ABCC1 p.Pro1003Ala
| Predicted by SNAP2: | A: N (78%), C: D (53%), D: N (57%), E: N (57%), F: D (63%), G: N (57%), H: D (59%), I: N (61%), K: N (53%), L: D (63%), M: D (59%), N: N (87%), Q: N (66%), R: N (53%), S: N (78%), T: N (66%), V: N (61%), W: D (66%), Y: D (59%), |
| 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
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.
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ABCC1 p.Pro1003Ala 14722114:58:908
status: NEW139 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.
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ABCC1 p.Pro1003Ala 14722114:139:325
status: NEW147 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.
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ABCC1 p.Pro1003Ala 14722114:147:52
status: NEW148 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.
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ABCC1 p.Pro1003Ala 14722114:148:65
status: NEW150 [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).
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ABCC1 p.Pro1003Ala 14722114:150:267
status: NEW159 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).
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ABCC1 p.Pro1003Ala 14722114:159:136
status: NEW162 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.
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ABCC1 p.Pro1003Ala 14722114:162:36
status: NEW