ABCMdb

PMID: 15180328

Hirouchi M, Suzuki H, Itoda M, Ozawa S, Sawada J, Ieiri I, Ohtsubo K, Sugiyama Y
Characterization of the cellular localization, expression level, and function of SNP variants of MRP2/ABCC2.
Pharm Res. 2004 May;21(5):742-8., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC2 p.Val417Ile ABCC2 p.Ala1450Thr The transport activity for E217betaG, LTC4, and DNP-SG, normalized by the expression level of MRP2, was similar between the wild-type, V417I, and A1450T MRP2s. Login to comment 8 ABCC2 p.Ser789Phe The transport activity of S789F MRP2 was slightly higher than that of wild-type MRP2. Login to comment 9 ABCC2 p.Val417Ile ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr However, the expression level of S789F and A1450T MRP2 proteins was significantly lower compared with the wild-type and V417I MRP2. Login to comment 10 ABCC2 p.Val417Ile ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr In addition, although the wild-type and V417I MRP2 were exclusively localized in the apical membrane, S789F and A1450T MRP2 were located in the apical membrane and also in the intracellular compartment. Login to comment 12 ABCC2 p.Val417Ile ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr These results suggest that the most frequently observed V417I substitution may not affect the in vivo function of MRP2, whereas the much less frequently observed S789F and A1450T may be associated with the reduced in vivo function. Login to comment 25 ABCC2 p.Val417Ile Among them, only G1249A is associated with amino acid alterations, from Val to Ile at amino acid position 417 (19). Login to comment 26 ABCC2 p.Arg768Trp ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr Only one respective heterozygote subject was observed out of 48 volunteers for C2302T (exon 18, Arg768Trp), C2366T (exon 18, Ser789Phe), and G4348A (exon 31, Ala1450Thr) (19), and their allele frequency was calculated to be 1%. Login to comment 27 ABCC2 p.Arg768Trp Among these three kinds of less frequently observed SNPs, C2302T (exon 18, Arg768Trp) has been identified as the mutation responsible for DJS (21). Login to comment 43 ABCC2 p.Arg768Trp ABCC2 p.Val417Ile ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr We have generated the following four kinds of missense mutations reported previously: Val417Ile, Arg768Trp, Ser789Phe, and Ala1450Thr. Login to comment 86 ABCC2 p.Val417Ile ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr The expression level of V417I, S789F, and A1450T MRP2 was 82% and 80%, 23% and 25%, and 18% and 8%, respectively, in duplicate experiments (Fig. 1). Login to comment 87 ABCC2 p.Arg768Trp R768W MRP2 showed no staining, presumably due to rapid degradation as previously reported (18). Login to comment 92 ABCC2 p.Arg768Trp As a positive control to determine the localization of MRP2, we also determined the localization of R768W MRP2, which has been reported to be responsible for the sideration of the DJS (18). Login to comment 93 ABCC2 p.Arg768Trp In our experimental system, R768W MRP2 was located in the intracellular compartment of LLC-PK1 cells, which is consistent with the previous report (18). Login to comment 94 ABCC2 p.Val417Ile ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr V417I MRP2 showed the same localization as wild-type MRP2 (Fig. 2), whereas two variants (S789F and A1450T MRP2s) were expressed not only at the apical surface, but also intracellularly (Fig. 2). Login to comment 95 ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr The fraction of A1450T MRP2 located intracellularly was higher than that of S789F MRP2 (Fig. 2). Login to comment 100 ABCC2 p.Val417Ile ABCC2 p.Ser789Phe As shown in Fig. 4, no significant difference was observed between the wild-type and V417I MRP2s, whereas S789F MRP2 showed approximately a 1.4-to 2.0-fold higher uptake of three substrates compared with the wild-type MRP2 (Fig. 4). Login to comment 101 ABCC2 p.Ala1450Thr For A1450T, the expression level was too low to detect the transport function of E217betaG (Fig. 3d), whereas the normalized uptake of DNP-SG and LTC4 was almost identical to that of the wild-type (Figs. 4b and 4c). Login to comment 102 ABCC2 p.Ser789Phe Moreover, the kinetic parameters for the transport of [3 H]DNP-SG by wild-type and S789F MRP2s were determined (Fig. 5). Login to comment 122 ABCC2 p.Ala1450Thr The uptake of E217betaG by A1450T was too low to be normalized by the expression level (Fig. 3d). Login to comment 123 ABCC2 p.Val417Ile ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr Key: ᭺, tTA; ᭹, wt-MRP2; ᮀ, V417I; ᭿, S789F; ᭝, A1450T. Login to comment 124 ABCC2 p.Ser789Phe nmol·min-1 ·mg-1 protein, for the membrane vesicles expressing the wild-type and S789F MRP2s, respectively. Login to comment 125 ABCC2 p.Ser789Phe The transport activity of S789F MRP2 under linear conditions, calculated by correcting the Vmax value by the protein expression level prior to the division by the Km value, was approximately 1.3-fold higher than that of the wild type. Login to comment 136 ABCC2 p.Val417Ile ABCC2 p.Ser789Phe V417I and S789F MRP2s showed similar substrate specificity for glucuronide- and glutathione-conjugates to that of wild-type MRP2 (Figs. 3 and 4). Login to comment 137 ABCC2 p.Ala1450Thr ABCC2 p.Ala1450Thr In contrast, the transport activity of glutathione-conjugates (LTC4 and DNP-SG) in membrane vesicles expressing A1450T MRTP2 was 1/4 to 1/5 of that in the membrane vesicles expressing wild-type MRP2, whereas A1450T MRP2-mediated transport of E217betaG was much less than wild-type MRP2-mediated transport of this glucuronide- conjugate (Figs. 3 and 4). Login to comment 140 ABCC2 p.Ser789Phe In addition, kinetic parameters for the transport of [3 H]DNP-SG were compared between wild-type and S789F MRP2s. Login to comment 142 ABCC2 p.Ser789Phe The analysis revealed that the Km and the corrected Vmax values of [3 H]DNP-SG for S789F MRP2-mediated transport are approximately 43% and 57% that of wild-type MRP2, respectively. Login to comment 143 ABCC2 p.Ser789Phe Consequently, the transport activity of S789F MRP2, defined as the corrected Vmax/Km, is approximately 1.3-fold higher than that of wild-type MRP2, which is consistent with its transport activity defined as the initial velocity for the uptake of ligands divided by the ligand concentration in the medium (Fig. 4). Login to comment 145 ABCC2 p.Val417Ile Wild-type and V417I MRP2s exhibited apical localization, which is consistent with the localization under physiological conditions. Login to comment 146 ABCC2 p.Val417Ile Moreover, the expression level of V417I MRP2 in isolated membrane vesicles was the same as that of wild-type MRP2. Login to comment 147 ABCC2 p.Val417Ile Together with the finding that the transport activity of V417I MRP2 is the same as that of wild-type MRP2, this suggests that it is possible that these frequently found SNPs may not affect the disposition of substrate drugs among individuals. Login to comment 148 ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr In contrast, S789F and A1450T MRP2s were expressed not only at the apical surface, but also in the intracellular compartment. Login to comment 150 ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr It is possible that the low expression of S789F and A1450T MRP2s may be due to the degradation of the protein synthesized in the infected cells. Login to comment 151 ABCC2 p.Arg768Trp Indeed, both R768W MRP2 mutant and MRP2 mutant, which lacks R1392 and M1393 found in DJS patients, were core glycosylated and localized predominantly within the endoplasmic reticulum (17,18). Login to comment 153 ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr It is possible that the two variants examined in the current study (S789F and A1450T) are also degraded in the same manner. Login to comment 156 ABCC2 p.Ser789Phe Saturation of the ATP-dependent uptake of [3 H]DNP-SG was determined using the membrane vesicles from LLC-PK1 cells expressing wild-type (squares) and S789F MRP2s (circles). Login to comment 161 ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr S789F and A1450T MRP2s is low compared with that of the wild type. Login to comment 162 ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr Due to this low expression level on the apical surface, it is possible that the function of S789F and A1450T MRP2s under in vivo conditions is much lower compared with that of wild-type MRP2. Login to comment 165 ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr Of the 48 Japanese subjects studied, both S789F and A1450T SNPs were only found in one heterozygous subject and, consequently, their allele frequency was calculated to be only 1%. Login to comment 167 ABCC2 p.Ser789Phe ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr ABCC2 p.Ala1450Thr Because S789F and A1450T MRP2s are located within the first and second nucleotide binding domains, respectively, where the DJS mutations are frequently located, it is possible that S789F and A1450T SNPs are associated with the pathogenesis of DJS. Login to comment 169 ABCC2 p.Val417Ile It is suggested that the most frequently observed amino acid substitution (V417I) may not affect the drug disposition mediated by MRP2. Login to comment 170 ABCC2 p.Ser789Phe ABCC2 p.Ala1450Thr Although the transport activity per MRP2 molecule was not significantly decreased in much less frequently observed S789F and A1450T MRP2s, their expression level was significantly lower than wild-type MRP2, which may be associated with their intracellular localization. Login to comment