ABCMdb

PMID: 17494643

Letourneau IJ, Slot AJ, Deeley RG, Cole SP
Mutational analysis of a highly conserved proline residue in MRP1, MRP2, and MRP3 reveals a partially conserved function.
Drug Metab Dispos. 2007 Aug;35(8):1372-9. Epub 2007 May 9., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC1 p.Pro1150Ala We previously described a mutation in cytoplasmic loop 7 (CL7) of MRP1, Pro1150Ala, which reduced leukotriene C4 (LTC4) transport but increased 17beta-estradiol 17beta-D-glucuronide (E217betaG) and methotrexate (MTX) transport. Login to comment 2 ABCC1 p.Pro1150Ala Vanadate-induced trapping of [␣-32 P]8N3ADP by the Pro1150Ala mutant in the absence of substrate was also greatly reduced compared with wild-type MRP1 suggesting an uncoupling of ATP hydrolysis and transport activity. Login to comment 5 ABCC1 p.Pro1150Ala ABCC2 p.Pro1158Ala As observed for MRP1-Pro1150Ala, LTC4 transport by MRP2-Pro1158Ala was decreased. Login to comment 6 ABCC1 p.Pro1150Ala However, E217betaG and MTX transport was comparable with that of wild-type MRP2 rather than increased as was observed for MRP1-Pro1150Ala. Login to comment 8 ABCC1 p.Pro1150Ala MTX transport by MRP3-Pro1147Ala was also increased but to a lesser extent than for MRP1-Pro1150Ala. Login to comment 39 ABCC1 p.Pro1150Ala Compared with wild-type MRP1, MRP1-Pro1150Ala displayed decreased levels of LTC4, estrone sulfate, and GSH transport but substantially increased levels of E217betaG and methotrexate (MTX) transport. Login to comment 41 ABCC1 p.Pro1150Ala Although no difference in ATP binding was detected, the ability of MRP1-Pro1150Ala to trap 8N3ADP under hydrolytic conditions in the presence of sodium orthovanadate (but in the absence of substrate) was severely diminished. Login to comment 42 ABCC1 p.Pro1150Ala Because the ability to detect vanadate-inducible trapped [␣-32 P]8N3ADP complexes is often considered an indicator of the ATPase activity of a protein (Urbatsch et al., 1995), our observations suggested that either ATP hydrolysis by the MRP1-Pro1150Ala mutant was reduced or that the release of ADP may be enhanced (Koike et al., 2004). Login to comment 43 ABCC1 p.Pro1150Ala It was also observed that although the apparent Km(ATP) for both wild-type MRP1 and MRP1-Pro1150Ala was the same during LTC4 transport, the Km(ATP) for the mutant transporter was reduced more than 4-fold during E217betaG transport. Login to comment 44 ABCC1 p.Pro1150Ala This change in ATP dependence suggests that E217betaG, but not LTC4, interacts with the MRP1-Pro1150Ala mutant in a way that increases its apparent affinity for ATP. Login to comment 45 ABCC1 p.Pro1150Ala The complexity of the MRP1-Pro1150Ala mutant phenotype, together with the high conservation of this Pro residue among ABCC family members (Fig. 1), has prompted us to investigate whether the corresponding residues in the MRP1 homologs, MRP2 and MRP3, are also functionally important. Login to comment 57 ABCC1 p.Pro1150Ala Generation of the MRP1-Pro1150Ala mutant has been described previously (Koike et al., 2004). Login to comment 59 ABCC2 p.Pro1158Ala The template for generating the MRP2-Pro1158Ala mutant was created by subcloning a 2.2-kb ApaI/ClaI fragment into pGEM-7Zf(ϩ) (Promega, Madison, WI). Login to comment 62 ABCC2 p.Pro1158Ala Mutagenesis was performed according to the manufacturer`s instructions with the following sense primers (substituted nucleotides are underlined, and in both cases, the substitution created a BstUI restriction site, which was used to confirm the successful introduction of the nucleotide substitutions): MRP2-Pro1158Ala, 5Ј-C ACC AGG TCC GCG ATC TAC TCT C-3Ј (BstUI) and MRP3-Pro1147Ala, 5Ј-GTC AGC CGC TCC GCG ATC TAC TCC C-3Ј (BstUI). Login to comment 63 ABCC2 p.Pro1158Ala The MRP2-Pro1158Ala mutation was subcloned back into pcDNA3.1(-) MRP2 as a 1.75-kb Bsu36I/SfiI fragment and the MRP3-Pro1147Ala mutation was subcloned into pcDNA3.1(ϩ) MRP3 as a 1.24-kb AgeI/SacII fragment (Oleschuk et al., 2003). Login to comment 119 ABCC1 p.Pro1150Ala We showed previously that the MRP1-Pro1150Ala mutant was expressed in transfected HEK293T cells at levels comparable to those for wild-type MRP1 (Koike et al., 2004). Login to comment 120 ABCC2 p.Pro1158Ala To determine whether this expression was also true for the corresponding MRP2-Pro1158Ala and MRP3-Pro1147Ala mutants, these mutations were created in pcDNA3.1(Ϯ)-based MRP2 and MRP3 expression vectors and then transfected into HEK293T cells. Login to comment 122 ABCC1 p.Pro1150Ala ABCC2 p.Pro1158Ala As shown in Fig. 2, all three Pro to Ala mutants (MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala) were expressed at levels comparable to those of their corresponding wild-type proteins, confirming that the Pro residue at this position is not required for expression of the MRP proteins in the plasma membrane of mammalian cells. Login to comment 129 ABCC1 p.Pro1150Ala As we reported previously, LTC4 transport by MRP1-Pro1150Ala was decreased by approximately 50% (Fig. 3A) (Koike et al., 2004). Login to comment 130 ABCC2 p.Pro1158Ala For MRP2-Pro1158Ala, LTC4 transport was also decreased by ϳ50% whereas it was significantly increased (1.8-fold) for MRP3-Pro1147Ala (Fig. 3A). Login to comment 137 ABCC2 p.Pro1158Ala In contrast to LTC4 uptake, E217betaG transport by MRP2 and MRP3 was unaffected by the Pro1158Ala and Pro1147Ala mutations, respectively (Fig. 3B). Login to comment 138 ABCC1 p.Pro1150Ala On the other hand, E217betaG transport by MRP1-Pro1150Ala was significantly increased (2-fold) relative to wild-type MRP1 as expected (Fig. 3B) (Koike et al., 2004). Login to comment 139 ABCC1 p.Pro1150Ala Also as expected for MRP1-Pro1150Ala, MTX transport was significantly increased by approximately ϳ3-fold (Fig. 3C) (Koike et al., 2004). Login to comment 140 ABCC2 p.Pro1158Ala In contrast, MTX transport by the MRP2-Pro1158Ala mutant remained the same as that of wild-type MRP2 (Fig. 3C), whereas FIG. 2. Login to comment 141 ABCC1 p.Pro1150Ala ABCC2 p.Pro1158Ala Protein expression levels of mutants MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala and their corresponding wild-type proteins. Login to comment 148 ABCC1 p.Pro1150Ala ABCC2 p.Pro1158Ala Vesicular transport of 3 H-labeled organic anions by mutants MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala. Login to comment 158 ABCC1 p.Pro1150Ala MTX transport by the MRP3-Pro1147Ala mutant was increased relative to that of wild-type MRP3 but to a lesser extent than was observed for MRP1-Pro1150Ala (1.5-fold versus 3-fold) (Fig. 3C). Login to comment 160 ABCC2 p.Pro1158Ala To determine whether the reduced LTC4 transport by MRP2-Pro1158Ala was associated with reduced substrate binding, [3 H]LTC4 photolabeling of this mutant was compared with that of wild-type MRP2. Login to comment 162 ABCC1 p.Pro1150Ala As shown previously, [3 H]LTC4 photolabeling of MRP1 was not affected by the Pro1150Ala mutation (Fig. 4A) (Koike et al., 2004). Login to comment 163 ABCC2 p.Pro1158Ala Similarly, MRP2-Pro1158Ala was photolabeled by [3 H]LTC4 to the same extent as wild-type MRP2 despite the reduced LTC4 transport activity of the mutant (Fig. 4B). Login to comment 165 ABCC1 p.Pro1150Ala ABCC2 p.Pro1158Ala Mutations MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala Do Not Affect [␥-32 P]8N3ATP Binding but Decrease Vanadate-Induced Trapping of [␣-32 P]8N3ADP. Login to comment 166 ABCC1 p.Pro1150Ala We previously showed that photolabeling of MRP1-Pro1150Ala by [␣-32 P]8N3ATP was comparable to that of wild-type MRP1 (Koike et al., 2004). Login to comment 167 ABCC2 p.Pro1158Ala When MRP2-Pro1158Ala and MRP3-Pro1147Ala were photolabeled with [␥-32 P]8N3ATP, the intensity of the signals observed with the mutant and wild-type proteins was also comparable (Fig. 5), suggesting that ATP can bind to the Pro mutants as well as it does to their wild-type counterparts. Login to comment 169 ABCC1 p.Pro1150Ala As reported previously, when MRP1-Pro1150Ala was exposed to vanadate (1 mM) and [␣-32 P]8N3ATP under hydrolytic conditions (37°C), the trapping of [␣-32 P]8N3ADP was very low compared with that of wild-type MRP1 (Fig. 6A) (Koike et al., 2004). Login to comment 170 ABCC2 p.Pro1158Ala ABCC2 p.Pro1158Ala When the MRP2-Pro1158Ala and MRP3-Pro1147Ala mutants were examined under the same conditions, a significant decrease (50%) in the level of vanadate-induced 8N3ADP trapping by MRP2-Pro1158Ala was also observed (Fig. 6B), whereas no trapping of nucleotide was detected with either wild-type MRP3 or its Pro1147Ala mutant (data not shown). Login to comment 175 ABCC1 p.Pro1150Ala ABCC2 p.Pro1158Ala Thus, 8N3ADP trapping by the MRP3 mutant was decreased as observed with the MRP1-Pro1150Ala and MRP2-Pro1158Ala mutants, although under different conditions. Login to comment 186 ABCC1 p.Pro1150Ala The complex phenotype of the MRP1-Pro1150Ala mutant and the fact that Pro1150 is highly conserved in the ABCC subfamily suggested to us that this residue might serve an important functional role in other members of this class of ABC transporters (Koike et al., 2004). Login to comment 188 ABCC1 p.Pro1150Ala For this reason, the consequences of replacing the Pro residues in MRP2 and MRP3 corresponding to MRP1-Pro1150 with Ala on the transport and nucleotide binding properties of the mutant proteins were examined. Login to comment 189 ABCC1 p.Pro1150Ala As described earlier, the MRP1-Pro1150Ala mutant showed substrate-selective changes in its transport activity (Koike et al., 2004). Login to comment 192 ABCC1 p.Pro1150Ala In contrast, the reduced LTC4 transport by MRP1-Pro1150Ala was associated with a decreased Vmax for LTC4 transport, whereas no change in Km(ATP) was observed (Koike et al., 2004). Login to comment 197 ABCC1 p.Pro1150Ala As for MRP1-Pro1150Ala, the transport activities of the MRP2 and MRP3 Pro mutants were altered in a substrate-selective manner, demonstrating the conserved functional importance of a Pro residue at the NH2-proximal end of CL7. Login to comment 208 ABCC1 p.Pro1150Ala ABCC2 p.Pro1158Ala Thus, although 8N3ATP binding to MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala remained unchanged, the level of vanadate-induced 8N3ADP trapping was very low for all three mutant transporters. Login to comment 221 ABCC1 p.Gly1161Pro ABCC1 p.Glu1157Leu Thus, the Glu1157Leu/ Gly1161Pro mutant displayed both decreased vanadate-induced 8N3ADP trapping as well as decreased 8N3ATP photolabeling at both NBDs (Ren et al., 2006). Login to comment 223 ABCC1 p.Gly1161Pro ABCC1 p.Glu1157Leu Unfortunately, the Glu1157Leu/Gly1161Pro mutant was not tested with other substrates, so it is not known whether the decreased transport activity of this mutant was substrate-selective. Login to comment