ABCMdb

PMID: 14722114

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 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC1 p.Pro1113Ala All MRP1-Pro mutants except P1113A were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. Login to comment 7 ABCC1 p.Pro1150Ala Significant substrate-specific changes in the ATP dependence of transport and binding by the P1150A mutant were also observed. Login to comment 46 ABCC1 p.Pro1113Ala 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 47 ABCC1 p.Pro478Ala In addition, five mutants containing substitutions of Pro residues predicted to be in or proximal to the TM helices of MSD2 (TM6-Pro343 , TM8-Pro448 , TM10-Pro557 , and TM11-Pro595 ) and MSD3 (TM14-Pro1088 ) exhibited significantly reduced transport of five structurally diverse organic anion substrates of MRP1, whereas TM9 mutant P478A showed a selective reduction in LTC4 and GSH transport and an increase in E217betaG, E13SO4, and MTX transport. Login to comment 49 ABCC1 p.Pro1150Ala The altered transport activity of P1150A was associated with significant changes in the ATP dependence of E217betaG but not LTC4 transport, suggesting that CL7 may play a differential role in coupling the activity of the MRP1 NBDs to the translocation of different substrates across the membrane. Login to comment 57 ABCC1 p.Pro478Ala ABCC1 p.Pro323Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro464Ala ABCC1 p.Pro600Ala ABCC1 p.Pro343Ala ABCC1 p.Pro359Ala Proline substitutions were generated in the pBluescriptSK(ϩ) and pGEM-3Z plasmids above according to the manufacturer`s instructions with the following mutagenic primers (substituted nucleotides are underlined): MSD2 Pro mutants, P323A, 5Ј-GTG TTA TAC AAG ACC TTT GGC GCC TAC TTC CTC ATG AGC-3Ј; P343A, 5Ј-G ATG ATG TTT TCC GGG GCG CAG ATC TTA AAG TTG C-3Ј; P359A, 5Ј-G AAT GAC ACG AAG GCC GCA GAC TGG CAG GG-3Ј; P448A, 5Ј-G ATC TGG TCA GCC GCC CTG CAA GTC ATC CTT GC-3Ј; P464A, 5Ј-G CTG AAT CTG GGC GCT TCC GTC CTG GCT GG-3Ј; P478A, 5Ј-G GTC CTC ATG GTG GCC GTC AAT GCT GTG-3Ј; P557A, 5Ј-CC TGG GTC TGC ACG GCC TTT CTG GTG GCC-3Ј; P595A, 5Ј-C AAC ATC CTC CGG TTT GCC CTG AAC ATT CTC C-3Ј; P600A, 5Ј-CCC CTG AAC ATT CTC GCG ATG GTC ATC TABLE I Conservation of MRP1 MSD2 and MSD3 Pro residues in human ABCC family members Sequences are from Swiss-Prot/TrEMBL entries P33527 (MRP1/ ABCC1), O15438 (MRP3,/ABCC3), Q92887 (MRP2,/ABCC2), O95255 (MRP6/ ABCC6), Q09428 (SUR1/ABCC8), O60706 (SUR2/ABCC9), Q8NHX7 (MRP7/ABCC10), O15439 (MRP4/ABCC4), O15440 (MRP5/ABCC5), and P13569 (CFTR/ABCC7). Login to comment 58 ABCC1 p.Pro1113Ala ABCC1 p.Pro1150Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1191Ala ABCC1 p.Pro1003Ala ABCC8 p.Ala478Pro ABCC4 p.Ala600Pro ABCC2 p.Pro1068Ala ABCC4 p.Leu557Pro ABCC4 p.Pro1121Ala ABCC4 p.Pro1121Ala ABCC8 p.Gln1060Pro ABCC5 p.Gly595Pro 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 115 ABCC1 p.Pro478Ala ABCC1 p.Pro323Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro464Ala ABCC1 p.Pro600Ala ABCC1 p.Pro343Ala ABCC1 p.Pro359Ala The locations of the Pro residues mutated in the present 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 (P323A, P343A, P359A, P448A, P464A, P478A, P557A, P595A, and P600A) 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 123 ABCC1 p.Pro478Ala ABCC1 p.Pro323Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala ABCC1 p.Pro359Ala [3 H]LTC4 uptake by the MSD2 mutants P323A and P359A was moderately reduced (by ϳ25%), whereas uptake by the P343A, P448A, P478A, P557A, and P595A TM mutants was substantially reduced (by 55-70%). Login to comment 124 ABCC1 p.Pro464Ala ABCC1 p.Pro600Ala [3 H]LTC4 uptake by the P464A and P600A mutants was comparable with wild-type MRP1 (Fig. 2A). Login to comment 126 ABCC1 p.Pro478Ala ABCC1 p.Pro323Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala ABCC1 p.Pro359Ala As shown in Fig. 2B, ATP-dependent uptake levels of this substrate by mutants P323A and P359A relative to wild-type MRP1 were moderately reduced (by ϳ30%), whereas uptake by mutants P343A, P448A, P557A, and P595A was substantially reduced (by 75-90%), and uptake by the P478A mutant was increased 1.5-fold. Login to comment 127 ABCC1 p.Pro464Ala ABCC1 p.Pro600Ala In contrast, as observed for [3 H]LTC4 uptake, [3 H]E217betaG uptake by the P464A and P600A mutants was comparable with wild-type MRP1. Login to comment 129 ABCC1 p.Pro323Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala ABCC1 p.Pro359Ala Relative to wild-type MRP1, [3 H]MTX uptake by the P323A and P359A mutants was moderately reduced (by 30-40%), whereas uptake levels of the P343A, P448A, P557A, and P595A mutants were substantially reduced (by 75-90%). Login to comment 130 ABCC1 p.Pro478Ala ABCC1 p.Pro464Ala ABCC1 p.Pro600Ala In contrast, [3 H]MTX uptake by the P464A and P478A mutants was moderately increased by ϳ30% relative to wild-type MRP1, whereas uptake by the P600A mutant was comparable with wild-type MRP1 (Fig. 2C). Login to comment 132 ABCC1 p.Pro478Ala ABCC1 p.Pro323Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro464Ala ABCC1 p.Pro600Ala ABCC1 p.Pro343Ala ABCC1 p.Pro359Ala Relative to wild-type MRP1, GSH uptake by six of the nine MSD2 Pro mutants (P323A, P343A, P448A, P478A, P557A, and P595A) was substantially reduced (60-93%), whereas GSH uptake levels by the P359A, P464A, and P600A mutants were comparable with wild-type MRP1 (Table II). Login to comment 133 ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala GSH-stimulated [3 H]E13SO4 uptake by the MSD2 Pro mutants was also measured and found to be moderately reduced in the P343A and P448A mutants (by 30-40%) and substantially reduced in the P557A and P595A mutants (by 75-85%). Login to comment 134 ABCC1 p.Pro478Ala ABCC1 p.Pro323Ala ABCC1 p.Pro464Ala ABCC1 p.Pro600Ala ABCC1 p.Pro359Ala In contrast, levels of [3 H]E13SO4 uptake by the P478A mutant were increased 1.5-fold, and those of the P323A, P359A, P464A, and P600A mutants were comparable with wild-type MRP1. Login to comment 139 ABCC1 p.Pro1113Ala ABCC1 p.Pro1150Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1191Ala ABCC1 p.Pro1003Ala ABCC1 p.Pro1068Ala ABCC1 p.Pro1088Ala ABCC1 p.Pro1060Ala ABCC1 p.Pro1121Ala 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 140 ABCC1 p.Pro478Ala ABCC1 p.Pro323Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro464Ala ABCC1 p.Pro600Ala ABCC1 p.Pro343Ala ABCC1 p.Pro359Ala TABLE II Summary of effects of MSD2 Pro substitutions on MRP1 vesicular transport activities Substrate % wild-type MRP1 activitya TM6 ECL3 P359A TM8 P448A ECL4 P464A TM9 P478A TM10 P557A TM11 P323A P343A P595A P600A LTC4 75 35 75 40 100 45 35 30 100 E217betaG 70 10 70 25 100 155 10 Ͻ5 100 MTX 60 20 70 20 135 125 25 10 100 GSH 40 35 100 15 100 20 Ͻ10 10 100 E13SO4 100 70 100 60 100 155 25 15 100 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. 2 and text). Login to comment 143 ABCC1 p.Pro1113Ala The exception was P1113A which was expressed at levels that were ϳ20% those of wild-type MRP1. Login to comment 144 ABCC1 p.Pro1113Ala 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 147 ABCC1 p.Pro1150Ala ABCC1 p.Pro1003Ala ABCC1 p.Pro1068Ala ABCC1 p.Pro1088Ala As shown in Fig. 4A, [3 H]LTC4 uptake levels of the P1003A, P1068A, P1088A, and P1150A mutants were ϳ40-50% less than those of wild-type MRP1, whereas LTC4 uptake by the other five MSD3 Pro mutants was similar to wild-type MRP1. Login to comment 148 ABCC1 p.Pro1113Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1191Ala ABCC1 p.Pro1003Ala ABCC1 p.Pro1068Ala ABCC1 p.Pro1088Ala ABCC1 p.Pro1060Ala ABCC1 p.Pro1121Ala 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 149 ABCC1 p.Pro1150Ala In marked contrast, uptake of [3 H]E217betaG by the P1150A mutant was 2.2-fold higher than wild-type MRP1 (Fig. 4B). Login to comment 150 ABCC1 p.Pro1113Ala ABCC1 p.Pro1150Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1191Ala ABCC1 p.Pro1003Ala ABCC1 p.Pro1068Ala ABCC1 p.Pro1088Ala ABCC1 p.Pro1060Ala ABCC1 p.Pro1121Ala [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 151 ABCC1 p.Pro1150Ala ABCC1 p.Pro1150Ala To determine whether Ala substitution of Pro1150 also affected the transport of other folic acid derivatives, [3 H]leucovorin uptake by the P1150A mutant was assessed. Login to comment 152 ABCC1 p.Pro1150Ala As shown in Fig. 4D, levels of [3 H]leucovorin uptake by wild-type and P1150A mutant MRP1 were similar, indicating that the enhanced MTX transport activity caused by the Pro1150 substitution is quite substrate-specific. Login to comment 154 ABCC1 p.Pro1150Ala ABCC1 p.Pro1068Ala ABCC1 p.Pro1088Ala Relative to wild-type MRP1, apigenin-stimulated GSH uptake by the P1068A, P1088A, and P1150A mutants was significantly reduced (by 50-70%), whereas uptake by the other six MSD3 Pro FIG. 4. Login to comment 159 ABCC1 p.Pro1113Ala ABCC1 p.Pro1150Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1191Ala ABCC1 p.Pro1003Ala ABCC1 p.Pro1068Ala ABCC1 p.Pro1088Ala ABCC1 p.Pro1060Ala ABCC1 p.Pro1121Ala 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 161 ABCC1 p.Pro1120Ala mutants was similar to wild-type MRP1 except for P1120A, which was 1.4-fold higher. Login to comment 162 ABCC1 p.Pro1113Ala ABCC1 p.Pro1150Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1191Ala ABCC1 p.Pro1003Ala ABCC1 p.Pro1068Ala ABCC1 p.Pro1088Ala ABCC1 p.Pro1060Ala ABCC1 p.Pro1121Ala 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 ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1121Ala ABCC1 p.Pro1121Ala ABCC1 p.Pro1121Ala 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 ABCC1 p.Pro1113Ala 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 168 ABCC1 p.Pro1120Ala ABCC1 p.Pro1121Ala 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 169 ABCC1 p.Pro1113Ala 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 ABCC1 p.Pro1113Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1121Ala 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 ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala 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 ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1121Ala 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 ABCC1 p.Pro1113Ala Expression levels of MRP1 mutants containing an Ala substitution of Pro1113 . Login to comment 175 ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1121Ala ABCC1 p.Pro1121Ala 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 ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1121Ala ABCC1 p.Pro1121Ala 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 ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1121Ala ABCC1 p.Pro1121Ala 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 185 ABCC1 p.Pro1150Ala ABCC1 p.Pro478Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala ABCC1 p.Pro1088Ala The horizontal white scale bar in the image represents 20 ␮m. Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Uptake by MRP1 Pro Mutants-MSD2 TM mutants P343A, P448A, P478A, P557A, and P595A, MSD3 CL7 mutant P1150A, and TM14 mutant P1088A whose [3 H]LTC4 or [3 H]E217betaG transport properties were substantially altered relative to wild-type MRP1 were further characterized by kinetic analyses (Table IV). Login to comment 189 ABCC1 p.Pro478Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala The apparent Km(LTC4) values for MSD2 TM mutants P343A, P448A, P478A, and P557A (range 39-63) were all somewhat lower than wild-type MRP1 (72-115 nM) except for P595A (TM6), which was increased by nearly 5-fold (485 versus 115 nM). Login to comment 191 ABCC1 p.Pro478Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro343Ala Consequently, the overall LTC4 transport efficiency (Vmax/Km) of the P343A, P448A, P478A, and P557A mutants was moderately reduced (range 3.03 to 4.08) compared with wild-type MRP1 (5.83, 5.84). Login to comment 192 ABCC1 p.Pro595Ala The LTC4 transport efficiency of the P595A mutant (0.49) was reduced to an even greater extent (more than 11-fold), because of both a marked decrease in apparent uptake affinity for this substrate and a decrease in Vmax. Login to comment 193 ABCC1 p.Pro1150Ala ABCC1 p.Pro1088Ala The apparent Km(LTC4) values for the MSD3 mutants P1088A and P1150A were also somewhat decreased (55 and 40 nM, respectively) compared with wild-type MRP1 (72 nM), as were their Vmax(LTC4) values (by ϳ50%), yielding overall LTC4 transport efficiencies (Vmax/Km) for these two mutants of 4.15 and 4.55, respectively, compared with a value of 5.86 for wild-type MRP1. Login to comment 194 ABCC1 p.Pro1150Ala The apparent Km(E217betaG) for the MSD3 P1150A mutant, which showed significantly enhanced E217betaG transport, was decreased 4-fold (0.24 versus 1.02 ␮M for wild-type MRP1). Login to comment 195 ABCC1 p.Pro1088Ala In contrast, the apparent Km(E217betaG) value for P1088A was increased 1.4-fold (1.39 ␮M). Login to comment 196 ABCC1 p.Pro1150Ala ABCC1 p.Pro1088Ala On the other hand, the Vmax(E217betaG) values of P1150A and P1088A (160 and 165 pmol mg-1 min-1 , respectively) were comparable with wild-type MRP1 (176 pmol mg-1 min-1 ). Login to comment 197 ABCC1 p.Pro1150Ala ABCC1 p.Pro1088Ala Thus, the overall E217betaG transport efficiency of P1150A was enhanced ϳ4-fold whereas that of P1088A was reduced by 35%, largely because of changes in the apparent uptake affinity of the mutant proteins for this conjugated estrogen. Login to comment 198 ABCC1 p.Pro1150Ala ABCC1 p.Pro478Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala ABCC1 p.Pro1088Ala Photolabeling of Wild-type and Pro Mutant MRP1 Proteins with [3 H]LTC4-To investigate further whether the reduced [3 H]LTC4 transport activity of the TM mutants P343A, P448A, P478A, P557A, P595A, and P1088A and the CL7 mutant P1150A was associated with a decrease in substrate binding, photolabeling experiments were carried out with this intrinsically photoactivable arachidonic acid derivative. Login to comment 199 ABCC1 p.Pro595Ala As shown in Fig. 7, a radiolabeled protein band of 190 kDa was readily detectable in [3 H]LTC4 photolabeled membrane vesicles prepared from cells expressing all of the mutants except P595A. Login to comment 200 ABCC1 p.Pro595Ala Thus the P595A mutation completely abrogates photolabeling by LTC4, whereas the other mutations caused no or only a moderate reduction in photolabeling compared with wild-type MRP1. Login to comment 201 ABCC1 p.Pro1150Ala ABCC1 p.Pro1150Ala ATP Dependence of LTC4 and E217betaG Transport by MRP1-P1150A-To explore further the mechanism of enhanced E217betaG transport by the P1150A mutant, the ATP dependence of E217betaG uptake by membrane vesicles expressing this mutant MRP1 was investigated by measuring initial rates of up- FIG. 7. Login to comment 204 ABCC1 p.Pro1150Ala ABCC1 p.Pro1150Ala ABCC1 p.Pro478Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala ABCC1 p.Pro1088Ala Radiolabeled vesicles enriched for wild-type MRP1 (WT-MRP1) and MRP1 mutants P343A, P448A, P1088A, P1150A, and empty vector control (pcDNA3.1(-)) are shown in the upper panel, and radiolabeled vesicles enriched for WT-MRP1 and mutants P478A, P557A, P595A, P1150A, and pcDNA3.1(-) are shown in the lower panel. Login to comment 205 ABCC1 p.Pro1150Ala ABCC1 p.Pro1150Ala ABCC1 p.Pro478Ala ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala ABCC1 p.Pro1088Ala ABCC1 p.Pro1088Ala TABLE IV Kinetic parameters of vesicular LTC4 and E217beta G uptake by selected Pro mutants of MRP1 Km Vmax Vmax/ Km Mutant:WT nM pmol mg-1 min-1 ϫ10-3 Vmax/Km LTC4 WT-MRP1 72 422 5.86 1.0 P343A 50 203 4.06 0.7 P448A 39 155 3.97 0.7 P1088A 55 229 4.15 0.7 P1150A 40 182 4.55 0.8 WT-MRP1 115 674 5.85 1.0 P478A 49 160 3.24 0.6 P557A 63 191 3.02 0.5 P595A 485 239 0.49 0.1 E217betaG WT-MRP1 1017 176 0.17 1.0 P1088A 1390 165 0.12 0.7 P1150A 243 160 0.66 3.9 take of this conjugated organic anion at different ATP concentrations (Fig. 8A). Login to comment 206 ABCC1 p.Pro1150Ala The ATP dependence of LTC4 transport by P1150A was also measured for comparison (Fig. 8B). Login to comment 207 ABCC1 p.Pro1150Ala In the case of E217betaG transport (Fig. 8A), the Km(ATP) and Vmax values were 108 ␮M and 295 pmol mg-1 min-1 and 483 ␮M and 142 pmol mg-1 min-1 for P1150A and wild-type MRP1, respectively. Login to comment 208 ABCC1 p.Pro1150Ala In the case of LTC4 transport (Fig. 8B), the Km(ATP) and Vmax values were 97 ␮M and 90 pmol mg-1 min-1 and 109 ␮M and 170 pmol mg-1 min-1 for P1150A and wild-type MRP1, respectively. Login to comment 209 ABCC1 p.Pro1150Ala Thus the MRP1-P1150A mutant showed a significantly lower (nearly 5-fold) Km (higher apparent affinity) for ATP than wild-type MRP1 when supporting transport of E217betaG. Login to comment 210 ABCC1 p.Pro1150Ala In contrast, when supporting LTC4 transport, the Km(ATP) of P1150A and wild-type MRP1 were similar. Login to comment 211 ABCC1 p.Pro1150Ala ABCC1 p.Pro1150Ala 8-Azido-[␣-32 P]ATP Binding and Orthovanadate-induced Trapping of 8-Azido-[␣-32 P]ADP by MRP1-P1150A-To investigate further the ATP binding and hydrolysis properties of the MRP1-P1150A mutant, we used a 32 P-labeled photoactivable nucleotide analog to evaluate its relative ability to bind ATP. Login to comment 212 ABCC1 p.Pro1150Ala As shown in Fig. 9A, at 4 °C 8-azido-[␣-32 P]ATP photolabeled a 190-kDa protein corresponding to MRP1 in membrane proteins prepared from both wild-type and P1150A mutant MRP1-transfected cells. Login to comment 214 ABCC1 p.Pro1150Ala In contrast, orthovanadate-induced trapping of 8-azido-[␣-32 P]ADP at 37 °C by the P1150A mutant was markedly reduced compared with wild-type MRP1 (Fig. 9B). Login to comment 220 ABCC1 p.Pro1113Ala 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 ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala 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 226 ABCC1 p.Pro1150Ala ATP dependence of [3 H]E217betaG and [3 H]LTC4 transport by wild-type and P1150A mutant MRP1 proteins. Login to comment 227 ABCC1 p.Pro1150Ala Initial rates of [3 H]E217betaG uptake (A) and [3 H]LTC4 uptake (B) by wild-type MRP1 (f) and the P1150A mutant (Œ) were measured for 1 min in the presence of 10 different concentrations of ATP (2 ␮M to 4 mM). Login to comment 231 ABCC1 p.Pro1150Ala 8-Azido-[␣-32 P]ATP binding and orthovanadate-induced trapping of 8-azido-[␣-32 P]ADP by wild-type and P1150A mutant MRP1 proteins. Login to comment 237 ABCC1 p.Pro1113Ala ABCC1 p.Pro1113Ala ABCC1 p.Pro1120Ala ABCC1 p.Pro1121Ala 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 ABCC1 p.Pro1120Ala ABCC1 p.Pro1121Ala 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 245 ABCC1 p.Pro478Ala ABCC1 p.Pro323Ala Between these two extremes of transport activity were mutants P323A and P478A. Login to comment 246 ABCC1 p.Pro478Ala Replacement of Pro323 at the TM6 membrane-cytosol interface with Ala caused a significant and relatively specific loss of GSH transport, whereas Ala substitution of Pro478 (TM9) was associated with a substantial loss of LTC4 and GSH transport on the one hand, and a moderate increase in E217betaG, MTX, and E13SO4 transport on the other. Login to comment 247 ABCC1 p.Pro478Ala ABCC1 p.Pro323Ala The partial and substrate-selective alterations in transport activity observed for the P323A and P478A mutants provide evidence that although these Pro residues are not essential for function, they may be involved in some substrate-specific interactions with the transporter. Login to comment 248 ABCC1 p.Pro448Ala ABCC1 p.Pro557Ala ABCC1 p.Pro595Ala ABCC1 p.Pro343Ala The almost total loss of MRP1 transport activity of the MSD2 TM mutants P343A, P448A, P557A, and P595A does not distinguish between a structural or dynamic role for these Pro residues but does confirm that these highly conserved residues are required for transport. Login to comment 249 ABCC1 p.Pro595Ala However, all but one of these mutants (P595A) could still be labeled with [3 H]LTC4, indicating that if the Pro substitutions caused any structural changes, they did not disrupt the binding site for this substrate. Login to comment 261 ABCC1 p.Pro595Ala However, of these four MSD2 TM Pro mutants that display a global and substantial loss of transport activity, only one of them (P595A) showed a complete loss of photolabeling by LTC4 and a major decrease in uptake affinity for this substrate. Login to comment 264 ABCC1 p.Pro1088Ala In contrast to the MSD2 mutants, none of the nine MSD3 Pro mutants examined showed a comparable global decrease in transport activity, although Ala substitution of Pro1088 (at the membrane cytosol interface of TM14) and to a lesser extent Pro1003 (in ECL6) did cause a significant reduction in the transport of all five organic anion substrates tested. Login to comment 265 ABCC1 p.Pro1088Ala The significantly reduced transport activity of the P1088A mutant, together with our previous findings that non-conservative mutations of polar residues in the same region of TM14 alter MRP1 transport activity, confirm that the TM14-cytosol interface is a particularly important region for MRP1 activity (25, 29).3 Other MSD3 Pro mutants showed more substrate-selective changes in transport activity. Login to comment 266 ABCC1 p.Pro1120Ala For example, Ala substitution of Pro1120 (in TM15) caused a moderate and selective loss of E217betaG and MTX uptake. Login to comment 273 ABCC1 p.Pro1068Ala ABCC1 p.Pro1060Ala Ala substitution of Pro1060 (in CL6) caused a moderate and selective loss of E217betaG transport, and Ala substitution of Pro1068 (in CL6) caused a moderate overall loss of organic anion transport except for E13SO4 transport, which remained comparable with wild-type MRP1. Login to comment 276 ABCC1 p.Pro1068Ala ABCC1 p.Pro1068Ala ABCC1 p.Pro1068Ala ABCC1 p.Pro1060Ala ABCC1 p.Pro1060Ala ABCC1 p.Pro1060Ala That structural changes are introduced into CL6 by replacement of Pro1060 or Pro1068 with Ala is evident from the loss of immunoreactivity of the P1060A and P1068A mutants with mAb MRPm5 which maps to amino acids 1063-1072.4 However, the moderate changes in the transport properties of the P1060A and P1068A mutants, and the fact that mAb MRPm5 does not inhibit MRP1 transport activity, suggest that considerable mobility of this loop can be accommodated without loss of MRP1 activity.4 CL7 is also predicted to be extensively ␣-helical and, like CL6, contains two Pro residues. Login to comment 280 ABCC1 p.Pro1150Ala The P1150A mutant showed a moderate but significant decrease in LTC4, GSH, and E13SO4 transport but, remarkably, also exhibited a substantial increase in E217betaG and MTX transport. Login to comment 281 ABCC1 p.Pro1150Ala The decrease in LTC4 transport caused by the P1150A mutation was associated with a 2.5-fold decrease in Vmax, whereas the increase in E217betaG transport was associated with a 4-fold increase in uptake affinity (decreased Km). Login to comment 282 ABCC1 p.Pro1150Ala These observations indicate that the structural change introduced by Ala substitution of Pro1150 in some way selectively enhances the apparent uptake affinity of MRP1 for the glucuronidated estrogen while at the same time diminishes the efficiency of LTC4 transport. Login to comment 283 ABCC1 p.Pro1150Ala Intriguingly, the P1150A mutation also affected the apparent ATP dependence of organic anion transport in a substrate-selective way. Login to comment 284 ABCC1 p.Pro1150Ala Thus, the P1150A mutation caused a 5-fold increase in apparent affinity (decreased Km) for ATP and 2-fold increase in Vmax when supporting E217betaG transport. Login to comment 285 ABCC1 p.Pro1150Ala However, for LTC4 transport, the Km(ATP) of P1150A was unchanged, and a 2-fold decrease in Vmax was observed. Login to comment 286 ABCC1 p.Pro1150Ala Furthermore, whereas the P1150A mutant appeared to bind azido-ATP as well as wild-type MRP1, it showed substantially reduced vanadate- and BeF-induced trapping of azido-ADP. Login to comment 289 ABCC1 p.Pro1150Ala This could contribute, at least in part, to the moderately reduced ability of the P1150A mutant to transport LTC4, because previous studies have indicated that ATP binding/ hydrolysis at NBD2 plays a dominant role in the transport of this substrate (46, 56). Login to comment 290 ABCC1 p.Pro1150Ala However, it is not immediately obvious how reduced ATP binding/hydrolysis activity at NBD2 can be reconciled with the enhanced ability of the P1150A mutant to transport E217betaG and MTX. Login to comment 291 ABCC1 p.Pro1150Ala One possibility is that ADP release from NBD2 of the P1150A mutant after hydrolysis is so rapid that the efficiency of vanadate-induced trapping of ADP is reduced, but this cannot explain the substrate-specific changes observed. Login to comment 294 ABCC1 p.Pro1150Ala MTX transport by the P1150A mutant was increased to an even greater extent than E217betaG transport. Login to comment 297 ABCC1 p.Pro1150Ala These structural differences are certain to affect the H-bonding capabilities of the two drugs, but whether or not they are related to the differing ability of the P1150A mutant to transport these two substrates awaits the availability of a more refined structure of MRP1. Login to comment