ABCMdb

ABCC1 p.Arg1249Ala

Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (95%), E: D (91%), F: D (85%), G: D (91%), H: D (80%), I: D (85%), K: D (80%), L: D (91%), M: D (80%), N: D (91%), P: D (91%), Q: D (66%), S: D (80%), T: D (91%), V: D (85%), W: D (91%), 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,

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[hide] Arginine 383 is a crucial residue in ABCG2 biogene... Biochim Biophys Acta. 2009 Jul;1788(7):1434-43. Epub 2009 May 3. Polgar O, Ediriwickrema LS, Robey RW, Sharma A, Hegde RS, Li Y, Xia D, Ward Y, Dean M, Ozvegy-Laczka C, Sarkadi B, Bates SE
Arginine 383 is a crucial residue in ABCG2 biogenesis.
Biochim Biophys Acta. 2009 Jul;1788(7):1434-43. Epub 2009 May 3., [PMID:19406100]

Abstract [show]
ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by functional assay, while the R383A mutant exhibited detectable but significantly decreased levels compared to wild-type, partial retention in the ER and altered glycosylation. Efflux of the ABCG2-substrates mitoxantrone and pheophorbide a was observed. Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. In addition, substitution with the positively charged lysine resulted in significantly decreased protein expression levels in HEK cells, while retaining function. In conclusion, arginine 383 is a crucial residue for ABCG2 biogenesis, where even the most conservative mutations have a large impact.

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No. Sentence Comment
223 For example, the R1249A mutant of MRP1 has been described as having altered substrate specificity. Login to comment

[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
9 Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Login to 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). Login to comment
168 To investigate the role of charged amino acids in GSH-dependent photolabeling of azido AG-A to MRP1, we replaced the arginine at position 1249 which is proximal to the C-terminus of TM17 of MRP1 with Ala (R1249A). Login to comment
169 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. Login to comment
190 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. Login to comment
193 However, the transport activities of MRP1 R1249A were considerably reduced, suggesting that Arg1249 was important for the LTC4 transporting activity of MRP1. Login to comment
196 The level of photolabeling of MRP1 R1249A with [125 I]azido AG-A was increased only 1.6-fold in the presence of 10 mM GSH compared to a 10-fold increase observed with wild-type MRP1. Login to comment
198 To test the ability of MRP1 R1249A to confer drug resistance, wild-type MRP1 and the MRP1 R1249A mutant cDNAs were cloned into the mammalian expression vector pCIneo and transfected into pig kidney LLC-PK1 cells. Login to comment
202 Transfection of the pCIneo empty vector or the MRP1 R1249A mutant resulted in no VCR resistant clones. Login to comment
203 However, limited dilution of G418 resistant mass populations transfected with the MRP1 R1249A mutant in the medium without VCR resulted in several clones expressing MRP1 R1249A (Figure 7A). Login to comment
205 As shown in Figure 7B, the MRP1 R1249A mutant impaired the ability to confer resistance to VCR on LLC-PK1 cells. Login to comment
206 To determine if the inability of the R1249A mutant to confer drug resistance might be due to an inability of the mutant protein to reach the cell membrane, we assessed the cellular localization of MRP1 R1249A by immunofluorescence using MRPm6 mAb. As shown in Figure 7C, the MRP1 R1249A mutant was localized to the plasma membrane in a manner similar to that of wild-type MRP1. Login to comment
207 Thus, the inability of R1249A to confer drug resistance was not due to impaired trafficking of the protein. Login to comment
225 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. Login to comment
259 Mutation of this FIGURE 7: Effect of the R1249A mutation in MRP1 on drug resistance in LLC-PK1 cells. Login to comment
260 (A) Expression of MRP1 and the MRP1 R1249A mutant in LLC-PK1 cells. Login to comment
261 Crude membranes (50 µg of protein) prepared from LLC-PK1 cells transfected with an expression vector encoding MRP1 or MRP1 R1249A, or with a control empty vector (pCIneo), were analyzed on 7.5% SDS-PAGE. Login to comment
264 LLC-PK1 cells expressing MRP1 (b) or MRP1 R1249A (0), or the cells transfected with an empty vector [pCIneo (O)], were exposed to the indicated concentrations of VCR, and the survival rates were determined. Login to comment
268 Indirect immunofluorescent staining of MRP1 and MRP1 R1249A expressed in LLC-PK1 cells was carried out with the MRPm6 mAb and an FITC-conjugated secondary antibody. Login to comment
271 arginine 1249 to alanine almost completely impaired the LTC4 transport activity of MRP1 and abrogated the ability of MRP1 to confer VCR resistance. Login to comment
273 The inability of R1249A to confer VCR resistance was not due to alterations in MRP1 trafficking since the mutant MRP1 was still localized to the plasma membrane. Login to comment

[hide] The MRP-related and BCRP/ABCG2 multidrug resistanc... Curr Drug Metab. 2004 Feb;5(1):21-53. Haimeur A, Conseil G, Deeley RG, Cole SP
The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation.
Curr Drug Metab. 2004 Feb;5(1):21-53., [PMID:14965249]

Abstract [show]
Several members of different families of the ATP-binding cassette (ABC) superfamily of transport proteins are capable of transporting an extraordinarily structurally diverse array of endo- and xenobiotics and their metabolites across cell membranes. Together, these transporters play an important role in the absorption, disposition and elimination of these chemicals in the body. In tumor cells, increased expression of these drug transporters is associated with resistance to multiple chemotherapeutic agents. In this review, current knowledge of the biochemical, physiological and pharmacological properties of nine members of the multidrug resistance protein (MRP)-related ABCC family (MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, MRP7, ABCC11 and ABCC12) as well as the G family member, ABCG2/BCRP, are summarized. A focus is placed on the structural similarities and differences of these drug transporters as well as the molecular determinants of their substrate specificities and transport activities. Factors that regulate expression of the MRP-related proteins and ABCG2/BCRP are also reviewed.

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Sentences [show]
No. Sentence Comment
276 Additional mutations of charged residues that lead to defective MRP1 transport activity (or altered drug interactions) have been described (e.g. TM16 Arg1202Gly and TM17 Arg1249Ala) but because only one or two substrates were tested in these studies, it is not possible to know whether the altered activity is relatively substrate selective or more global in nature [167, 168]. Login to comment

[hide] Mutational analysis of ionizable residues proximal... J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18. Situ D, Haimeur A, Conseil G, Sparks KE, Zhang D, Deeley RG, Cole SP
Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter.
J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18., 2004-09-10 [PMID:15208328]

Abstract [show]
The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.

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No. Sentence Comment
229 Unlike Arg1202 and Glu1204 , substitution of Arg1197 and Arg1249 with oppositely charged residues did not adversely affect expression of MRP1 but instead caused a substantial reduction in transport activity. Our observations with respect to the R1249D mutant are consistent with those of Ren et al. (19) who reported that Ala substitution of Arg1249 impaired MRP1-mediated LTC4 transport and reduced vincristine resistance. Login to comment

[hide] GSH inhibits trypsinization of the C-terminal half... J Biol Chem. 2005 Feb 18;280(7):6231-7. Epub 2004 Dec 3. Ren XQ, Furukawa T, Nakajima Y, Takahashi H, Aoki S, Sumizawa T, Haraguchi M, Kobayashi M, Chijiiwa K, Akiyama S
GSH inhibits trypsinization of the C-terminal half of human MRP1.
J Biol Chem. 2005 Feb 18;280(7):6231-7. Epub 2004 Dec 3., 2005-02-18 [PMID:15579473]

Abstract [show]
MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an approximately 35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L(0) region of MRP1. Thus the L(0) region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates.

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No. Sentence Comment
219 We had previously mutated Arg1249 to Ala (32) and therefore tested this mutant for the generation of the C2 fragment following trypsin digestion. Login to comment

[hide] Functional role of arginine 375 in transmembrane h... Mol Pharmacol. 2008 Oct;74(4):964-71. Epub 2008 Jul 8. El-Sheikh AA, van den Heuvel JJ, Krieger E, Russel FG, Koenderink JB
Functional role of arginine 375 in transmembrane helix 6 of multidrug resistance protein 4 (MRP4/ABCC4).
Mol Pharmacol. 2008 Oct;74(4):964-71. Epub 2008 Jul 8., [PMID:18612080]

Abstract [show]
Multidrug resistance protein (MRP) 4 transports a variety of endogenous and xenobiotic organic anions. MRP4 is widely expressed in the body and specifically localized to the renal apical proximal tubule cell membrane, where it mediates the excretion of these compounds into urine. To characterize the MRP4 substrate-binding site, the amino acids Phe368, Phe369, Glu374, Arg375, and Glu378 of transmembrane helix 6, and Arg998 of helix 12, localized in the intracellular half of the central pore, were mutated into the corresponding amino acids of MRP1 and MRP2. Membrane vesicles isolated from human embryonic kidney 293 cells overexpressing these mutants showed significantly reduced methotrexate (MTX) and cGMP transport activity compared with vesicles that expressed wild-type MRP4. The only exception was substitution of Arg375 with serine, which had no effect on cGMP transport but significantly decreased the affinity of MTX. Substitution of the same amino acid with a positively charged lysine returned the MTX affinity to that of the wild type. Furthermore, MTX inhibition of MRP4-mediated cGMP transport was noncompetitive, and the inhibition constant was increased by introduction of the R375S mutation. A homology model of MRP4 showed that Arg375 and Arg998 face right into the central aqueous pore of MRP4. We conclude that positively charged amino acids in transmembrane helices 6 and 12 contribute to the MRP4 substrate-binding pocket.

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No. Sentence Comment
230 Moreover, alanine substitution of Arg1249 in MRP1 (also corresponding to R998A) impaired MRP1-mediated LTC4 transport and reduced vincristine resistance (Ren et al., 2002). Login to comment