ABCMdb

ABCC1 p.Pro1121Ala

Predicted by SNAP2:	A: N (53%), C: N (66%), D: D (71%), E: D (80%), F: D (59%), G: D (63%), H: D (71%), I: D (59%), K: D (80%), L: D (66%), M: D (59%), N: D (63%), Q: D (71%), R: D (80%), S: D (59%), T: D (63%), V: D (53%), W: D (80%), Y: D (75%),
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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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
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
168 On the other hand, the third mutant, P1120A/P1121A, was expressed at levels that were at least 60% those of wild-type MRP1. 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
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
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
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
239 Given the very substantial structural change that would be introduced by simultaneous substitution of two adjacent Pro residues (33, 49), it is somewhat surprising that the TM15 double Pro mutant P1120A/P1121A was expressed at levels comparable with wild-type MRP1, and its transport activity was also similar to the wild-type protein (results not shown). Login to comment