ABCMdb

ABCC1 p.Pro1113Ala

Predicted by SNAP2:	A: D (80%), C: D (75%), D: D (85%), E: D (91%), F: D (91%), G: D (75%), H: D (85%), I: D (85%), K: D (91%), L: D (91%), M: D (85%), N: D (85%), Q: D (80%), R: D (91%), S: D (75%), T: D (80%), V: D (85%), W: D (75%), Y: D (85%),
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, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Identification of proline residues in the core cyt... J Biol Chem. 2004 Mar 26;279(13):12325-36. Epub 2004 Jan 13. Koike K, Conseil G, Leslie EM, Deeley RG, Cole SP
Identification of proline residues in the core cytoplasmic and transmembrane regions of multidrug resistance protein 1 (MRP1/ABCC1) important for transport function, substrate specificity, and nucleotide interactions.
J Biol Chem. 2004 Mar 26;279(13):12325-36. Epub 2004 Jan 13., 2004-03-26 [PMID:14722114]

Abstract [show]
Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette transporter that confers resistance to drugs and mediates the transport of organic anions. MRP1 has a core structure of two membrane spanning domains (MSDs) each followed by a nucleotide binding domain. This core structure is preceded by a third MSD with five transmembrane (TM) helices, whereas MSD2 and MSD3 each contain six TM helices. We investigated the consequences of Ala substitution of 18 Pro residues in both the non-membrane and TM regions of MSD2 and MSD3 on MRP1 expression and organic anion transport function. All MRP1-Pro mutants except P1113A were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. In addition, five mutants containing substitutions of Pro residues in or proximal to the TM helices of MSD2 (TM6-Pro(343), TM8-Pro(448), TM10-Pro(557), and TM11-Pro(595)) and MSD3 (TM14-Pro(1088)) exhibited significantly reduced transport of five organic anion substrates. In contrast, mutation of Pro(1150) in the cytoplasmic loop (CL7) linking TM15 to TM16 caused a substantial increase in 17beta-estradiol-17-beta-(D-glucuronide) and methotrexate transport, whereas transport of other organic anions was reduced or unchanged. Significant substrate-specific changes in the ATP dependence of transport and binding by the P1150A mutant were also observed. Our findings demonstrate the importance of TM6, TM8, TM10, TM11, and TM14 in MRP1 transport function and suggest that CL7 may play a differential role in coupling the activity of the nucleotide binding domains to the translocation of different substrates across the membrane.

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No. Sentence Comment
4 All MRP1-Pro mutants except P1113A were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. Login to comment
46 We found that all but one of the singly substituted MRP1 Pro mutants (P1113A) could be expressed in HEK cells at levels comparable with the wild-type protein. Login to comment
58 Position MRP1 MRP3 MRP2 MRP6 SUR1 SUR2 MRP7 MRP4 MRP5 CFTR MSD2 323 Pro Ser Met Ser Gly Gly Arg Lys Thr Trp 343 Pro Pro Pro Pro Pro Asn Pro Pro Pro Pro 359 Pro Pro Tyr Pro Phe Phe Pro Ala Asn Glu 448 Pro Pro Val Leu Pro Pro Pro Pro Pro Pro 464 Pro Pro Pro Pro Val Ser Val Ile Pro Ala 478 Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu 557 Pro Pro Pro Thr Pro Pro Pro Ser Val Gly 595 Pro Pro Pro Ala Pro Pro Pro Thr Ala Ala 600 Pro Pro Pro Pro Ser Phe Pro Phe Pro Phe MSD3 1003 Pro Ala Ser Pro Ala Lys Gln Gly Gln 1060 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 1068 Pro Pro Pro Pro Pro Pro Pro Pro Pro Lys 1088 Pro Ala Pro Pro Pro Pro Pro Pro Pro Pro 1113 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 1120 Pro Leu Ile Leu Leu Leu Pro Val Gly Val 1121 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 1150 Pro Pro Pro Ser Pro Pro Pro Pro Pro Pro 1191 Pro Pro Ser Pro Phe Phe Ala Leu Leu Leu AGC AGC-3Ј; and MSD3 Pro mutants B P1003A, 5Ј-C TGG ACT GAT GAC GCC ATC GTC AAC GGG-3Ј; P1060A, 5Ј-C CTG CGG TCA GCC ATG AGC TTC-3Ј; P1068A, 5Ј-C TTT GAG CGG ACC GCG AGT GGG AAC C-3Ј; P1088A, 5Ј-C TCC ATG ATC GCG GAG GTC ATC-3Ј; P1113A, 5Ј-CTG CTG GCC ACG GCC ATC GCC GCC-3Ј; P1120A, 5Ј-GCC ATC ATC ATC GCG CCC CTT GG-3Ј; P1121A, 5Ј-C ATC ATC ATC CCG GCG CTT GGC CTC ATC-3Ј; P1150A, 5Ј-G GTC AGC CGC TCC GCG GTC TAT TCC C-3Ј; P1191A, 5Ј-C CAG AAG GCC TAT TAC GCT AGC ATC GTG GCC AAC-3Ј; P1120A/P1121A, 5Ј-C GCC ATC ATC ATC GCA GCG CTT GGC CTC ATC TAC TTC-3Ј. Login to comment
139 The locations of the MSD3 Pro residues mutated in this study are highlighted, and the approximate boundaries of the TM helices are indicated by dashed lines. B, shown is a representative immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (P1003A, P1060A, P1068A, P1088A, P1113A, P1120A, P1121A, P1150A, and P1191A) MRP1 cDNAs. MRP1 proteins were detected with mAb QCRL-1, and relative levels of expression estimated by densitometry are indicated; equal protein loading was confirmed by Amido Black staining of the polyvinylidene difluoride membrane and is shown below the blot. Login to comment
143 The exception was P1113A which was expressed at levels that were ϳ20% those of wild-type MRP1. Login to comment
144 This finding suggests that Ala substitution of Pro1113 (which is predicted to be located in ECL7 connecting TM14 to TM15) in some way impairs the expression or stability of MRP1 (Fig. 3B). Login to comment
148 Levels of [3 H]E217betaG uptake by six of the nine MSD3 mutants (P1003A, P1060A, P1068A, P1088A, P1120A, and P1121A) were moderately reduced (by 30-55%), whereas uptake of this substrate by the P1113A and P1191A mutants was comparable with wild-type MRP1. Login to comment
150 [3 H]MTX uptake by the P1150A mutant was also dramatically increased (ϳ6-fold) whereas uptake of this antifolate by the remaining eight MSD3 Pro mutants was either moderately reduced (by 50-60% for P1068A, P1088A, and P1120A) or comparable with wild-type MRP1 (P1003A, P1060A, P1113A, P1121A, and P1191A) (Fig. 4C). Login to comment
159 TABLE III Summary of effects of MSD3 Pro substitutions on MRP1 vesicular transport activities Substrate % wild-type MRP1 activitya ECL6 P1003A CL6 TM14 P1088A ECL7 P1113A TM15 CL7 P1060A P1068A P1120A P1121A P1150A P1191A LTC4 60 90 60 50 100 100 110 50 90 E217betaG 65 50 55 45 90 60 70 220 90 MTX 80 80 40 45 100 50 85 620 100 GSH 80 95 50 30 120 140 80 35 100 E13SO4 75 75 90 50 120 90 80 65 60 a The values shown are means of triplicate determinations in a single experiment and are representative of results obtained in 2-3 independent experiments (for details, see legend to Fig. 4 and text). Login to comment
162 GSH-stimulated E13SO4 uptake by the P1003A, P1060A, P1088A, P1150A, and P1191A mutants was moderately reduced (by 25-50%), whereas uptake of this sulfated estrogen by the other four MSD3 Pro mutants (P1068A, P1113A, P1120A, and P1121A) was comparable with wild-type MRP1. Login to comment
165 On the other hand, despite its apparent importance for MRP1 protein expression, Pro1113 (ECL7) does not seem to be involved. Expression of MRP1 Mutant Proteins P1113A, P1113A/ P1120A, and P1113A/P1121A Is Reduced, but Membrane Localization and MRP1 mRNA Levels Are Comparable with Wild-type MRP1-To explore further the reduced levels of expression caused by Ala substitution of Pro1113 , two double mutants were created to determine whether Ala substitution of an additional Pro residue in relatively close proximity to Pro1113 (Pro1120 and Pro1121 ) might restore MRP1 expression by a compensatory change in structure (P1113A/P1120A and P1113A/P1121A); a third mutant, P1120A/P1121A, was also generated and included in these experiments. Login to comment
167 The results show that the expression levels of the two Pro1113 double mutants were reduced by more than 80% as was observed for the single P1113A mutant. Login to comment
169 These results indicate that mutation of Pro1113 to Ala is consistently associated with a marked reduction in MRP1 protein expression levels that cannot be compensated for by additional Pro substitutions nearby. Login to comment
170 Despite the decreased expression levels of the three P1113A containing MRP1 mutants, indirect immunofluorescence confocal microscopy of intact transfected HEK293T cells showed that all of the Pro1113 mutants were correctly routed to the plasma membrane as was the P1120A/P1121A mutant (Fig. 6). Login to comment
171 To gain further insight into the mechanism by which the Ala substitution of Pro1113 results in decreased MRP1 expression, steady state MRP1 mRNA levels of the three double mutants and the single P1113A mutant were determined by Northern blot analysis of total RNA isolated from transfected cells expressing the mutant proteins (Fig. 5B). Login to comment
172 Levels of P1113A, P1113A/P1120A, P1113A/1121A, and P1120/P1121A mutant MRP1 mRNAs were found to be comparable with those of wild-type MRP1 mRNA, suggesting that the low expression of MRP1 mutants containing a Pro1113 3 Ala substitution is caused by some event that occurs after transcription. Login to comment
174 Expression levels of MRP1 mutants containing an Ala substitution of Pro1113 . Login to comment
175 A, membrane vesicle proteins (1 and 2 ␮g) prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (P1113A, P1113A/ P1120A, P1113A/P1121A, and P1120A/P1121A) MRP1 cDNAs were immunoblotted, and MRP1 was detected with mAb QCRL-1. Login to comment
182 Confocal laser-scanning fluorescence micrographs of HEK293T cells transfected with wild-type and P1113A, P1113A/ P1120A, P1113A/P1121A, and P1120A/P1121A mutant MRP1 cDNA constructs. Login to comment
183 HEK293T cells were transfected with WT-MRP1 (A), MRP1 mutants P1113A (B), P1113A/P1120A (C), P1113A/P1121A (D), P1120A/P1121A (E), and 48 h later, cells were stained with mAb QCRL-3 and processed for confocal fluorescence microscopy. Login to comment
220 In the present study, we found that the MRP1 mutant containing an Ala substitution of Pro1113 in MSD3 was also poorly expressed. Login to comment
224 Consistent with this conclusion is the observation that incubation of P1113A-transfected HEK293T cells at a reduced temperature (30 °C) significantly enhances the levels of plasma membrane expression of the P1113A mutant protein (results not shown). Login to comment
237 However, the structural change introduced by substitution of Pro1113 in MRP1 could not be compensated for by introducing another structural change by replacing nearby Pro residues in TM15 (Pro1120 and Pro1121 ) because the double mutants P1113A/P1120A and P1113A/ P1121A were also poorly expressed. 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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Sentences [show]
No. Sentence Comment
402 Substitution of 18 proline residues in both nonmembrane and transmembrane regions of TMD1 and TMD2 showed that all MRP1-Pro mutants except Pro1113Ala were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. Login to comment