ABCMdb

PMID: 11429411

Zhang DW, Cole SP, Deeley RG
Identification of a nonconserved amino acid residue in multidrug resistance protein 1 important for determining substrate specificity: evidence for functional interaction between transmembrane helices 14 and 17.
J Biol Chem. 2001 Sep 14;276(37):34966-74. Epub 2001 Jun 27., 2001-09-14 [PubMed]

Sentences

No. Mutations Sentence Comment

11 ABCC1 p.Thr1242Ala As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/ T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C4 and E217betaG. Login to comment 66 ABCC1 p.Thr1242Ala To generate a MRP1E1089Q/T1242A double mutation, pGEM-MRP1-(2730-4832) containing mutation MRP1E1089Q was digested with StuI and XbaI to yield a 4.1-kilobase pair fragment comprised of nucleotides 2730-3758 of MRP1E1089Q attached to the vector fragment, and this fragment was then ligated to a 1.1-kilobase pair StuI-XbaI fragment encompassing nucleotides 3758-4832 of MRP1T1242A. Login to comment 134 ABCC1 p.Thr1242Ala ABCC1 p.Thr1242Cys ABCC1 p.Thr1242Ser Thus, Thr1242 was mutated to Ala, Cys, Ser, Leu, Lys, and Asp. Login to comment 137 ABCC1 p.Thr1242Ala After normalization for differences in expression levels, substitution of Thr1242 with Ala, Cys, Ser, Leu, and Lys decreased the ability of MRP1 to transport E217betaG by more than 2-fold relative to the wild type protein with no significant effect on LTC4 FIG. 2. Time course of ATP-dependent [3 H]LTC4 and [3 H]E217betaG uptake by membrane vesicles prepared from HEK293 stable transfectants expressing wild type mrp1 or A1239 mutant proteins. Login to comment 161 ABCC1 p.Thr1242Ala Consistent with the results obtained with the mrp1 mutant, substitution of Thr1242 with Ala in MRP1 decreased the normalized Vmax value for the mutant ϳ2-fold relative to wild type MRP1. Login to comment 167 ABCC1 p.Thr1242Ala In MRP1, substitution of Thr1242 with Ala increased the IC50 value from 15 to 134 ␮M. These results are independent of protein expression levels and provide strong evidence that the increase or decrease in E217betaG transport by mrp1A1239T and MRP1T1242A, respectively, is at least partially attributable to changes in the affinity of the proteins for this substrate. Login to comment 196 ABCC1 p.Glu1089Gln ABCC1 p.Glu1089Gln ABCC1 p.Thr1242Ala ABCC1 p.Thr1242Ala Consequently, a double mutation of MRP1 was also made, in which Glu1089 was replaced with Gln and Thr1242 was substituted with Ala (E1089Q/T1242A). Login to comment 203 ABCC1 p.Glu1089Gln Similarly, introduction of the E1089Q mutation into MRP1T1242A increased resistance to vincristine and VP-16 despite the fact that the single TM14 mutation in the human protein has been shown to decrease resistance to both of these drugs (29). Login to comment 204 ABCC1 p.Glu1089Gln However, the E1089Q mutation eliminated the ability of MRP1T1242A to confer resistance to anthracyclines (Table III), as observed previously when this amino acid substitution was introduced into wild type MRP1 as a single mutation (29). Login to comment 228 ABCC1 p.Thr1242Ala Replacement of Thr1242 with Ala, Ser, Cys, Leu, and Lys decreased the ability of the protein to transport E217betaG 2-3-fold without significantly affecting LTC4 transport. Login to comment 234 ABCC1 p.Thr1242Ala The values shown represent the means Ϯ S.D. of relative resistance factors determined from 3-6 independent experiments. Resistance factors normalized for differences in the levels of mutant proteins expressed in the transfectant populations used are shown in parentheses. Transfectant Drug (relative resistance factor) Vincristine VP-16 Doxorubicin Epirubicin HEKMRP1 18.6 Ϯ 3.1 (18.6) 18.9 Ϯ 2.0 (18.9) 6.7 Ϯ 0.9 (6.7) 9.3 Ϯ 0.4 (9.3) n ϭ 6 n ϭ 6 n ϭ 6 n ϭ 6 HEKMRP1T1242A 14.5 Ϯ 0.9 (10.8) 13.6 Ϯ 1.2 (10.1) 2.6 Ϯ 0.2 (2.0) 3.2 Ϯ 0.3 (2.4) n ϭ 5 n ϭ 5 n ϭ 5 n ϭ 5 HEKMRP1T1242C 9.7 Ϯ 0.3 (9.7) 10.7 Ϯ 0.3 (10.7) 3.1 Ϯ 0.2 (3.1) 3.3 Ϯ 0.2 (3.3) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1T1242S 9.1 Ϯ 0.5 (11.4) 9.6 Ϯ 0.7 (11.9) 2.5 Ϯ 0.5 (3.1) 2.6 Ϯ 0.4 (3.2) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1T1242L 10.7 Ϯ 3.3 (11.9) 10.4 Ϯ 1.2 (10.7) 2.8 Ϯ 0.9 (2.9) 2.9 Ϯ 3.9 (3.0) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1T1242D 6.5 Ϯ 1.2 (9.3) 7.0 Ϯ 0.5 (10.0) 2.3 Ϯ 0.2 (2.5) 2.4 Ϯ 0.1 (2.6) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1T1242K 5.4 Ϯ 0.3 (11.5) 4.8 Ϯ 0.3 (12.0) 1.5 Ϯ 0.2 (3.7) 2.1 Ϯ 0.5 (4.1) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1E1089Q/T1242A 18.3 Ϯ 5.3 (26.9) 17.8 Ϯ 1.5 (26.2) Ͻ1 Ͻ1 n ϭ 5 n ϭ 5 n ϭ 3 n ϭ 3 transport. Login to comment 239 ABCC1 p.Thr1242Asp The consequences of mutating mrp1A1239 and MRP1T1242, with the exception of the T1242D mutation, are qualitatively similar to those we observed following mutation of the highly conserved Trp1246 , which abolished drug resistance and E217betaG transport (30). Login to comment 248 ABCC1 p.Thr1242Cys ABCC1 p.Thr1242Ser However, substitution of Thr1242 with either Cys or Ser, which retain hydrogen bonding capability, decreased the ability of the protein to transport E217betaG and to confer drug resistance. Login to comment 252 ABCC1 p.Thr1242Ala In contrast, the IC50 for LTC4 transport by MRP1 T1242A was ϳ130 ␮M, a value similar to that obtained for wild type mrp1, while the IC50 of mrp1A1239T decreased to ϳ30 ␮M. It has been proposed that the transport of anionic/cationic substrates by MRP1 is facilitated by cationic/anionic acid residues present in the transmembrane helices (39). Login to comment 257 ABCC1 p.Thr1242Lys However, we found in this study that mutation of Thr1242 to Lys did not enhance LTC4 transport and actually decreased transport of E217betaG. Login to comment 264 ABCC1 p.Thr1242Ala Transfectants tested were HEKmrp1 (f), HEKmrp1A1239T (Œ), and HEKmrp1Q1086E/A1239T (q) (C and D) and HEKMRP1 (Ⅺ), HEKMRP1T1242A (‚), and HEKMRP1E1089Q/T1242A (E) (E and F). Login to comment 266 ABCC1 p.Thr1242Asp Only substitution of Thr1242 with Asp affected transport of both E217betaG transport and, albeit to a lesser extent, LTC4. Login to comment 268 ABCC1 p.Thr1242Ala Having found previously that mutation of Glu1089 in TM14 of MRP1 to Gln not only markedly reduced that ability of the protein to confer resistance to anthracyclines but also, to a lesser extent, to vincristine and VP-16 (29), we investigated the effect of combining this mutation, and the reciprocal mutation in the murine protein, with the A1239T and T1242A mutations of mrp1 and MRP1, respectively. 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