ABCMdb

ABCC1 p.Pro595Ala

Predicted by SNAP2:	A: D (75%), C: D (80%), D: D (91%), E: D (91%), F: D (91%), G: D (80%), H: D (91%), I: D (85%), K: D (91%), L: D (91%), M: D (85%), N: D (91%), Q: D (91%), R: D (91%), S: D (71%), T: D (80%), V: D (85%), W: D (91%), Y: D (91%),
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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57 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
115 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 [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
126 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
129 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
132 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 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
140 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
185 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 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
192 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
198 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 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 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
204 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 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
248 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 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 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

[hide] A molecular understanding of ATP-dependent solute ... Cancer Metastasis Rev. 2007 Mar;26(1):15-37. Chang XB
A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.
Cancer Metastasis Rev. 2007 Mar;26(1):15-37., [PMID:17295059]

Abstract [show]
Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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113 In addition, the P595A mutation in TM11 exhibited significantly reduced transport of five organic anion substrates [75]. Login to comment

[hide] Molecular mechanism of ATP-dependent solute transp... Methods Mol Biol. 2010;596:223-49. Chang XB
Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.
Methods Mol Biol. 2010;596:223-49., [PMID:19949927]

Abstract [show]
Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Over-expression of ATP-binding cassette transporters, such as P-glycoprotein, breast cancer resistance protein and/or multidrug resistance-associated protein 1 (MRP1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs across the cell membrane barrier. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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104 Mutations of C43S in TM1 (112); P343A, K332L and K332D in TM6 (113, 114); W445A and P448A in TM8 (113, 115); T550A, T556A and P557A in TM10 (113, 116); N590A, F594A, P595A, N597A, S604A and S605A in TM11 (113, 117, 118); E1089Q, E1089A, E1089L, E1089N, K1092, S1097 and N1100 in TM14 (119, 120); R1197K in TM16 (121); Y1236F, T1241A, T1242A, T1242C, T1242S, T1242L, Y1243F, N1245A, W1246C, W1246A, W1246F, W1246Y or R1249K in TM17 (121-124) significantly affect MRP1 function. Login to comment