1249A.
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ABCC1 p.Arg1222Met 12450376:169:126
status: NEW190 Figure 6A shows that the expression level of MRP1 R1222M was comparable to that of wild-type MRP1, whereas the expression level of MRP1 R1249A was higher than that of wild-type MRP1.
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ABCC1 p.Arg1222Met 12450376:190:50
status: NEW192 As shown in Figure 6B, MRP1 R1222M transported LTC4 as efficiently as wild-type MRP1.
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ABCC1 p.Arg1222Met 12450376:192:28
status: NEW195 As shown in Figure 6C, GSH stimulated [125 I]azido AG-A binding to MRP1 R1222M as efficiently as wild-type MRP1.
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ABCC1 p.Arg1222Met 12450376:195:72
status: NEW225 Membrane vesicles (25 µg of protein) expressing MRP1 R1222M or MRP1 R1249A as indicated were incubated with 1.37 nM [3H]LTC4 at 37 °C in 50 µL of transport buffer as described in the legend of Figure 3 in the presence or absence of 4 mM ATP at the indicated periods.
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ABCC1 p.Arg1222Met 12450376:225:58
status: NEW229 Membrane vesicles (100 µg of protein) expressing MRP1, MRP1 R1249M, and MRP1 R1222M were photolabeled with [125I]azido AG-A in the absence or presence of the indicated concentrations of GSH as described in the legend ofo Figure 2.
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ABCC1 p.Arg1222Met 12450376:229:82
status: NEW