ABCMdb

PMID: 14561746

Campbell JD, Koike K, Moreau C, Sansom MS, Deeley RG, Cole SP
Molecular modeling correctly predicts the functional importance of Phe594 in transmembrane helix 11 of the multidrug resistance protein, MRP1 (ABCC1).
J Biol Chem. 2004 Jan 2;279(1):463-8. Epub 2003 Oct 15., 2004-01-02 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC1 p.Phe594Ala Substitution of Phe594 with Ala substantially reduced or eliminated the transport of five organic anion substrates by MRP1 and abrogated the binding of leukotriene C4. Login to comment 7 ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr On the other hand, the conservatively substituted F594W and F594Y mutants remained transport competent, although significant substrate- and substitution-specific changes were observed. Login to comment 51 ABCC1 p.Phe594Ala To test this hypothesis, we used site-directed mutagenesis to replace MRP1-Phe594 with Ala, Trp, and Tyr and examined the transport and organic anion binding properties of these mutants. Login to comment 69 ABCC1 p.Phe594Ala ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr Phe594 substitutions were generated in the pBluescriptSK(ϩ) plasmid above according to the manufacturer`s instructions with the following mutagenic primers (substituted nucleotides are underlined): F594A, 5Ј-G TTC AAC ATC CTC CGG GCT CCC CTG AAC ATT CTC C-3Ј; F594W, 5Ј-C TTG TTC AAC ATC CTC CGC TGG CCC CTG AAC ATT CTC CCC-3Ј; and F594Y, 5Ј-G TTC AAC ATC CTC CGC TAT CCC CTG AAC ATT CTC C-3Ј. Login to comment 99 ABCC1 p.Phe594Ala ABCC1 p.Phe594Ala ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr LTC4 Transport and Photo-labeling Is Eliminated by Ala Substitution of Phe594 -As shown in Fig. 2A, all three mutants generated (F594A, F594W, and F594Y) were expressed at levels 60-100% of those of wild-type MRP1 in transfected HEK cells. Login to comment 102 ABCC1 p.Phe594Ala ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr After correcting for differences in MRP1 protein expression levels, ATP-dependent LTC4 uptake by the F594A mutant was reduced by more than 90%, whereas uptake by the F594W and F594Y mutants was comparable with that by wild-type MRP1. Login to comment 103 ABCC1 p.Phe594Ala To further investigate the loss of LTC4 transport by the F594A mutant, protein-labeling experiments were carried out with this intrinsically photoactivatable arachidonic acid derivative. Login to comment 111 ABCC1 p.Phe594Ala ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr A, representative immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (F594A, F594W, and F594Y) MRP1 cDNAs. Login to comment 121 ABCC1 p.Phe594Ala In contrast, no such band is detectable in photolabeled vesicles from cells expressing comparable levels of the F594A mutant. Login to comment 122 ABCC1 p.Phe594Ala This indicates that this non-conservative substitution of Phe594 abrogates photolabeling by LTC4 and, hence, binding of this substrate to MRP1, a finding that is consistent with the complete loss of LTC4 transport activity by the F594A mutant. Login to comment 123 ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr The conservatively substituted Phe594 mutants F594W and F594Y could still be photolabeled by [3 H]LTC4, although photolabeling was reduced, by ϳ50% when corrected for differences in MRP1 protein expression. Login to comment 128 ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr For some substrates, uptake by the F594W mutant was up to 1.5-fold higher than that by wild-type MRP1 (GSH) and substantially higher than that by the F594Y mutant (E13SO4 and GSH). Login to comment 129 ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr For other substrates the opposite effects were observed, i.e. vesicular uptake by the F594Y mutant was higher than that by wild-type MRP1 (E217betaG and MTX) and even higher than that by the F594W mutant (E217betaG and MTX). Login to comment 130 ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr For example, E217betaG uptake by F594W was ϳ30% of wild-type MRP1, whereas vesicular uptake of this glucuronide conjugate by F594Y was ϳ1.4-fold higher than that by wild-type MRP1 (Fig. 3A). Login to comment 131 ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr In contrast, GSH uptake by the F594Y mutant was just 15% of that of wild-type MRP1, whereas uptake of this tripeptide by the F594W mutant was ϳ1.6-fold higher than that of wild-type MRP1. Login to comment 161 ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr On the other hand, conservative substitutions of Phe594 with either Tyr or Trp had little effect on LTC4 transport but, in some cases, caused some significant changes in the transport of at least two of the four other MRP1 organic anion substrates tested. Login to comment 162 ABCC1 p.Phe594Ala ABCC1 p.Phe594Trp ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr ABCC1 p.Phe594Tyr Thus, although the aromatic properties of the residue at position 594 are critical for retaining overall activity (i.e. F594 (wild-type), F594W, and F594Y are active but F594A is not), the addition of hydrogen bonding capacity to the amino acid side chain (as in F594W and F594Y) also influences substrate specificity, as shown previously for the polar aromatic residues at positions 553, 1198, 1243, and 1246. Login to comment