ABCMdb

PMID: 18336795

Letourneau IJ, Nakajima A, Deeley RG, Cole SP
Role of proline 1150 in functional interactions between the membrane spanning domains and nucleotide binding domains of the MRP1 (ABCC1) transporter.
Biochem Pharmacol. 2008 Apr 15;75(8):1659-69. Epub 2008 Feb 5., 2008-04-15 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC1 p.Pro1150Ala We previously described a mutant, MRP1-Pro1150Ala, which exhibits selectively increased estradiol glucuronide (E217bG) and methotrexate transport as well as altered interactions with ATP. Login to comment 6 ABCC1 p.Pro1150Ala All four mutants exhibited a phenotype similar to MRP1-Pro1150Ala with respect to organic anion transport and [g32 P]8N3ATP photolabeling. Login to comment 10 ABCC1 p.Pro1150Ala [a32 P]8N3ADP trapping by MRP1-Pro1150Ala could be increased by using Ni2+ instead of Mg2+ , and by decreasing temperature; however, the transport properties of the mutant remained unchanged. Login to comment 27 ABCC1 p.Pro1150Ala Compared to wild-type MRP1, the Pro1150Ala mutant displayed decreased levels of LTC4, estrone sulfate, and GSH transport but substantially increased levels of E217bG and methotrexate (MTX) transport. Login to comment 28 ABCC1 p.Pro1150Ala The increased E217bG transport by MRP1-Pro1150Ala was associated with a 5-fold decrease in apparent Km(E217bG) and 4-fold decrease in Km(ATP) during E217bG transport. Login to comment 29 ABCC1 p.Pro1150Ala However, the apparent Km(ATP) values for wild-type MRP1 and MRP1-Pro1150Ala were the same during LTC4 transport. Login to comment 30 ABCC1 p.Pro1150Ala The interaction of the MRP1-Pro1150Ala mutant with nucleotide was further investigated using the 32 P-labeled photoaffinity ligand 8N3ATP. Login to comment 32 ABCC1 p.Pro1150Ala In the case of MRP1-Pro1150Ala, vanadate-induced trapping of [a32 P]8N3ADP was greatly diminished relative to wild-type MRP1, while photolabeling of this mutant with [g32 P]8N3ATP under nonhydrolytic conditions was unchanged [11]. Login to comment 49 ABCC1 p.Pro1150Ala Site-directed mutagenesis The generation of the MRP1-Pro1150Ala mutant expression construct has been described previously [11]. Login to comment 52 ABCC1 p.Pro1150Leu ABCC1 p.Pro1150Gly ABCC1 p.Pro1150Val ABCC1 p.Pro1150Ile Mutagenesis was performed according to the manufacturer`s instructions with the following sense primers (the substituted nucleotides causing the mutation are underlined; silent nucleotide substitutions added to introduce or disrupt a restriction site are in bold; other nucleotide substitutions are in lowercase typeface; and diagnostic restriction enzymes are indicated in parentheses): MRP1-Pro1150Gly (50 -G TCG GTC AGC CGG TCg GGG GTC TAT TCC C-30 ) (BsrFI), MRP1-Pro1150Ile (50 -C AGC CGC TCC ATC GTC TAC TCC CAT TTC AAC-30 ) (AccI), MRP1- Pro1150Leu (50 -CG GTC AGC CGG TCC CTC GTC TAC TCC CAT TTC-30 ) (AccI) and MRP1-Pro1150Val (50 -CG GTC AGC CGG TCC GTG GTC TAT TCC C-30 ) (RsrII). Login to comment 76 ABCC1 p.Pro1150Ala Statistical comparisons of the IC50 values for wild-type MRP1 and MRP1-Pro1150Ala were carried out using a paired Student t-test and considered significant when p < 0.05. Login to comment 95 ABCC1 p.Pro1150Ala Mutation of Pro1150 affects the trypsin sensitivity of MRP1 To determine whether the functional impact of the MRP1-Pro1150 mutation was associated with changes in MRP1 structure, limited trypsin digests of wild-type MRP1 and the MRP1-Pro1150Ala mutant were performed side-by-side and the fragments probed by immunoblotting with MAbs against the NH2-proximal (N1, N3) (MAb MRPr1) and COOH-proximal (C1, C2) (MAb MRPm6) halves of MRP1 (Fig. 1A). Login to comment 96 ABCC1 p.Pro1150Ala As shown in Fig. 1B and C, the Pro1150Ala mutant was more resistant to trypsin digestion than wild-type MRP1 as evident from the persistence of the full-length protein at high trypsin:protein ratios. Login to comment 102 ABCC1 p.Pro1150Ala Immunoblots of the membrane vesicles prepared after transient expression in HEK293T cells revealed that none of the four mutations affected levels of MRP1 expression relative to wild-type MRP1 b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 5 9 - 1 6 6 9 1661 as shown previously for the Pro1150Ala mutant (Fig. 2A) [11,13]. Login to comment 105 ABCC1 p.Pro1150Ala As shown in Fig. 2B-D, the transport properties of all four mutants were similar to that described previously for MRP1-Pro1150Ala [11,13], viz., the mutants exhibited an approximately 50% decrease in LTC4 transport (Fig. 2B), a 2-fold increase in E217bG transport (Fig. 2C), and a 3-fold increase in MTX transport (Fig. 2D). Login to comment 108 ABCC1 p.Pro1150Ala As observed previously, [3 H]LTC4 photolabeling of the Pro1150Ala mutant was similar to that of wild-type MRP1 (Fig. 3A) [11]. Login to comment 109 ABCC1 p.Pro1150Leu ABCC1 p.Pro1150Gly ABCC1 p.Pro1150Val ABCC1 p.Pro1150Ile Levels of [3 H]LTC4 labeling of the Pro1150Gly, Pro1150Ile, Pro1150Leu and Pro1150Val mutants were also comparable to wild-type MRP1 (Fig. 3A). Login to comment 110 ABCC1 p.Pro1150Ala We next examined whether the binding of other MRP1 substrates (e.g. E217bG and MTX) was affected by Ala substitution of Pro1150 . Login to comment 112 ABCC1 p.Pro1150Ala As shown in Fig. 3B, E217bG inhibited [3 H]LTC4 photolabeling of wild-type MRP1 and the MRP1-Pro1150Ala mutant in a comparable concentration dependent fashion. Login to comment 113 ABCC1 p.Pro1150Ala [3 H]LTC4 photolabeling of wild-type and Pro1150Ala mutant MRP1 was also similarly inhibited by MTX (Fig. 3C). Login to comment 114 ABCC1 p.Pro1150Ala These results indicate that binding of LTC4, E217bG and MTX to wild-type MRP1 and the MRP1-Pro1150Ala mutant is similar, and thus suggest that the changes observed in the transport of these substrates are not due to changes in their initial binding to MRP1. Login to comment 115 ABCC1 p.Pro1150Ala Comparable experiments were not carried out for the other Pro1150 mutants because their transport activities were so similar to those of Pro1150Ala and accordingly, would be expected to exhibit a similar pattern of substrate competition. Login to comment 118 ABCC1 p.Pro1150Ala To test this, E217bG transport by the MRP1-Pro1150Ala mutant was examined in the presence of MK571, S-decyl-GSH, BAY u9773 and LY465803. Login to comment 121 ABCC1 p.Pro1150Ala As shown in Fig. 4, MK571, S-decyl-GSH, BAY u9773 and LY465803 inhibited MRP1-mediated E217bG transport with IC50 values Fig. 1 - Limited trypsin digestion of wild-type MRP1 and Pro1150Ala mutant MRP1. Login to comment 127 ABCC1 p.Pro1150Ala b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 5 9 - 1 6 6 91662 which were similar for both wild-type MRP1 and the MRP1-Pro1150Ala mutant ( p > 0.05). Login to comment 128 ABCC1 p.Pro1150Ala The effect of the four modulators on E217bG transport of the other Pro1150 mutants was not tested because their transport activities were so similar to those of Pro1150Ala that it would be reasonable to expect them to exhibit similar patterns of inhibitor sensitivity. Login to comment 130 ABCC1 p.Pro1150Ala Pro1150 mutants interact similarly with 32 P-labeled nucleotide We have reported previously that MRP1-Pro1150Ala and wild-type MRP1 bind similar levels of 8N3ATP but vanadate-induced trapping of 8N3ADP by the mutant is greatly reduced [11]. Login to comment 144 ABCC1 p.Pro1150Leu ABCC1 p.Pro1150Gly ABCC1 p.Pro1150Ile b i o c h e m i c a l p h a r m a c o l o g y 5 ( 2 0 0 8 ) 1 6 5 9 - 1 6 6 9 1663 (5 mM) to MRP1 was not affected by mutation of Pro1150 to Gly, Ile, Leu or Val. Login to comment 145 ABCC1 p.Pro1150Ala Even when photolabeling was carried out with a broader range of [g32 P]8N3ATP concentrations, no differences in photolabeling of the Pro1150Ala mutant and wild-type MRP1 were observed (results not shown). Login to comment 146 ABCC1 p.Pro1150Ala When nucleotide binding was determined under hydrolysis conditions (37 8C) and in the presence of vanadate, the Pro1150Ala mutant showed a substantial reduction (approximately 70%) in levels of trapped [a32 P]8N3ADP as expected [11]. Login to comment 147 ABCC1 p.Pro1150Leu ABCC1 p.Pro1150Gly ABCC1 p.Pro1150Val ABCC1 p.Pro1150Ile Under these conditions, the Pro1150Gly, Pro1150Ile and Pro1150Leu mutants exhibited similar levels of [a32 P]8N3ADP trapping (50-70% reduction) (Fig. 5B), while trapping by the Pro1150Val mutant was only slightly reduced (20%) relative to wild-type MRP1 (Fig. 5B). Login to comment 149 ABCC1 p.Pro1150Ala However, as observed when ATP was used, E217bG uptake by MRP1-Pro1150Ala in the presence of 8N3ATP was still 2-fold higher than uptake by wild-type MRP1 (results not shown). Login to comment 150 ABCC1 p.Pro1150Ala To exclude the possibility that diminished interaction between the mutant transporter and vanadate was responsible for the decreased [a32 P]8N3ADP trapping observed, the effect of different concentrations of vanadate on E217bG transport by the Pro1150Ala mutant was compared with wild-type MRP1. Login to comment 151 ABCC1 p.Pro1150Ala The IC50 for vanadate-mediated inhibition of E217bG transport was $0.5 mM for wild-type MRP1 and $1 mM for the Pro1150Ala mutant (results not shown). Login to comment 154 ABCC1 p.Pro1150Ala E217bG decreases vanadate-induced [a32 P]8N3ADP trapping by wild-type and Pro1150Ala mutant MRP1 It is generally believed that the presence of substrates can stimulate the ATPase activity of ABC transporters and can thereby enhance levels of vanadate-induced trapping of ADP. Login to comment 156 ABCC1 p.Pro1150Ala Because E217bG transport by the MRP1-Pro1150Ala mutant was substantially increased, it was of interest to determine if ADP trapping by the wild-type and mutant proteins might be differentially influenced by this substrate. Login to comment 157 ABCC1 p.Pro1150Ala ABCC1 p.Pro1150Ala Consequently, [a32 P]8N3ADP trapping experiments with the Pro1150Ala mutant were repeated in the presence of Fig. 4 - Effect of MRP1 modulators on E217bG transport by wild-type MRP1 and MRP1-Pro1150Ala. Login to comment 160 ABCC1 p.Pro1150Ala Open symbols represent data obtained with vesicles prepared from cells expressing wild-type MRP1 and filled symbols represent data obtained with vesicles prepared from cells expressing the MRP1-Pro1150Ala mutant. Login to comment 172 ABCC1 p.Pro1150Ala Trapping of [a32 P]8N3ADP by the MRP1-Pro1150Ala mutant was also decreased in the presence of E217bG. Login to comment 175 ABCC1 p.Pro1150Ala ABCC1 p.Pro1150Ala Release of 8N3ADP from MRP1-Pro1150Ala is enhanced relative to wild-type MRP1 While the decreased trapping of 8N3ADP by MRP1-Pro1150Ala could be due to reduced ATP hydrolysis, it might also be due to enhanced release of the dinucleotide. Login to comment 178 ABCC1 p.Pro1150Ala At 23 8C, lower levels of vanadate-trapped [a32 P]8N3ADP by wild-type MRP1 were observed; however, at 37 8C, the amount of [a32 P]8N3ADP trapped by the Pro1150Ala mutant was 40% that of wild-type MRP1 while at 23 8C, the amount trapped increased to 90% of wild-type MRP1. Login to comment 179 ABCC1 p.Pro1150Ala To determine if the increased trapping of [a32 P]8N3ADP at 23 8C by MRP1-Pro1150Ala was associated with any changes in its transport activity relative to wild-type MRP1, vesicular transport assays were performed at both 23 and 37 8C. Login to comment 180 ABCC1 p.Pro1150Ala As shown in Fig. 7B, E217bG transport by the Pro1150Ala mutant was 2-to 3-fold higher than wild-type MRP1 at both temperatures. Login to comment 181 ABCC1 p.Pro1150Ala Thus, no differences in relative transport activity were observed at 23 8C despite comparable levels of [a32 P]8N3ADP trapping (and implied ATP hydrolysis) by wild-type and Pro1150Ala mutant MRP1 at this lower temperature. Login to comment 182 ABCC1 p.Pro1150Ala ABCC1 p.Pro1150Ala The possibility that reduced vanadate-induced trapping by MRP1-Pro1150Ala at 37 8C was due to differences in the ability of wild-type MRP1 and MRP1-Pro1150Ala mutant to interact with divalent metal cations was also explored. Login to comment 183 ABCC1 p.Pro1150Ala Thus, Mn2+ , Co2+ , Ni2+ , Zn2+ and Cd2+ were tested for their ability to support vanadate-induced trapping of [a32 P]8N3ADP by the wild-type and MRP1-Pro1150Ala mutant proteins, and compared to the physiological cation Mg2+ . Login to comment 187 ABCC1 p.Pro1150Ala With most divalent cations that supported [a32 P]8N3ADP trapping, the MRP1-Pro1150Ala mutant still trapped significantly less dinucleotide than wild-type MRP1 (!65% decrease). Login to comment 188 ABCC1 p.Pro1150Ala Interestingly, when Ni2+ was used, the difference in trapping levels between the mutant and wild-type MRP1 proteins was reduced, with the trapping signal for MRP1-Pro1150Ala increasing from approximately 40% of wild-type MRP1 in the presence of Mg2+ to approximately 70% in the presence of Ni2+ (Fig. 7A, bottom panel). Login to comment 189 ABCC1 p.Pro1150Ala E217bG vesicular uptake measured under these conditions was approximately 20-fold lower in the presence of Ni2+ compared to Mg2+ , but the >2-fold difference in E217bG transport activity between wild-type MRP1 and the MRP1-Pro1150Ala mutant was still observed (Fig. 7B). Login to comment 197 ABCC1 p.Pro1150Ala Thus, replacement of Pro1150 with alanine is a non-conservative substitution since the small hydrophobic side chain of alanine can support the formation of a-helices although it does not necessarily result in the straightening of the a-helix [26,28,29]. Login to comment 199 ABCC1 p.Pro1150Ala ABCC1 p.Pro1150Gly In contrast, substitution of Pro1150 with glycine may be considered a conservative substitution because glycine, like Fig. 6 - Effect of E217bG on vanadate-induced trapping of [a32 P]8N3ADP by wild-type and Pro1150Ala mutant MRP1. Login to comment 201 ABCC1 p.Pro1150Ala Relative levels of vanadate-induced [a32 P]8N3ADP trapping are indicated in italics and have been corrected where necessary to take into account differences in expression of the Pro1150Ala mutant relative to wild-type MRP1. Login to comment 204 ABCC1 p.Pro1150Ala Although the tryptic digestion pattern of the Pro1150Ala mutant differed from that of the wild-type protein, indicating that loss of Pro1150 does introduce some change in the structure of MRP1, we observed no differences in expression levels of the various MRP1-Pro1150 mutants. Login to comment 210 ABCC1 p.Pro1150Ala Similarly, inhibition of [3 H]LTC4 photolabeling by E217bG and MTX was comparable for the wild-type and MRP1-Pro1150Ala proteins. Login to comment 211 ABCC1 p.Pro1150Ala Therefore, it may be concluded that the substrate selective increases in E217bG and MTX transport activities are not due to substantial changes in initial Fig. 7 - Effects of temperature and divalent cations on vanadate-induced [a32 P]8N3ADP trapping and E217bG transport by wild-type and Pro1150Ala mutant MRP1. Login to comment 216 ABCC1 p.Pro1150Ala HEK (black bars), wild-type-MRP1 (open bars) and MRP1-Pro1150Ala (grey bars). Login to comment 220 ABCC1 p.Pro1150Ala Furthermore, despite the marked differences in their chemical structures, specificity and mode of inhibitory action, the unchanged IC50 values for the MRP1 modulators MK571, BAY u9773, S-decylGSH and LY465803 also suggest that recognition of these compounds is unaffected by the MRP1-Pro1150Ala mutation. Login to comment 221 ABCC1 p.Pro1150Val Although none of the amino acids could replace Pro1150 with respect to the substrate specificity of MRP1, it is interesting that MRP1-Pro1150Val exhibited a substantially smaller decrease in vanadate-induced [a32 P]8N3ADP trapping than the other mutants. Login to comment 229 ABCC1 p.Pro1150Ala In contrast to LTC4, however, we now find that E217bG decreases [a32 P]8N3ADP trapping by both wild-type MRP1 and the Pro1150Ala mutant. Login to comment 231 ABCC1 p.Pro1150Ala Trapping of [a32 P]8N3ADP by the MRP1-Pro1150Ala mutant was also decreased in the presence of E217bG. Login to comment 235 ABCC1 p.Pro1150Ala To explain the decreased vanadate-induced [a32 P]8N3ADP trapping by the MRP1-Pro1150Ala mutant, we hypothesized that rather than decreasing ATP hydrolysis, the mutation was enhancing the release of ADP following ATP hydrolysis, most of which is known to occur at NBD2 [9,33]. Login to comment 238 ABCC1 p.Pro1150Ala We found that by carrying out the reactions at 23 8C instead of 37 8C (which would presumably increase the occupancy time of 8N3ADP in NBD2), [a32 P]8N3ADP trapping by MRP1-Pro1150Ala could be increased to levels comparable to that of wild-type MRP1 (Fig. 7). Login to comment 244 ABCC1 p.Pro1150Ala Although Co2+ and Mn2+ supported vanadate-induced [a32 P]8N3ADP trapping by wild-type MRP1 to a similar or higher level than Ni2+ , they were unable to increase [a32 P]8N3ADP trapping by the MRP1-Pro1150Ala mutant to the same extent as Ni2+ (result not shown). Login to comment 246 ABCC1 p.Pro1150Ala Overall, our data clearly indicate that the changes in the transport activities of MRP1-Pro1150Ala and other Pro1150 mutants are not tightly linked to the reduced vanadate-induced 8N3ADP trapping observed. Login to comment