ABCMdb

PMID: 18300232

Janke D, Mehralivand S, Strand D, Godtel-Armbrust U, Habermeier A, Gradhand U, Fischer C, Toliat MR, Fritz P, Zanger UM, Schwab M, Fromm MF, Nurnberg P, Wojnowski L, Closs EI, Lang T
6-mercaptopurine and 9-(2-phosphonyl-methoxyethyl) adenine (PMEA) transport altered by two missense mutations in the drug transporter gene ABCC4.
Hum Mutat. 2008 May;29(5):659-69., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC4 p.Glu757Lys ABCC4 p.Thr1142Met ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile A total of four variants (Y556C, E757K, V776I, and T1142M) exhibited a 20% to 40% reduced expression level compared to the wild type. Login to comment 8 ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile Compared to wild-type MRP4, the transmembrane variant V776I, revealed a significant lower activity in 6-MP transport, while the amino acid exchange Y556C in the WalkerB motif displayed significantly higher transport of PMEA. Login to comment 11 ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile Carriers of the rare MRP4 variants Y556C and V776I may have altered disposition of MRP4 substrates. Login to comment 34 ABCC4 p.Gly187Trp ABCC4 p.Lys304Asn ABCC4 p.Val854Phe ABCC4 p.Tyr556Cys ABCC4 p.Arg531Gln ABCC4 p.Val776Ile We also sequenced large regions of the MRP4 gene of these individuals and identified 74 genetic variations, among them 10 missense mutations (I18L, G187W, K304N, R531Q, Y556C, E757 K, V776I, V854F, I866 V, and T1142 M). Login to comment 54 ABCC4 p.Val776Ile ABCC4 p.Val776Ile ABCC4 p.Val776Ile Untagged hMRP4(wild-type,V776I) Construct for Xenopus laevis Oocyte Expression MRP4.pcDNA3.1/Hygro was AccI/XhoI-digested and a 160-bp fragment containing the MRP4 stop codon was cloned into the AccI/XhoI site of MRP4(wild-type, V776I).EGFP-pSGEM obtaining MRP4(wild-type, V776I)-pSGEM without an EGFP tag, respectively. Login to comment 107 ABCC4 p.Gly187Trp ABCC4 p.Thr1142Met ABCC4 p.Lys304Asn The following primer pairs were applied to amplify SNP-containing fragments by PCR: * rs11568658 (c.559G4T, Gly187Trp): 50 -Biotin-CCTCTTTT ATTTCAGGCACTTCG-30 , * 50 -TGCAGCTTACCTGATCAAACTTGT-30 (117 bp) * rs2274407 (c.912G4T, Lys304Asn): 50 -Biotin-ACGATGA TTTTGCTTGCACT-30 , * 50 - CGTGAGCCACTTTATCTGGT-30 (146 bp) * rs11568644 (c.3425C4T, Thr1142Met): 50 -GGGCACTTAG GAACCTGTTTTGT-30 , * 50 -Biotin-CTCTTGTAAGGCATTCCACAGTTC-30 (101 bp). Login to comment 110 ABCC4 p.Gly187Trp ABCC4 p.Thr1142Met ABCC4 p.Lys304Asn Procedures of Pyrosequencing were performed according to the manufacturer`s instructions using the PSQ 96 SNP Reagent Kit (Biotage AB) and following sequencing primers: rs11568658 (Gly187Trp): 50 -CCTGTGGTTGTCTTCC-30 and rs2274407 (Lys304Asn): 50 -CTGTACTCTCTTTCAG-30 for reverse assay, rs11568644 (Thr1142Met), 50 -TGGATCCCTTTAATGAGC-30 for forward assay. Login to comment 164 ABCC4 p.Gly487Glu ABCC4 p.Gly187Trp ABCC4 p.Arg820Ile ABCC4 p.Val854Phe ABCC4 p.Tyr556Cys A summary of all MRP4 protein mutations examined, their distribution within the protein, their frequencies and their functional prediction scores (SIFT/PolyPhen/Grantham) are listed in Supplementary Table S1 and Supplementary Figure S3. A total of six of them (G187W, G487E, Y556C, R820I, V854F, and T1142 M) are located either within transmembrane regions or near the ATP-binding domain of MRP4 and were predicted to have a functional effect by the computer-based algorithms (Supplementary Table S2). Login to comment 165 ABCC4 p.Lys1081Asn Moreover, a mutation was introduced into the critical WalkerA motif of MRP4 ATP-binding site 2 (K1081N) to provide an additional, negative control for the efflux experiments. Login to comment 167 ABCC4 p.Lys1081Asn In fact, substrate efflux in the absence or presence (Supplementary Fig. S4) of extracellular added unlabeled 500 mM 6-MP or 5 mM PMEA from oocytes expressing the MRP4(K1081N) mutant was not different from efflux of control oocytes, verifying that a functional WalkerA motif is necessary for MRP4, and providing a nonfunctional MRP4 control. Login to comment 170 ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile However, the mutation Y556C located in the WalkerB motif resulted in significantly increased [3 H] PMEA efflux, while the mutation V776I, located in the transmembrane region, led to a significantly decreased [14 C] 6-MP transport activity compared to wild-type (Supplementary Figs. S5 and S6). Login to comment 171 ABCC4 p.Gly487Glu ABCC4 p.Arg820Ile The G487E and R820I mutations resulted in a nonsignificant decrease of both, [14 C] 6-MP and [3 H] PMEA efflux (Supplementary Fig. S5). Login to comment 175 ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile The MRP4 protein variants E757 K, V776I, and T1142 M showed a 20-30% reduced protein expression and Y556C exhibited a 40% reduced protein expression compared to wild type, respectively. Login to comment 182 ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile Quantification of the fluorescence intensity demonstrated a 20-30% reduction in the surface expression for the variants E757 K, V776I, and T1142 M, and a 40% reduction in the surface expression of Y556C (Fig. 4B). Login to comment 183 ABCC4 p.Lys1081Asn All other variants including MRP4 (K1081N) had a fluorescence intensity similar to wild-type MRP4. Login to comment 185 ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile To investigate intracellular localization of MRP4 wild-type and variants (Y556C, E757 K, V776I, and T1142 M) with a reduced protein level, we prepared thin cryostat sections (12 mm) from oocytes. Login to comment 188 ABCC4 p.Gly487Glu ABCC4 p.Arg820Ile ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile Comparison ofTransport to Cell Surface Expression Y556C caused a two to three times higher [3 H] PMEA efflux, and V776I, G487E, and R820I caused a 25-30% decrease in [14 C] 6-MP efflux compared to wild-type when transport activity was normalized using the MRP4 expression levels (Fig. 5). Login to comment 189 ABCC4 p.Gly487Glu ABCC4 p.Arg820Ile ABCC4 p.Val776Ile The G487E and R820I mutations had a similar effect on [3 H] PMEA efflux, whereas the V776I did not (Fig. 5). Login to comment 190 ABCC4 p.Gly187Trp ABCC4 p.Lys304Asn Hepatic MRP4 Expression in Relation to MRP4 Protein Variants Genomic DNA samples corresponding to 285 liver samples of Caucasian origin were screened for three frequent Caucasian MRP4 mutations: c.559G4T (G187W), c.912G4T (K304N), and c.3425C4T (T1142 M) (Supplementary Table S1) [Gradhand et al., in press]. Login to comment 201 ABCC4 p.Tyr556Cys ÃPo0.05 for MRP4(Y556C).EGFP variant vs. wild-type. Login to comment 209 ABCC4 p.Tyr556Cys ÃPo0.05 for MRP4(Y556C) variant vs. wild-type. Login to comment 211 ABCC4 p.Gly187Trp ABCC4 p.Lys304Asn MRP4 protein expression in samples carrying nonsynonymous SNPs relative to the control group taken as 100% was as follows: G187W (69%, n 5 7, all heterozygous), K304N (101%, n 5 12, 11 heterozygous, one homozygous), and T1142 M (60%, n 5 4, all heterozygous). Login to comment 229 ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile MRP4 proteins variants Y556C, E757 K, V776I, and T1142 M were expressed at lower levels (range 20-40% of wild type) compared to MRP4 wild-type protein. Login to comment 233 ABCC4 p.Val776Ile The maximal decrease in overall 6-MP transport activity caused by any one of the 10 MRP4 mutations was approximately 50% (V776I) and it was partly due to a reduction of the protein`s expression. Login to comment 234 ABCC4 p.Val776Ile The transmembrane variant V776I was not computationally predicted to be deleterious (by SIFT, PolyPhen, and Grantham). Login to comment 236 ABCC4 p.Tyr556Cys With the exception of Y556C, the effects of all other MRP4 variants on PMEA transport ranged from none to moderate (o25% decrease). Login to comment 237 ABCC4 p.Tyr556Cys Y556C exhibited the greatest reduction in MRP4 protein expression (40%), and it caused an increase in specific PMEA (two- to three-fold of wild type) and 6-MP (1.5-fold of wild type) transport activity (Fig. 5). Login to comment 238 ABCC4 p.Tyr556Cys This may be due to an alteration of a region (nucleotide binding domain 1 [NBD1], WalkerB) affecting ATP-binding caused by the radical chemical change at this position indicated by the high Grantham value (Grantham value was 194) (Supplementary Table S1) and by a different drug binding of 6-MP and PMEA in the transmembrane domains of MRP4 Y556C that can induce differential long-range conformational changes in the NBDs, such that these compounds stimulate ATPase activity by decreasing the distance between the Walker A, B, and LSGGQ motif sequences. Login to comment 246 ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile It is possible that MRP4 mutations like Y556C and V776I may affect tissue accumulation and elimination of nucleoside-based agents, including PMEA, 6-MP, or other antiviral agents. Login to comment 247 ABCC4 p.Lys304Asn With the exception of K304N, MRP4 nonsynonymous SNPs reported in our previous study [Gradhand et al., in press] have been found to be heterozygous in the DNA samples screened. Login to comment 248 ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile In addition, four variants have been found as singletons, including the mutation leading to Y556C and V776I substitution (Supplementary Table S1). Login to comment 250 ABCC4 p.Gly187Trp ABCC4 p.Lys304Asn In this study, the more common variants G187W, K304N, and I866 V exhibited normal functional activity, consistent with the observation that common variants are less likely to exhibit altered function than are rare variants. Login to comment 251 ABCC4 p.Gly487Glu ABCC4 p.Arg820Ile ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile In contrast, variants of MRP4 having an influence on functional activity (G487E, Y556C, V776I, and R820I) were present at allelic frequencies o1%. Login to comment 253 ABCC4 p.Tyr556Cys Only for Y556C (WalkerB, consensus sequence ''YLLDD``), the human MRP4 wild-type residue is highly conserved compared to sequences of MRP4 orthologs and other ABCC homologs. Login to comment 257 ABCC4 p.Gly487Glu ABCC4 p.Gly187Trp ABCC4 p.Arg820Ile ABCC4 p.Val854Phe ABCC4 p.Tyr556Cys When SIFT and PolyPhen were used to evaluate the MRP4 nonsynonymous SNPs, both predicted the same variants as deleterious (G187W, G487E, Y556C, R820I, V854F, and TABLE 1. Login to comment 258 ABCC4 p.Glu757Lys ABCC4 p.Gly487Glu ABCC4 p.Gly187Trp ABCC4 p.Thr1142Met ABCC4 p.Arg820Ile ABCC4 p.Lys304Asn ABCC4 p.Val854Phe ABCC4 p.Tyr556Cys ABCC4 p.Val776Ile ABCC4 p.Ile866Val Conservation of the MRP4 Polymorphic AminoAcids Among Di¡erent ABCC Orthologs and HomologsÃ Protein Speciesa G187W K304N G487E Y556C E757K V776I R820I V854F I866V T1142M MRP4 Human G K G Y E V R V I T Mouse G K G Y E V R I V T Rat G K G Y G V R I L S MRP1 Human K Q D Y K ^ N C F S Mouse K Q D Y F A ^ V F S Rat K Q D Y ^ G N V V S MRP2 Human K K G Y S G R L V S Mouse R K G Y S G R L V S Rat K K G Y S G R L I S MRP3 Human R Q C F L G R L V S Rat R Q C F S G R I V S MRP5 Human ^ ^ A Y D L R V S T Mouse ^ ^ A Y D L R V S T Rat ^ ^ A Y D L R V S T MRP6 Human K G T Y G H S V V S Mouse K G T Y H G N G V T Rat K G T Y G N G V V T ÃAligned using ClustalW (www.ebi.ac.uk/clustalw). Login to comment 264 ABCC4 p.Gly187Trp ABCC4 p.Lys304Asn In our human liver study we found no significant influence of three MRP4 missense mutations (G187W, K304N, and T1142 M) on its hepatic expression. Login to comment 266 ABCC4 p.Tyr556Cys It is noteworthy, that MRP4 protein expression in one liver sample carrying Y556C showed a 40% reduction relative to the control group [Gradhand et al., in press]. Login to comment