ABCMdb

PMID: 16415113

Zhang DW, Nunoya K, Vasa M, Gu HM, Cole SP, Deeley RG
Mutational analysis of polar amino acid residues within predicted transmembrane helices 10 and 16 of multidrug resistance protein 1 (ABCC1): effect on substrate specificity.
Drug Metab Dispos. 2006 Apr;34(4):539-46. Epub 2006 Jan 13., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC1 p.Thr556Ala ABCC1 p.Thr550Ala Although mutation of Asn1208 was without effect, two of six mutations in TM10, T550A and T556A, modulated the drug resistance profile of MRP1 without affecting transport of leukotriene C4, 17beta-estradiol 17-(beta-D-glucuronide) (E217betaG), and glutathione. Login to comment 7 ABCC1 p.Thr556Ala ABCC1 p.Thr550Ala Mutation T550A increased vincristine resistance but decreased doxorubicin resistance, whereas mutation T556A decreased resistance to etoposide (VP-16) and doxorubicin. Login to comment 34 ABCC1 p.Tyr568Ala ABCC1 p.Tyr568Ser ABCC1 p.Tyr568Trp ABCC1 p.Tyr568Phe In TM10, Thr550 , Thr552 , Thr556 , Thr564 , and Thr570 were individually mutated to Ala, and Tyr568 was mutated to Ala, Ser, Phe, and Trp. Login to comment 37 ABCC1 p.Asn1208Ala Mutation N1208A in TM16 had no detectable effect on the function of MRP1. Login to comment 44 ABCC1 p.Thr564Ala Mutation T564A was generated using the Transformer Site-Directed Mutagenesis kit (BD Biosciences Clontech, Palo Alto, CA). Login to comment 48 ABCC1 p.Thr556Ala ABCC1 p.Thr550Ala ABCC1 p.Tyr568Ala ABCC1 p.Tyr568Ser ABCC1 p.Tyr568Trp ABCC1 p.Tyr568Phe ABCC1 p.Asn1208Ala ABCC1 p.Thr570Ala ABCC1 p.Thr552Ala Mutations T550A, T552A, T556A, Y568A, Y568S, Y568F, Y568W, T570A, and N1208A were generated using the Quikchange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Login to comment 50 ABCC1 p.Thr556Ala ABCC1 p.Thr550Ala ABCC1 p.Tyr568Ala ABCC1 p.Tyr568Ser ABCC1 p.Tyr568Trp ABCC1 p.Tyr568Phe ABCC1 p.Asn1208Ala ABCC1 p.Thr570Ala ABCC1 p.Thr552Ala Oligonucleotides bearing mismatched bases at the residues to be mutated (underlined) were synthesized by ACGT Corp. with the following sequences: T550A, 5Ј-GTCAGCCGTGGG- CGCCTTCACCTGGGT-3Ј; T552A, 5Ј-CGTGGGCACCTTCGCCTGGGTC- TGCAC-3Ј; T556A, 5Ј-CACCTGGGTCTGCGCGCCCTTTCTGGT-3Ј; Y568A, 5Ј-TGCACATTTGCCGTCGCCGTGACCATTGACGA-3Ј; Y568S, 5Ј-TGCACATTTGCCGTCTCCGTGACCATTGACGA-3Ј; Y568F, 5Ј-TGC- ACATTTGCCGTCTTCGTGACCATTGACGA-3Ј; Y568W, 5Ј-TGCACAT- TTGCCGTCTGGGTGACCATTGACGA-3Ј; T570A, (5Ј-TGCCGTCTACG- TGGCCATTGACGAGAACAAC-3Ј; and N1208A, 5Ј-GCCGTGCGGCTG- GAGTGTGTGGGCGCC-3Ј. Login to comment 120 ABCC1 p.Tyr568Ala ATP-dependent transport of [3 H]E217betaG was also examined (Fig. 3, D to F), only replacement of Tyr568 with Ala decreased the transport efficiency by approximately 60%, indicating that the polar and/or the bulky aromatic side chain of the residue at position 568 is important for the ability of MRP1 to transport E217betaG. Login to comment 121 ABCC1 p.Tyr568Ser ABCC1 p.Tyr568Trp ABCC1 p.Tyr568Phe Effect of Mutations Y568S, Y568F, and Y568W on the Transport of [3 H]LTC4 and [3 H]E217betaG by MRP1. Login to comment 122 ABCC1 p.Tyr568Ala Because replacement of Tyr568 by Ala selectively decreased transport of E217betaG, this residue was also mutated to Ser, Phe, and Trp. Login to comment 126 ABCC1 p.Tyr568Ala ABCC1 p.Tyr568Ser Like mutation Y568A, substitution of Tyr568 with Ser did not affect the transport of LTC4 but decreased E217betaG transport. Login to comment 129 ABCC1 p.Tyr568Ala Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Transport by Wild-Type and Y568A Mutant MRP1. Login to comment 130 ABCC1 p.Tyr568Ala To more precisely determine the influence of mutation Y568A on the ability of MRP1 to transport E217betaG, we compared kinetic parameters for the wild-type and mutant proteins (Fig. 5). Login to comment 131 ABCC1 p.Tyr568Ala For wild-type MRP1 and Y568A, the Km and normalized Vmax values for LTC4 uptake were essentially identical. Login to comment 132 ABCC1 p.Tyr568Ala Linear regression using a Hanes-Woolf transformation yielded values of 115 nM and 143 nM, and 76.8 pmol/mg/min and 64 pmol/mg/min, for the Km and Vmax values of wild-type and Y568A proteins, respectively (Fig. 5, B and C). Login to comment 147 ABCC1 p.Thr550Ala Substitution T550A reduced resistance to doxorubicin approximately 2-fold and increased resistance to vincristine approximately 4.5-fold, but had no effect on VP-16 resistance. Login to comment 148 ABCC1 p.Thr556Ala In contrast, mutation T556A reduced the resistance to both VP-16 and doxorubicin approximately 2-fold without affecting vincristine resistance. Login to comment 151 ABCC1 p.Thr556Ala ABCC1 p.Thr550Ala ABCC1 p.Tyr568Ala We have shown that mutations T550A and T556A both affect the ability of MRP1 to confer drug resistance, whereas mutation Y568A only influenced the transport of E217betaG. Login to comment 164 ABCC1 p.Glu1204Leu ABCC1 p.Arg1197Glu ABCC1 p.Arg1197Lys Conversely, replacement of Glu1204 with Leu or Arg1197 with Glu or Lys affected either substrate specificity or overall transport activity of MRP1 (Situ et al., 2004). Login to comment 165 ABCC1 p.Trp1198Ala Mutation of Trp1198 to Ala also dramatically decreased overall transport activity (Koike et al., 2002). Login to comment 206 ABCC1 p.Thr550Ala The increase in resistance to vincristine observed with the T550A mutation may be attributable to the smaller size of the Ala side chain that favors transport of the larger drug. Login to comment 208 ABCC1 p.Thr550Ala Like the T550A mutation, these mutations decreased resistance to VP-16 and increased resistance to vincristine. Login to comment 233 ABCC1 p.Tyr568Ala However, kinetic analysis showed that the nonconservative mutation Y568A increased the apparent Km for E217betaG approximately 5-fold, without altering Vmax. Login to comment