ABCMdb

PMID: 11721885

Conrad S, Kauffmann HM, Ito K, Deeley RG, Cole SP, Schrenk D
Identification of human multidrug resistance protein 1 (MRP1) mutations and characterization of a G671V substitution.
J Hum Genet. 2001;46(11):656-63., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC1 p.Gly671Val J Hum Genet (2001) 46:656-663 (c) Jpn Soc Hum Genet and Springer-Verlag 2001 ORIGINAL ARTICLE Identification of human multidrug resistance protein 1 (MRP1) mutations and characterization of a G671V substitution Silke Conrad · Hans-Martin Kauffmann · Ken-ichi Ito Roger G. Deeley · Susan P.C. Cole · Dieter Schrenk S. Conrad · H.-M. Kauffmann · D. Login to comment 6 ABCC1 p.Gly671Val One of these mutations (G671V) was of special interest because it is located near the first nucleotide binding domain. Login to comment 45 ABCC1 p.Gly671Val Site-directed mutagenesis The mutation of Gly671 to Val was generated using the sense primer 5ЈP-CTCCATCCCCGAAGTGGCTTTGGTGGC CG-3Ј (substituted nucleotides are bold and underlined) with the U.S.E. mutagenesis kit (Amersham Pharmacia Biotech, Quebec, Canada), according to the manufacturer`s instructions. Login to comment 78 ABCC1 p.Gly671Val ABCC1 p.Arg433Ser Mutations in the MRP1 gene and median mRNA expression levels of samples with different MRP1 genotypes Position Location SNPs Change FRE EXPa Intron 7 809ϩ54 C/A Intronic 2.8 101 Exon 8b 825 T/C Silent 2.8 51 Intron 8 1040ϩ13 T/C Intronic 16.7 95 Exon 10 1299 G/T Arg433Ser 1.4 156 Intron 11b 1474-48 C/T Intronic 2.8 146 Intron 11 1474-8 T/C Intronic 9.7 72 Intron 12 1678-9 DEL T Intronic 11.1 106 Exon 13b 1684 T/C Silent 8.3 106 Exon 13 1704 C/T Silent 2.8 59 Exon 16 2012 G/T Gly671Val 2.8 43 Intron 20 2736-36 T/C Intronic 2.8 132 Intron 20 2736-18 CC/TT Intronic 2.8 132 Intron 20 2736-6 T/C Intronic 2.8 132 Intron 21 2871ϩ17 G/A Intronic 1.4 132 Intron 26 3819ϩ7 G/A Intronic 1.4 124 Exon 28b 4002 G/A Silent 2.8 95 Intron 30 4487ϩ5 A/T Intronic 2.8 137 Intron 30 4487ϩ18 A/G Intronic 1.4 142 Intron 30 4487ϩ28 C/G Intronic 2.8 137 Densitometrical analysis was performed with TINA software (Raytest, Straubenhardt, Germany), average relative MRP1 expression was set as 100% FRE, allelic frequency in %; EXP, expression level; SNP, single-nucleotide polymorphism a The average expression level of all samples was 100 Ϯ 55 b Recently described by Ito et al. 2001b Fig. 2. Login to comment 88 ABCC1 p.Gly671Val These mutations include heterozygous silent mutations in exon 8 and 13 and one heterozygous mutation in exon 16 that could alter the amino acid sequence (G671V). Login to comment 91 ABCC1 p.Gly671Val These results, together with the localization of G671V close to the first ATP-binding site and the highly conserved nature of Gly671 among ABC proteins (Figure 2) prompted us to analyze the functional importance of this amino acid. Login to comment 92 ABCC1 p.Gly671Val This was accomplished by introducing the G671V substitution into the pcDNA3.1(-)wt-MRP1K expression vector by site-directed mutagenesis, followed by a transient transfection of the construct into HEKSV293T cells. Login to comment 96 ABCC1 p.Gly671Val Transport activity of wild-type and mutant G671V-MRP1 in transiently transfected human embryonic kidney (HEKSV293T) cells. Login to comment 97 ABCC1 p.Gly671Val ABCC1 p.Gly671Val A Vesicles protein expression levels of pcDNA3.1(-) as control, pcDNA3.1(-)wt-MRP1k (wt-MRPk-1), and pcDNA3.1(-)-MRPk-1- G671V (G671V) were determined by immunoblotting with the MRP1-specific murine Mab QCRL-1. Login to comment 99 ABCC1 p.Gly671Val ABCC1 p.Gly671Val B Time course of E217 G uptake in membrane vesicles prepared from HEKSV293T cells transiently transfected with wild-type MRP1 (wt-MRPk-1, solid square), mutant (G671V-MRPk-1, triangle), and empty control (pcDNA3.1(-), open square) cDNA expression vectors. Results shown are means (Ϯ SD) of triplicate determinations in a single experiment. Login to comment 101 ABCC1 p.Gly671Val C Time course of LTC4 uptake in membrane vesicles prepared from HEKSV293T cells transiently transfected with wild-type MRP1 (wt-MRPk-1, solid square), mutant (G671V-MRPk-1, triangle), and empty control (pcDNA3.1(-), open square) cDNA expression vectors. Results shown are means (Ϯ SD) of triplicate determinations in a single experiment. Login to comment 103 ABCC1 p.Gly671Val D Determination of Km and Vmax for ATP-dependent uptake of [3 H]LTC4 in membrane vesicles from transiently transfected HEKSV293T cells (wt-MRPk-1, solid square; G671V-MRPk-1, triangle). Login to comment 107 ABCC1 p.Gly671Val ABCC1 p.Gly671Val For the [3 H]LTC4 and [3 H]E217 G transport experiments shown in Figures 3B and C, the levels of uptake by G671V-MRP1 were comparable to wild-type MRP1. Login to comment 109 ABCC1 p.Gly671Val Our findings were supported by Eadie-Hofstee transformation (Figure 3D) of the data obtained from a ATP-dependent [3 H]LTC4 uptake experiment with various substrate concentrations, which yielded similar Km (175.8 and 151.0nM, respectively) and Vmax (107.9 and 111.3pmol/mg/min, respectively) values for G671V-MRP1 and wild-type MRP1, respectively. Login to comment 111 ABCC1 p.Gly671Val In accordance with previous findings, both wild-type MRP1 and G671V exhibited a markedly enhanced uptake of [3 H]estrone sulfate in the presence of GSH (Qian et al. 2001). Login to comment 113 ABCC1 p.Gly671Val Thus, the G671V mutant showed a phenotype similar to wild-type MRP1 (Figures 3 and 4) in all transport assays performed. Login to comment 118 ABCC1 p.Gly671Val ABCC1 p.Arg433Ser They are located in the second transmembrane spanning domain (R433S) and in the vicinity of the first ATP-binding site (G671V). Login to comment 119 ABCC1 p.Gly671Val G671V is located only six amino acids upstream from the conserved Walker A motif in the nucleotide binding domain, and previous studies have shown that mutations in and near the Walker A motifs can cause a decrease in transport activity (Gao et al. 2000; Szakacs et al. 2000; Ramjeesingh et al. 1999). Login to comment 133 ABCC1 p.Gly671Val Of all the mutations detected in the present study, we specifically examined the functional consequences of the G671V substitution for three reasons: (1) the reduced mRNA expression of the affected individuals; (2) the high conservation of Gly671 in several subfamily C ABC-transporters; and (3) the location of the exchange position near the essential Walker A motif of NBD1. Login to comment 136 ABCC1 p.Gly671Val Although organic anion transport appears unaffected, it is possible the G671V substitution could affect drug resistance, since the two properties are not always linked. Login to comment