ABCMdb

PMID: 21177244

Iram SH, Cole SP
Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2.
J Biol Chem. 2011 Mar 4;286(9):7202-13. Epub 2010 Dec 20., 2011-03-04 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC1 p.Lys513Ala Ala substitution of Lys513 , Lys516 , Glu521 , and Glu535 markedly reduced MRP1 levels. Login to comment 8 ABCC1 p.Lys503Ala Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Login to comment 9 ABCC1 p.Gly511Ile Studies with 32 P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1. Login to comment 47 ABCC1 p.Lys513Ala ABCC1 p.Lys503Ala ABCC1 p.Gly511Ile ABCC1 p.Glu535Asp ABCC1 p.Arg532Ala ABCC1 p.Lys516Arg ABCC1 p.Lys513Arg ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala To generate point mutations in CL5, plasmid pBluescriptSK(ϩ)BamHI/SpHI-MRP1 containing a 1.9-kb fragment from pcDNA3.1(-)-MRP1k was used as the template with the following mutagenic primers (substituted nucleotides are underlined): R501A, 5Ј-GAG CAA AGA CAA TGC GAT CAA GCT GAT G-3Ј; K503A, 5Ј-GAC AAT CGG ATC GCG CTG ATG AAC G-3Ј; E507A, 5Ј-GCT GAT GAA CGC AAT TCT CAA TGG G-3Ј; G511I, 5Ј-CTC AAT ATT ATC AAA GTG CTA AAG CTT TAT GCC TGG GAG CTG GC-3Ј; K513A, 5Ј-CTC AAT GGG ATC GCA GTG CTA AAG C-3Ј; K513R, 5Ј-CTC AAT GGG ATC AGA GTG CTA AAG C-3Ј; K516A, 5Ј-GAT CAA AGT GCT AGC GCT TTA TGC CTG-3Ј; K516R, 5Ј-GAT CAA AGT GCT AAG ACT TTA TGC CTG-3Ј; E521A, 5Ј-CTT TAT GCC TGG GCG CTG GCA TTC-3Ј; E521D, 5Ј-CTT TAT GCC TGG GAC CTG GCA TTC-3Ј; R532A, 5Ј-GTG CTG GCC ATT GCG CAG GAG GAG CT-3Ј; E535A, 5Ј-CAT CAG GCA GGA GGC CTT GAA GGT GCT GAA G-3Ј; and E535D, 5Ј- CAG GCA GGA GGA TCT GAA GGT GCT GAA G-3Ј. Login to comment 49 ABCC1 p.His1049Ala To create the CL6 mutation H1049A, a 2-kb XmaI fragment (containing nucleotides 2337-4322) in pGEM-3Z (Promega, Madison, WI) was used as the template with the mutagenic primer 5Ј-CCG CTG TCT GGC CGT GGA CCT GC-3Ј. Login to comment 51 ABCC1 p.Arg1367Ala ABCC1 p.His1364Ala To engineer mutations of His1364 and Arg1367 in NBD2, a 0.8-kb EcoRI/KpnI fragment of MRP1 in pBluescriptSK(ϩ) was used as the template with the following mutagenic primers (substituted nucleotides are underlined): H1364A, 5Ј-GAT CGG CCT GGC CGA CCT CCG C-3Ј; and R1367A, 5Ј-CTG CAC GAC CTC GCC TTC AAG ATC AC-3Ј. Login to comment 110 ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Glu521Ala Densitometric analysis of immunoblots showed that the K516A, E521A, and E535A mutants were consistently expressed very poorly (levels Ͻ10% of wild-type MRP1), whereas the levels of K513A were reduced by Ͼ70% (Fig. 2). Login to comment 112 ABCC1 p.Lys503Ala ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala On the other hand, levels of the four remaining CL5 Ala-substituted mutants (R501A, K503A, E507A, and R532A) were comparable with wild-type MRP1 (see Fig. 6A). Login to comment 116 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Lys516Ala ABCC1 p.Glu521Ala ABCC1 p.Glu521Ala A marked increase in the levels of the K513A, K516A, E521A, and E535A mutants was observed in cells incubated at the lower temperature; however, most of this increase was attributable to the underglycosylated form of the MRP1. Furthermore, despite the increase, the levels of K513A, K516A, E521A, and E535A FIGURE 2. Login to comment 118 ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Glu521Ala Shown is a representative immunoblot of whole cell lysates (WCL) (10 ␮g of protein/lane) prepared from HEK293T cells transfected with wild-type (WT-MRP1) and mutant (K513A, K516A, E521A, and E535A) cDNA expression vectors. Untransfected cells were used as a negative control. MRP1 proteins were detected with mAb QCRL-1 (top), and the relative levels, estimated by densitometry, are indicated below the blot (after correction for loading based on ␣-tubulin levels detected using an anti-␣-tubulin mAb (bottom)). Login to comment 122 ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Glu521Ala A, immunoblots of whole cell lysates (WCL) (10 ␮g of protein/lane) prepared from HEK293T cells incubated at 28 °C for 60 h after transfection with mutant (K513A, K516A, E521A, and E535A) and wild-type MRP1 cDNA expression vectors. Login to comment 135 ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Glu521Ala Thus, at 37 °C the MRP1 signals from cells expressing the K513A, E521A, and E535A mutants were very faint; however, the signals were found mostly at the plasma membrane as observed for wild-type MRP1 (Fig. 3B). Login to comment 137 ABCC1 p.Lys516Ala In contrast, K516A was not detectable at all in the HEK cells at 37 °C, suggesting that the misfolding of this mutant is so severe that none of this mutant protein escapes the ERAD pathways of the cell. Login to comment 138 ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Glu521Ala On the other hand, when cells were incubated at 28 °C, the amount of mutant proteins increased (more so for K513A and K516A than for E521A and E535A) (Fig. 3, A and C). Login to comment 140 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Lys516Ala ABCC1 p.Glu521Ala ABCC1 p.Glu521Ala Although the "rescue" of K516A and K513A was greater than for E521A and E535A (Fig. 3A), the retention of K516A and K513A in the endoplasmic reticulum was more pronounced, whereas a small amount of E521A and E535A could be detected at the plasma membrane (Fig. 3C). Login to comment 141 ABCC1 p.Glu535Asp ABCC1 p.Lys516Arg ABCC1 p.Lys513Arg ABCC1 p.Glu521Asp Expression and Transport Activity of "Same Charge" Mutants of Lys513 , Lys516 , Glu521 , and Glu535 -To determine whether it was the charge or another property of the amino acid at positions 513, 516, 521, and 535 that was critical for efficient plasma membrane expression of MRP1, same charge mutants (K513R, K516R, E521D, and E535D) were created and again expressed in HEK cells. Login to comment 142 ABCC1 p.Glu535Asp ABCC1 p.Lys516Arg ABCC1 p.Lys513Arg ABCC1 p.Glu521Asp At 37 °C, levels of the K513R mutant were comparable with wild-type MRP1 levels, whereas levels of the K516R, E521D, and E535D mutants were somewhat reduced (20-40%) (Fig. 4A). Login to comment 148 ABCC1 p.Glu535Asp ABCC1 p.Lys516Arg ABCC1 p.Lys513Arg ABCC1 p.Glu521Asp A and B, representative immunoblots of whole cell lysates (WCL) (10 ␮g of protein/lane) (A) and membrane vesicles (1 ␮g of protein/lane) (B) prepared from HEK293T cells transfected with wild-type (WT-MRP1) and mutant (K513R, K516R, E521D, and E535D) MRP1 cDNA expression vectors. Untransfected cells were used as a negative control. MRP1 levels were detected with mAb QCRL-1, and the relative protein expression levels, estimated by densitometry, are shown below the blots. Login to comment 158 ABCC1 p.His1049Ala ABCC1 p.Arg1367Ala ABCC1 p.His1364Ala To test this hypothesis, mutants H1049A, H1364A, and R1367A were generated, and the levels of the mutant proteins in HEK293T cells determined as described above. Login to comment 159 ABCC1 p.Lys516Ala ABCC1 p.Arg1367Ala As observed for the CL5 mutant K516A, the NBD2 mutant R1367A was not detectable by immunoblot analysis (Fig. 5C). Login to comment 160 ABCC1 p.His1049Ala ABCC1 p.His1364Ala The levels of the NBD2 mutant H1364A and the CL6 mutant H1049A were also reduced by Ͼ50%. Login to comment 163 ABCC1 p.Arg501Ala Effect of Ala substitution of Arg501 , Lys503 , Glu507 , and Arg532 on MRP1 Transport Activity-As mentioned earlier, levels of the Ala-substituted mutants of Arg501 , Lys503 , Glu507 , and Arg532 were comparable with that of wild-type MRP1 (Fig. 6A), and the mutant proteins all routed correctly to the plasma membrane (results not shown). Login to comment 164 ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala When their transport properties were examined, however, a 50-60% decrease in the [3 H]LTC4 and [3 H]E217betaG transport levels of the R501A, E507A, and R532A mutants was observed (Fig. 6, B and C). Login to comment 165 ABCC1 p.Lys503Ala In contrast, the transport activity of K503A was comparable with wild-type MRP1, and therefore this mutant was not investigated further. Login to comment 166 ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala The reduced E217betaG transport activity of the R501A, E507A, and R532A mutants was analyzed further by determining the kinetic parameters of uptake; the results of these experiments are summarized in Table 1. Login to comment 168 ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala In contrast, the Km (E217betaG) values for the R501A, E507A, and R532A mutants were increased 3-5-fold (18-25 ␮M). Login to comment 171 ABCC1 p.Gly511Ile Effect of Mutation G511I on MRP1 Expression and Function-Analyses of our homology model of MRP1 also suggested that substitution of the sterically unencumbered Gly511 with a bulky beta-branched amino acid like Ile could affect the geometry and/or mobility of CL5. Login to comment 173 ABCC1 p.Gly511Ile ABCC1 p.Gly511Ile As shown in Fig. 7A, substitution of Gly511 with Ile had no effect on MRP1 protein levels; however, transport of LTC4 and E217betaG by the G511I mutant was decreased by ϳ60 and 75%, respectively (Fig. 7, B and C). Login to comment 175 ABCC1 p.Gly511Ile The reduced transport activity of the G511I mutant could be caused by a reduction in substrate affinity or transport capacity. Login to comment 176 ABCC1 p.Gly511Ile Because of the very low levels of E217betaG transport by G511I, the apparent affinity (Km) of this mutant for E217betaG could not be determined directly by kinetic analysis. Login to comment 178 ABCC1 p.His1049Ala ABCC1 p.Arg1367Ala ABCC1 p.His1364Ala Homology models and levels of MRP1 mutants H1049A, H1364A, and R1367A. Login to comment 183 ABCC1 p.Gly511Ile As shown in Fig. 7D, E217betaG inhibited [3 H]LTC4 uptake by G511I and wild-type MRP1 with IC50 values that differed by 5-fold (40 versus 8 ␮M, respectively). Login to comment 184 ABCC1 p.Gly511Ile The ability of LTC4 to inhibit [3 H]E217betaG uptake by the G511I mutant relative to wild-type MRP1 was also reduced, as reflected by an ϳ3-fold difference in the IC50 values (Fig. 7E). Login to comment 185 ABCC1 p.Gly511Ile These observations support the conclusion that decreased transport by the G511I mutant is caused by a reduction in substrate affinity. Login to comment 186 ABCC1 p.Gly511Ile ABCC1 p.Gly511Ile ABCC1 p.Arg532Ala ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala ABCC1 p.Arg501Ala Photolabeling of MRP1 Mutants R501A, E507A, R532A, and G511I with [3 H]LTC4-To determine whether the decrease in LTC4 uptake by the R501A, E507A, R532A, and G511I mutants was associated with decreased binding of this substrate to MRP1, membrane vesicles enriched for wild-type or mutant MRP1 were photolabeled with [3 H]LTC4. Login to comment 187 ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala As shown in Fig. 8A, [3 H]LTC4 labeling of the R501A and E507A mutants was decreased by 50%, whereas labeling of the R532A mutant was decreased by 70%. Login to comment 188 ABCC1 p.Gly511Ile Labeling of the G511I mutant by [3 H]LTC4 was also reduced by 40% (Fig. 8B) (after correcting for differences in protein levels). Login to comment 189 ABCC1 p.Gly511Ile These relative levels of photolabeling correlated well with the relative LTC4 transport activities of these mutants and, in the case of G511I, with the reduced affinity for this substrate suggested by the competition experiments (Fig. 7E). Login to comment 190 ABCC1 p.Gly511Ile ABCC1 p.Glu507Ala Photolabeling of Mutants E507A and G511I with 8-Azido- [␣-32 P]ATP and Orthovanadate- and BeF-induced Trapping of 8-Azido-[␣-32 P]ADP-The precise role of the CLs in coupling the ATPase activity of ABC transporters to the translocation of their substrates is not yet fully understood (12, 42). Login to comment 192 ABCC1 p.Gly511Ile ABCC1 p.Glu507Ala For this reason, the interactions of the transport-deficient E507A and G511I mutants with ATP were examined. Login to comment 195 ABCC1 p.Gly511Ile ABCC1 p.Glu507Ala Next, the catalytic activity of the E507A and G511I mutants was examined by measuring the amount of azido-[32 P]ADP trapped by orthovanadate under conditions permissive for ATP hydrolysis (26). Login to comment 196 ABCC1 p.Gly511Ile ABCC1 p.Glu507Ala As shown in Fig. 9B, [32 P]ADP trapping by the G511I mutant was reduced by 40%, whereas trapping by the E507A mutant was comparable to wild-type MRP1. Login to comment 199 ABCC1 p.Lys503Ala ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala Levels and organic anion transport activity of MRP1 mutant proteins R501A, K503A, E507A, and R532A. Login to comment 200 ABCC1 p.Lys503Ala ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala A, shown is a representative immunoblot of membrane vesicles (MV) (1 ␮g of protein/lane) prepared from HEK293T cells transfected with wild-type (WT-MRP1) and mutant (R501A, K503A, E507A, and R532A) cDNA expression vectors. Untransfected cells were used as a negative control. MRP1 was detected with mAb QCRL-1, and the relative protein expression levels were estimated by densitometry and are shown below the blot. Login to comment 204 ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala TABLE 1 Kinetic parameters of E217betaG uptake by MRP1 mutants R501A, E507A, and R532A Km and Vmax values for E217betaG uptake by membrane vesicles (4 ␮g of protein) prepared from HEK293T cells transfected with wild-type or mutant proteins were determined by measuring ATP-dependent uptake at eight different E217betaG concentrations (0.25-25 ␮M) for 1 min at 37 °C as described under "Experimental Procedures." Login to comment 207 ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala Transfectant Km Vmax a (Vmax/Km) ؋ 10-3 ␮M pmol/mg/min mg/liter/min WT-MRP1 5.4 1088 0.20 R501A 22.9 1274 0.05 E507A 18.1 1150 0.06 R532A 24.5 1031 0.04 a Vmax values have been corrected for differences in protein expression of the mutants relative to WT-MRP1 (Fig. 7A). Login to comment 210 ABCC1 p.Lys513Ala ABCC1 p.Lys503Ala ABCC1 p.Arg532Ala ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala ABCC1 p.Glu521Ala Of the charged amino acid mutants, only K503A exhibited properties comparable with those of wild-type MRP1, whereas the seven remaining Ala-substituted mutants were either poorly expressed (K513A, K516A, E521A, and E535A) or exhibited significantly reduced transport activity (R501A, E507A, and R532A). Login to comment 211 ABCC1 p.Gly511Ile LTC4 and E217betaG transport by the G511I mutant was also reduced, although their expression levels were comparable with wild-type MRP1. Together, these results illustrate the remarkable sensitivity of CL5 to mutation and thus establish its critical role in the expression and function of MRP1. Login to comment 213 ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Glu521Ala The substantially reduced levels of the K513A, K516A, E521A, and E535A mutants indicates that introducing a neutral cavity-creating Ala residue at these positions is not compatible with some aspect(s) of MRP1 biosynthesis or assembly, at least in HEK cells. When cells transfected with the FIGURE 7. Login to comment 214 ABCC1 p.Gly511Ile Protein expression, transport activity, and inhibition of [3 H]LTC4 and[3 H]E217betaG uptake by wild-type MRP1 and mutant G511I. Login to comment 215 ABCC1 p.Gly511Ile A, immunoblot of membrane vesicles (1 ␮g of protein/lane) prepared from HEK293T cells transfected with wild-type MRP1 and mutant G511I cDNA expression vectors. Login to comment 220 ABCC1 p.Gly511Ile D and E, membrane vesicles prepared from HEK293T cells transfected with wild-type (f, WT-MRP1) and mutant (Œ, G511I) MRP1 cDNA expression vectors were incubated for 1 min with either [3 H]LTC4 at 23 °C in the presence of E217betaG (0, 12.5, 25, and 50 ␮M) (D) or [3 H]E217betaG at 37 °C in the presence of LTC4 (0, 300, 600, and 900 nM) (E). Login to comment 223 ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Lys516Ala ABCC1 p.Glu521Ala Role of Cytoplasmic Loop 5 in MRP1 Expression and Function 7210 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 9•MARCH 4, poorly expressed mutant MRP1 cDNA expression vectors were incubated at a subphysiological temperature, marked increases in the levels of the K513A, K516A, E521A, and E535A mutants were observed, although they remained well below those of wild-type MRP1 and consisted of both mature and underglycosylated forms of the transporter. Login to comment 227 ABCC1 p.His1364Ala This hypothesis was supported by our observations that MRP1 expression is retained when the three CL5 residues are replaced with same charge amino acids but is lost or reduced when either the NBD2 residue Arg1367 or His1364 is replaced with Ala. Login to comment 231 ABCC1 p.His1364Glu ABCC1 p.Glu521His In light of this finding, it is perhaps not surprising that MRP1 levels remained low in the reciprocal double exchange mutant E521H/H1364E (data not shown). Login to comment 233 ABCC1 p.Glu535Ala ABCC1 p.His1049Ala If such interdomain interactions exist and are important for the proper folding and assembly of MRP1, it was reasonable to expect that, as we observed for the Ala substitution of Glu535 , replacing His1049 with Ala would also affect the levels of MRP1. Login to comment 234 ABCC1 p.Glu535Ala ABCC1 p.His1049Ala Indeed, this was the case, although the reduction in E535A protein levels was significantly greater (Ͼ90%) than the reduction observed for H1049A (60%). Login to comment 236 ABCC1 p.Gly511Ile ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala [3 H]LTC4 photolabeling of wild-type and MRP1 mutants R501A, E507A, R532A, and G511I. Login to comment 240 ABCC1 p.Gly511Ile ABCC1 p.Glu507Ala Azido-[32 P]ATP labeling and vanadate-induced trapping of azido-[32 P]ADP by E507A and G511I mutant MRP1 proteins. Login to comment 251 ABCC7 p.Arg258Gly ABCC7 p.Glu292Lys Similarly, a deletion mutation (⌬E278) and two missense mutations (R258G and E292K) in CFTR/ABCC7 that involve amino acids analogous to MRP1-Glu521 , -Arg501 , and -Glu535 , respectively, are associated with cystic fibrosis (48). Login to comment 253 ABCC1 p.Lys503Ala ABCC1 p.Arg532Ala ABCC1 p.Glu507Ala ABCC1 p.Arg501Ala Nevertheless, although the K503A mutant exhibited properties similar to wild-type MRP1, the LTC4 and E217betaG transport activities of R501A, E507A, and R532A were significantly reduced. Login to comment 263 ABCC1 p.Gly511Ile The fact that the G511I mutant was expressed at levels comparable with that of wild-type MRP1 suggests that even after the geometry of CL5 was altered, the integrity of its interface with NBD2 was not significantly altered. Login to comment 264 ABCC1 p.Gly511Ile Nevertheless, the G511I mutant showed reduced transport activity, which was associated with an apparent decrease in substrate binding, again implicating a direct or indirect role for this region in substrate recognition by MRP1. Login to comment 267 ABCC1 p.Gly511Ile ABCC1 p.Glu507Ala Although binding of azido-[32 P]ATP by the mutants was not affected, vanadate-induced trapping of azido-[32 P]ADP was moderately reduced for G511I but not for E507A. Login to comment 270 ABCC1 p.Glu507Leu ABCC1 p.Glu507Leu ABCC1 p.Gly511Pro ABCC1 p.Gly511Pro Relevant to our findings described here are the properties of a double MRP1 mutant containing a Leu substitution of Glu507 and a Pro substitution of Gly511 (E507L/ G511P) after expression in insect cells (reported by Ren et al. (49)). Login to comment 272 ABCC1 p.Gly511Ile ABCC1 p.Glu507Ala Nevertheless, these authors reported that the LTC4 transport activity of the double mutant was reduced by Ͼ80%, a larger decrease than we observed for either of the single E507A and G511I mutants. Login to comment