ABCMdb

PMID: 12388190

Oleschuk CJ, Deeley RG, Cole SP
Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3.
Am J Physiol Gastrointest Liver Physiol. 2003 Feb;284(2):G280-9. Epub 2002 Oct 9., [PubMed]

Sentences

No. Mutations Sentence Comment

59 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala Mutations for Trp1242 substitutions (underlined), silent DraI restriction sites (italicized), and their corresponding oligonucleotides were as follows: W1242A (5Ј-G CAG GTG ACA TTC GCT TTA AAC GCG ATG ATA CGA ATG ATG TCA G-3Ј), W1242C (5Ј-G CAG GTG ACA TTC GCT TTA AAC TGC ATG ATA CGA ATG ATG TCA G-3Ј), W1242F (5Ј-G CAG GTG Fig. 1. Login to comment 70 ABCC3 p.Trp1242Pro ABCC3 p.Trp1242Tyr G281CONSERVED ACA TTC GCT TTA AAC TTC ATG ATA CGA ATG ATG TCA G-3Ј), W1242Y (5Ј-G CAG GTG ACA TTC GCT TTA AAC TAC ATG ATA CGA ATG ATG TCA G-3Ј), and W1242P (5Ј-G CAG GTG ACA TTC GCT TTA AAC CCG ATG ATA CGA ATG ATG TCA G-3Ј). Login to comment 103 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr These included nonconservative substitutions with a nonaromatic nonpolar amino acid (Ala; W1242A-MRP3) and a nonaromatic polar amino acid (Cys; W1242C-MRP3), as well as conservative substitutions with aromatic polar (Tyr; W1242Y-MRP3) and nonpolar (Phe; W1242F-MRP3) amino acids. Login to comment 104 ABCC3 p.Trp1242Pro Trp1242 was also replaced with a ␣-helix-disrupting residue (Pro; W1242P-MRP3). Login to comment 106 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr As shown in Fig. 2, wild-type MRP3 and the four mutants (W1242A-, W1242C-, W1242F-, and W1242Y-MRP3) were expressed at similar levels. Login to comment 107 ABCC3 p.Trp1242Pro In contrast, the W1242P-MRP3 mutant was expressed at significantly lower levels, suggesting that this mutation affects the biogenesis or stability of the protein. Login to comment 108 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Pro ABCC3 p.Trp1242Tyr Mean expression levels of the mutant MRP3 proteins relative to wild-type MRP3 were as follows: W1242A, 1.1 Ϯ 0.3; W1242C, 1.0 Ϯ 0.2; W1242Y, 1.0 Ϯ 0.1; W1242F, 1.0 Ϯ 0.3; and W1242P, 0.6 Ϯ 0.2 (4-6 independent transfections). Login to comment 110 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Pro ABCC3 p.Trp1242Tyr Time courses of ATP-dependent [3 H]E217betaG uptake were determined for the W1242A-, W1242C-, W1242F-, W1242Y-, and W1242P-MRP3 mutants by using inside-out membrane vesicles prepared from transfected HEK293T cells (Fig. 3A). Login to comment 111 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr Unexpectedly, four of the five mutants (W1242A-, W1242C-, W1242F-, and W1242Y-MRP3) transported this glucuronide substrate at levels that were substantially higher than those of wild-type MRP3. Login to comment 112 ABCC3 p.Trp1242Pro In contrast, transport by W1242P-MRP3 was almost undetectable. Login to comment 113 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr At 3 min, [3 H]E217betaG uptake in membrane G282 CONSERVED TRP AND MRP3 (ABCC3) SUBSTRATE SPECIFICITY AJP-Gastrointest Liver Physiol • VOL 284 • FEBRUARY 2003 • www.ajpgi.org atUnivofNorthCarolina-AcqSrvcsonOctober,2012http://ajpgi.physiology.org/Downloadedfrom vesicles enriched for W1242A-, W1242C-, and W1242F-MRP3 was 2.5-to 3-fold higher than for wild-type MRP3, whereas [3 H]E217betaG uptake by the most conservatively substituted mutant, W1242Y-MRP3, was ϳ7-fold higher (Fig. 3B). Login to comment 120 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr [3 H]LTC4 uptake by the W1242A-, W1242F-, and W1242Y-MRP3 mutants was somewhat less than the wild-type MRP3 transport activity (Fig. 4A). Login to comment 121 ABCC3 p.Trp1242Cys At 5 min, levels of [3 H]LTC4 uptake by these three mutants were ϳ6270% those of wild-type MRP3, whereas uptake by the W1242C-MRP3 mutant was only 37% of wild-type levels (after subtraction of uptake by vector control membrane vesicles and normalization of mutant MRP3 protein levels to wild-type MRP3 protein levels) (Fig. 4B). Login to comment 122 ABCC3 p.Trp1242Pro [3 H]LTC4 uptake by the W1242P-MRP3 mutant was undetectable (not shown). Login to comment 123 ABCC3 p.Trp1242Pro Because of the consistently lower levels of W1242P-MRP3 expression and because this mutant did not transport either E217betaG or LTC4, it was not characterized further. Login to comment 128 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr At 10 min, ATP-dependent [3 H]MTX uptake levels by the W1242A, W1242C, W1242F, and W1242Y Fig. 3. Login to comment 130 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Pro ABCC3 p.Trp1242Tyr A: time course of ATP-dependent [3 H]E217betaG uptake in membrane vesicles prepared from HEK293T cells transfected with empty vector (ᮀ), wild-type MRP3 (s), and mutant [W1242A (), W1242C (}), W1242F (F), W1242Y (Œ), and W1242P (ƒ)] cDNA expression vectors. Login to comment 135 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Pro ABCC3 p.Trp1242Tyr Top: MRP3 expression in membrane vesicles prepared from human embryonic kidney (HEK) 293T cells transfected with empty vector [pcDNA3.1(ϩ)], wild-type (WT-MRP3), and mutant (W1242A, W1242C, W1242F, W1242Y, and W1242P) MRP3 cDNAs was determined by immunoblotting with MAb M3II-9, and relative levels of expression were estimated by densitometry. Login to comment 145 ABCC3 p.Trp1242Tyr Only the most conservatively substituted W1242Y-MRP3 mutant transports [3 H]leucovorin. Login to comment 147 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr To determine if Trp1242 substitutions also affected MRP3-mediated transport of the latter substrate, [3 H]leucovorin uptake into membrane vesicles prepared from transfected cells expressing wild-type MRP3 and W1242A-, W1242C-, W1242F-, and W1242Y-MRP3 mutants was examined. Login to comment 149 ABCC3 p.Trp1242Tyr Of the four Trp1242 mutants tested, only the most conservatively substituted mutant, W1242Y-MRP3, transported [3 H]leucovorin at levels comparable to those of wild-type MRP3. Login to comment 151 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr Time course of [3 H]methotrexate (MTX) uptake and MTX- mediated inhibition of [3 H]E217betaG uptake by wild-type and Trp1242 mutant MRP3 proteins. A: ATP-dependent uptake of [3 H]MTX was measured in membrane vesicles prepared from HEK293T cells transfected with empty pcDNA3.1(ϩ) vector (ᮀ), wild-type (s), and mutant [W1242A (), W1242C (}), W1242F (F), and W1242Y (Œ)] MRP3 cDNA expression vectors. Membrane vesicles were incubated at 37°C with 1 ␮M [3 H]MTX and ATP or AMP in transport buffer. Login to comment 161 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr Taurocholate uptake by the W1242A-, W1242C-, W1242F-, and W1242Y-MRP3 mutants was then examined and, in all cases, was not significantly different from uptake by wild-type MRP3 (Fig. 7B). Login to comment 162 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr Consistent with this observation, [3 H]E217betaG uptake by W1242A-, W1242C-, W1242F-, and W1242Y-MRP3 could still be inhibited by taurocholic acid (40-60%) at concentrations of 50 and 100 ␮M (Fig. 7C). Login to comment 170 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr Cells were transfected with empty vector [pcDNA3.1(ϩ)], wild-type, and mutant (W1242A, W1242C, W1242F, and W1242Y) MRP3 cDNA expression. Login to comment 171 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr C: membrane vesicles from cells expressing wild-type and W1242A, W1242C, W1242F, and W1242Y mutant MRP3 were incubated at 37°C with 400 nM [3 H]E217betaG in transport buffer and other components for 3 min in the absence (open bars) or presence of taurocholic acid (50 ␮M, grey bar; 100 ␮M, solid bar). Login to comment 173 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr [3 H]leucovorin uptake by wild-type and Trp1242 mutant MRP3 proteins. A: uptake of [3 H]leucovorin was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector [pcDNA3.1(ϩ)], wild-type, and Trp1242 mutant (W1242A, W1242C, W1242F, and W1242Y) MRP3 cDNA expression vectors. Membrane vesicles were incubated at 37°C with 250 ␮M [3 H]leucovorin and ATP or AMP in transport buffer for 20 min. Login to comment 193 ABCC1 p.Trp1246Ala ABCC2 p.Trp1254Ala ABCC3 p.Trp1242Ala Effects of nonconservative (Ala) and conservative (Tyr) substitutions of MRP1-Trp1246 , MRP2-Trp1254 , and MRP3-Trp1242 on transport activity of common substrates Transporter Substitution %Wild-type MRP Transport Activity LTC4 E217betaG MTX MRP1-Trp1246 Ala 100 Ͻ10 Ͻ10* Tyr 100 Ͻ10 10* MRP2-Trp1254 Ala Ͻ10 Ͻ10 14 Tyr 30 100 1 MRP3-Trp1242 Ala 70 250 20 Tyr 65 700 20 Data are from Ito et al. (17, 18) and the present study, with exceptions (*) noted (I. Letouneau, C. J. Oleschuk, R. G. Deeley, and S. P. C. Cole, unpublished observations). Login to comment 196 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Tyr Membrane vesicles from cells expressing wild-type MRP3 (WT-MRP3) and W1242A, W1242C, W1242F, and W1242Y mutant MRP3 were incubated at 37°C with 400 nM [3 H]E217betaG in transport buffer and other components for 3 min in the absence (open bars) or presence of glycocholate (50 ␮M, grey bar; 100 ␮M, solid bar). Login to comment 199 ABCC3 p.Trp1242Phe ABCC3 p.Trp1242Cys ABCC3 p.Trp1242Ala ABCC3 p.Trp1242Pro ABCC3 p.Trp1242Tyr Transport activities of variously substituted MRP3-Trp1242 mutants Substrate Transport Activity W1242A W1242C W1242F W1242Y W1242P E217betaG 1 1 1 11 222 LTC4 2 22 2 2 222 MTX 222 222 222 222 n.d. Login to comment 204 ABCC3 p.Trp1242Tyr However, the highest level of E217betaG uptake was observed with W1242Y-MRP3, which is the most conservatively substituted mutant with respect to steric bulk, aromaticity, and H-bonding capability of the lateral side chain. Login to comment 217 ABCC3 p.Trp1242Pro The loss of H-bonding and aromatic stacking interactions alone cannot fully account for the inactivity of the W1242P-MRP3 mutant, because the Ala, Cys, Phe, and Tyr mutants all retained some transport activity. Login to comment 229 ABCC3 p.Trp1242Tyr However, if they do, they must do so in a somewhat different way for leucovorin than for MTX, which may account for the difference in affinities of these compounds for MRP3 as well as for the ability of the W1242Y-MRP3 mutant to transport the former but not the latter drug. Login to comment