ABCMdb

ABCC1 p.Glu1204Leu

Predicted by SNAP2:	A: D (80%), C: D (71%), D: D (71%), F: D (91%), G: D (80%), H: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (85%), N: D (80%), P: D (91%), Q: D (75%), R: D (91%), S: D (85%), T: D (85%), V: D (85%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: D, C: D, D: N, 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] 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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5 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. Login to comment
8 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. Login to comment
118 Immunoblots of membrane vesicle proteins prepared from cells expressing the Glu1204 mutants E1204L and E1204D were carried out as described in A. TABLE I Summary of organic anion transport activity of MRP1 mutants with substitutions of ionizable amino acids in and proximal to TM13 to TM17 of MSD3 Mutation % Wild-type MRP1 transport activitya E217betaG LTC4 E1SO4 MTX GSH TM13 R1046D 115 70 80 120 NDb TM14 D1084R Ͻ10 Ͻ10 15 25 Ͻ10 D1084E 80 20 65 90 20 TM15 R1131E 70 50 80 60 ND TM16 R1197E Ͻ10 Ͻ10 Ͻ15 Ͻ10 ND R1197K 20 Ͻ25 Ͻ20 Ͻ10 ND R1202G 115 115 75 70 ND R1202L 115 120 50 110 ND E1204L Ͻ10 50 10 110 Ͻ25 E1204D 100 115 100 115 Ͻ25 TM17 R1249D Ͻ10 Ͻ15 Ͻ10 Ͻ10 ND R1249K Ͻ10 10 Ͻ15 Ͻ10 ND a The values shown are means of duplicate or triplicate determinations and are derived from Fig. 2, 4, and 5 (see figure legends for details). Login to comment
121 sion of MRP1 in HEK cell membranes was further explored by replacing Arg1202 with the hydrophobic Leu, and Glu1204 with Leu and Asp. Login to comment
124 Immunoblots showed that expression levels of the R1202G and R1202L mutants (Fig. 3B) and the E1204L and E1204D mutants (Fig. 3C) ranged from 80 to 225% of wild-type MRP1. Login to comment
130 In contrast to the neutrally substituted Arg1202 mutants, E217betaG uptake by the neutrally substituted Glu1204 mutant E1204L was Ͻ10% of wild-type MRP1 levels (Fig. 4E). Login to comment
131 In addition, LTC4 uptake by E1204L was reduced by 50% (Fig. 4F), and E1SO4 uptake was reduced by 90% (Fig. 4G). Login to comment
134 To determine whether the substrate-selective loss of transport function observed in the E1204L mutant was because of the loss of the acidic character or the change in the size of the side chain, organic anion uptake by the same-charge mutant, E1204D, was also assessed. Login to comment
137 As shown in Fig. 4I, both E1204L and E1204D exhibited a similar and substantial decrease in GSH transport levels (Ͼ75%). Login to comment
151 Effect of Glu1204 , Arg1197 , and Arg1249 Mutations on Photolabeling with [3 H]LTC4 and 8-Azido-[␣-32 P]ATP-In the next series of experiments, those same-charge or neutrally substituted mutants that showed substantially reduced transport activities (R1197K, E1204L, and R1249K) were further examined to determine whether their loss of transport activity was accompanied by a decrease in substrate binding. Login to comment
153 In contrast, [3 H]LTC4 labeling of the E1204L mutant was comparable with wild-type MRP1, despite the fact that transport of this organic anion by this mutant was substantially reduced. Login to comment
154 To determine whether the mutations of Arg1197 , Glu1204 , and Arg1249 that altered the transport properties of MRP1 also affected the interaction of the transporter with nucleotide, the ability of the R1197K, E1204L, and R1249K mutants to be photolabeled with 8-azido-[␣-32 P]ATP, both at 4 °C to minimize hydrolysis and at 37 °C in the presence of sodium vanadate to trap azido-ADP after hydrolysis, was examined (31, 32). Login to comment
159 8-Azido-[␣-32 P]ATP labeling of the transport-compromised E1204L mutant was also comparable with wild-type MRP1 (Fig. 6B). Login to comment
161 Because E1204L could still be photolabeled with LTC4 despite substantially reduced transport of this organic anion, [3 H]LTC4 photolabeling of the mutant protein after prior incubation with ATP and vanadate was examined to determine whether the increased trapping of azido-ADP by E1204L altered the substrate binding properties of MRP1. Login to comment
163 A similar decrease in [3 H]LTC4 labeling of the E1204L mutant was observed under the same conditions. Login to comment
172 E-H, uptake of 3 H-labeled organic anions by the membrane vesicles shown in Fig. 3C which were prepared from cells transfected with empty pcDNA3.1 vector (open bars), vector containing wild-type MRP1 cDNA (black bars), and the Glu1204 mutant E1204L and E1204D cDNAs (gray bars). Login to comment
192 A, membrane vesicle proteins (50 ␮g) prepared from cells expressing wild-type (WT-MRP1) and transport-compromised mutant MRP1 proteins (R1197K, E1204L, and R1249K) were incubated with [3 H]LTC4 (200 nM; 250 nCi) followed by UV cross-linking, SDS-PAGE, and fluorography. Login to comment
197 D, wild-type and E1204L mutant membrane vesicles (50 ␮g) were incubated in transport buffer containing 5 mM MgCl2 for 20 min in the absence (-) or presence (ϩ) of ATP (5 mM) and vanadate (1 mM), alone or in combination, and then incubated with [3 H]LTC4 (200 nM, 110 nCi) for a further 30 min followed by UV cross-linking, SDS-PAGE, and fluorography. Login to comment
205 Unlike the neutrally substituted Arg1202 mutants, transport of organic anions by the neutrally substituted Glu1204 mutant E1204L was substantially reduced or eliminated with the exception of MTX. Login to comment
206 Nevertheless, the substrate (LTC4)-binding site of E1204L remained intact. Furthermore, GSH transport remained very low, although other MRP1 transport activities of the same-charge E1204D mutant were comparable with wild-type MRP1. Login to comment
211 This was shown to be the case when the nucleotide interactions of the transport-compromised E1204L mutant were examined. Login to comment
223 Thus, inactivation of NBD2 abolishes transport by MRP1, but inactivation of NBD1 results in only a partial loss of activity. Our demonstration that vanadate-induced trapping of azido-ADP by the mutant E1204L protein (and specifically by NBD2) was substantially increased suggests that the mutation may impair the ability of NBD2 to release ADP after hydrolysis of ATP, which could in turn impair substrate translocation and/or release. Login to comment
224 Alternatively, the E1204L mutant may hydrolyze ATP and release ADP very rapidly in the absence of vanadate but be unable to proceed through a second catalytic cycle (36). Login to comment
228 Thus, the altered catalytic activity and impaired transport of the E1204L mutant suggests that Glu1204 (or at least the region in which it resides) could play a role in the signaling between the substrate translocation pathway and NBD2. Login to comment

[hide] Mutational analysis of polar amino acid residues w... Drug Metab Dispos. 2006 Apr;34(4):539-46. Epub 2006 Jan 13. Zhang DW, Nunoya K, Vasa M, Gu HM, Cole SP, Deeley RG
Mutational analysis of polar amino acid residues within predicted transmembrane helices 10 and 16 of multidrug resistance protein 1 (ABCC1): effect on substrate specificity.
Drug Metab Dispos. 2006 Apr;34(4):539-46. Epub 2006 Jan 13., [PMID:16415113]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) has a total of 17 transmembrane (TM) helices arranged in three membrane-spanning domains, MSD0, MSD1, and MSD2, with a 5 + 6 + 6 TM configuration. Photolabeling studies indicate that TMs 10 and 11 in MSD1 and 16 and 17 in MSD2 contribute to the substrate binding pocket of the protein. Previous mutational analyses of charged and polar amino acids in predicted TM helices 11, 16, and 17 support this suggestion. Mutation of Trp(553) in TM10 also affects substrate specificity. To extend this analysis, we mutated six additional polar residues within TM10 and the remaining uncharacterized polar residue in TM16, Asn(1208). Although mutation of Asn(1208) was without effect, two of six mutations in TM10, T550A and T556A, modulated the drug resistance profile of MRP1 without affecting transport of leukotriene C4, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), and glutathione. Mutation T550A increased vincristine resistance but decreased doxorubicin resistance, whereas mutation T556A decreased resistance to etoposide (VP-16) and doxorubicin. Although conservative mutation of Tyr(568) in TM10 to Phe or Trp had no apparent effect on substrate specificity, substitution with Ala decreased the affinity of MRP1 for E(2)17betaG without affecting drug resistance or the transport of other substrates tested. These analyses confirm that several amino acids in TM10 selectively alter the substrate specificity of MRP1, suggesting that they interact directly with certain substrates. The location of these and other functionally important residues in TM helices 11, 16, and 17 is discussed in the context of an energy-minimized model of the membrane-spanning domains of MRP1.

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164 Conversely, replacement of Glu1204 with Leu or Arg1197 with Glu or Lys affected either substrate specificity or overall transport activity of MRP1 (Situ et al., 2004). Login to comment

[hide] Transmembrane transport of endo- and xenobiotics b... Physiol Rev. 2006 Jul;86(3):849-99. Deeley RG, Westlake C, Cole SP
Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins.
Physiol Rev. 2006 Jul;86(3):849-99., [PMID:16816140]

Abstract [show]
Multidrug Resistance Proteins (MRPs), together with the cystic fibrosis conductance regulator (CFTR/ABCC7) and the sulfonylurea receptors (SUR1/ABCC8 and SUR2/ABCC9) comprise the 13 members of the human "C" branch of the ATP binding cassette (ABC) superfamily. All C branch proteins share conserved structural features in their nucleotide binding domains (NBDs) that distinguish them from other ABC proteins. The MRPs can be further divided into two subfamilies "long" (MRP1, -2, -3, -6, and -7) and "short" (MRP4, -5, -8, -9, and -10). The short MRPs have a typical ABC transporter structure with two polytropic membrane spanning domains (MSDs) and two NBDs, while the long MRPs have an additional NH2-terminal MSD. In vitro, the MRPs can collectively confer resistance to natural product drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and, under certain circumstances, alkylating agents. The MRPs are also primary active transporters of other structurally diverse compounds, including glutathione, glucuronide, and sulfate conjugates of a large number of xeno- and endobiotics. In vivo, several MRPs are major contributors to the distribution and elimination of a wide range of both anticancer and non-anticancer drugs and metabolites. In this review, we describe what is known of the structure of the MRPs and the mechanisms by which they recognize and transport their diverse substrates. We also summarize knowledge of their possible physiological functions and evidence that they may be involved in the clinical drug resistance of various forms of cancer.

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834 The distinct phenotypes associated with mutations of the highly conserved Arg1202 and Glu1204 are presumably caused by perturbations in the ␣-helical geometry of TM16 that contribute (depending on the substituting amino acid) to misfolding of MRP1 and, in the case of the neutral Glu1204 Leu mutant, disruption of the signaling between the TMs that comprise the substrate translocation pathway through the membrane and NBD2. Login to comment
833 The distinct phenotypes associated with mutations of the highly conserved Arg1202 and Glu1204 are presumably caused by perturbations in the ␣-helical geometry of TM16 that contribute (depending on the substituting amino acid) to misfolding of MRP1 and, in the case of the neutral Glu1204 Leu mutant, disruption of the signaling between the TMs that comprise the substrate translocation pathway through the membrane and NBD2. Login to comment
835 The distinct phenotypes associated with mutations of the highly conserved Arg1202 and Glu1204 are presumably caused by perturbations in the ␣-helical geometry of TM16 that contribute (depending on the substituting amino acid) to misfolding of MRP1 and, in the case of the neutral Glu1204 Leu mutant, disruption of the signaling between the TMs that comprise the substrate translocation pathway through the membrane and NBD2. Login to comment

[hide] Characterization and classification of ATP-binding... J Biol Chem. 2006 Nov 10;281(45):34503-14. Epub 2006 Sep 7. Matsumura Y, Ban N, Ueda K, Inagaki N
Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency.
J Biol Chem. 2006 Nov 10;281(45):34503-14. Epub 2006 Sep 7., [PMID:16959783]

Abstract [show]
The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency.

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251 For example, the E1204L mutation in TM-16 of MRP1(multidrug resistance associated protein 1) affects vanadate-induced nucleotide trapping as well as transport activity of the protein (41). Login to comment

[hide] Structure, function, expression, genomic organizat... Int J Toxicol. 2006 Jul-Aug;25(4):231-59. Choudhuri S, Klaassen CD
Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters.
Int J Toxicol. 2006 Jul-Aug;25(4):231-59., [PMID:16815813]

Abstract [show]
The ATP-binding cassette (ABC) transporters constitute a large family of membrane proteins, which transport a variety of compounds through the membrane against a concentration gradient at the cost of ATP hydrolysis. Substrates of the ABC transporters include lipids, bile acids, xenobiotics, and peptides for antigen presentation. As they transport exogenous and endogenous compounds, they reduce the body load of potentially harmful substances. One by-product of such protective function is that they also eliminate various useful drugs from the body, causing drug resistance. This review is a brief summary of the structure, function, and expression of the important drug resistance-conferring members belonging to three subfamilies of the human ABC family; these are ABCB1 (MDR1/P-glycoprotein of subfamily ABCB), subfamily ABCC (MRPs), and ABCG2 (BCRP of subfamily ABCG), which are expressed in various organs. In the text, the transporter symbol that carries the subfamily name (such as ABCB1, ABCC1, etc.) is used interchangeably with the corresponding original names, such as MDR1P-glycoprotein, MRP1, etc., respectively. Both nomenclatures are maintained in the text because both are still used in the transporter literature. This helps readers relate various names that they encounter in the literature. It now appears that P-glycoprotein, MRP1, MRP2, and BCRP can explain the phenomenon of multidrug resistance in all cell lines analyzed thus far. Also discussed are the gene structure, regulation of expression, and various polymorphisms in these genes. Because genetic polymorphism is thought to underlie interindividual differences, including their response to drugs and other xenobiotics, the importance of polymorphism in these genes is also discussed.

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410 Organic anion transport by Glu1204Leu substitution was substantially reduced although substrate binding by the transport- compromised Glu1204Leu mutant remained intact. Login to comment

[hide] Bindings of hMRP1 transmembrane peptides with dode... Biochim Biophys Acta. 2014 Jan;1838(1 Pt B):493-509. doi: 10.1016/j.bbamem.2013.10.012. Epub 2013 Oct 21. Abel S, Lorieau A, de Foresta B, Dupradeau FY, Marchi M
Bindings of hMRP1 transmembrane peptides with dodecylphosphocholine and dodecyl-beta-d-maltoside micelles: a molecular dynamics simulation study.
Biochim Biophys Acta. 2014 Jan;1838(1 Pt B):493-509. doi: 10.1016/j.bbamem.2013.10.012. Epub 2013 Oct 21., [PMID:24157718]

Abstract [show]
In this paper, we describe molecular dynamics simulation results of the interactions between four peptides (mTM10, mTM16, TM17 and KTM17) with micelles of dodecylphosphocholine (DPC) and dodecyl-beta-d-maltoside (DDM). These peptides represent three transmembrane fragments (TM10, 16 and 17) from the MSD1 and MSD2 membrane-spanning domains of an ABC membrane protein (hMRP1), which play roles in the protein functions. The peptide-micelle complex structures, including the tryptophan accessibility and dynamics were compared to circular dichroism and fluorescence studies obtained in water, trifluoroethanol and with micelles. Our work provides additional results not directly accessible by experiments that give further support to the fact that these peptides adopt an interfacial conformation within the micelles. We also show that the peptides are more buried in DDM than in DPC, and consequently, that they have a larger surface exposure to water in DPC than in DDM. As noted previously by simulations and experiments we have also observed formation of cation-pi bonds between the phosphocholine DPC headgroup and Trp peptide residue. Concerning the peptide secondary structures (SS), we find that in TFE their initial helical conformations are maintained during the simulation, whereas in water their initial SS are lost after few nanoseconds of simulation. An intermediate situation is observed with micelles, where the peptides remain partially folded and more structured in DDM than in DPC. Finally, our results show no sign of beta-strand structure formation as invoked by far-UV CD experiments even when three identical peptides are simulated either in water or with micelles.

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39 Indeed, mutations of the ionizable residues (for example, R1197E, R1202(G,L) and E1204L) have impact on protein expression, substrate binding and/or transport [37], whereas the mutation of a single tryptophan W1246A in TM17 affects the estradiol 17-b2;-D-glucuronide transport [36]. Login to comment