ABCMdb

PMID: 18775981

Grant CE, Gao M, DeGorter MK, Cole SP, Deeley RG
Structural determinants of substrate specificity differences between human multidrug resistance protein (MRP) 1 (ABCC1) and MRP3 (ABCC3).
Drug Metab Dispos. 2008 Dec;36(12):2571-81. Epub 2008 Sep 5., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCC1 p.Tyr440Phe It is noteworthy that substitution of Tyr440 with Phe, as found in MRP3, reduced LTC4 and GSH-stimulated estrone-3-sulfate transport without affecting transport of other substrates tested. Login to comment 138 ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu The most dramatic reduction in transport was found for one double mutation located in the G1 region, Y440F/I441L, which reduced LTC4 transport to less than 20% of wild-type levels (Fig. 3B). Login to comment 140 ABCC1 p.Ala481Gly ABCC1 p.Val482Ala A second double mutation, A481G/V482A, located in the G2 region also reduced LTC4 transport by 6-fold, and in this case, E217betaG transport was reduced to approximately one third of wild-type MRP1 levels (data not shown). Login to comment 142 ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu With the exception of one triple mutation, in which substitution of M443 with L was introduced into the LTC4 transport-deficient Y440F/I441L double mutant, no combination of mutations tested reduced LTC4 transport by more than 60 to 70% (data not shown). Login to comment 143 ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Met443Leu The Y440F/I441L/M443L virtually eliminated LTC4 transport (Fig. 3B) but also reduced E217betaG transport by approximately 80% (Fig. 3C). Login to comment 144 ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Met443Leu Transport of LTC4, E217betaG, E13SO4, and MTX by Y440F, I441L, and M443L MRP1 Mutant Proteins. Login to comment 156 ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Met443Leu The Y440F and M443L mutations each independently decreased initial rates of LTC4 transport by approximately 60 and 50%, respectively, whereas the I441L mutation had little or no effect (Fig. 3B). Login to comment 157 ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Met443Leu In contrast, E217betaG transport was decreased approximately 50% by both the I441L and M443L mutations but only 20% by the Y440F mutation (Fig. 3C). Login to comment 158 ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Met443Leu All three mutations significantly decreased E13SO4 transport in the presence of 2 mM S-methyl GSH, with the Y440F, I441L, and M443L mutations reducing transport at 1 min by approximately 65, 50, and 90%, respectively (Fig. 3D). Login to comment 175 ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu Kinetic Parameters of [3 H]LTC4 Transport by Y440F and I441L Single and Double Mutants. Login to comment 176 ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu We also determined the effects of the Y440F and I441L single and double mutations on the Km and Vmax for LTC4 transport. Login to comment 177 ABCC1 p.Tyr440Phe The Km and Vmax values obtained for wild-type MRP1 were 72 nM and 37 pmol/mg/min, respectively, whereas the Km for the Y440F mutation was 328 nM and the normalized Vmax was 41 pmol/mg/min (Fig. 4, A and B). Login to comment 179 ABCC1 p.Ile441Leu ABCC1 p.Ile441Leu ABCC1 p.Ile441Leu Although LTC4 transport by the I441L mutant was only marginally decreased at a fixed concentration of substrate, linear regression analysis indicated that the Km for LTC4 was increased approximately 2-fold (149 nM for the I441L mutant protein versus 72 nM for wild-type MRP1), again with no significant change in Vmax (normalized Vmax for the I441L mutation 42 versus 37 pmol/mg/min for the wild-type protein) (Fig. 4, A and B). Login to comment 193 ABCC1 p.Tyr440Phe ABCC1 p.Ala481Gly ABCC1 p.Ala481Gly ABCC1 p.Ala481Gly ABCC1 p.Ala481Gly ABCC1 p.Val482Ala ABCC1 p.Val482Ala ABCC1 p.Val482Ala ABCC1 p.Val482Ala ABCC1 p.Met443Leu ABCC1 p.Tyr490Phe ABCC1 p.Asn500Ser ABCC1 p.Asn500Ser ABCC1 p.Asn500Ser ABCC1 p.Met485Val ABCC1 p.Met485Val ABCC1 p.Met485Val ABCC1 p.Val479Leu ABCC1 p.Val479Leu ABCC1 p.Val479Leu ABCC1 p.Ser497Leu ABCC1 p.Ser497Leu ABCC1 p.Ser497Leu ABCC1 p.Thr487Met ABCC1 p.Met483Val ABCC1 p.Met483Val ABCC1 p.Met483Val ABCC1 p.Met483Val ABCC1 p.Asn506Ser ABCC1 p.Asn506Ser ABCC1 p.His494Gln ABCC1 p.His494Gln ABCC1 p.His494Gln ABCC1 p.Ala493Lys ABCC1 p.Ala493Lys ABCC1 p.Ala493Lys ABCC1 p.Lys488Arg ABCC1 p.Thr489Ala Mutations Included 23 M443L/Y440F/1441L 24 A481G/V482A/M483V/M485V 25 V479L/A481G/V482A/M483V/M485V 26 A493K/H494Q/S497L/N500S 27 V479L/A481G/V482A/M483V/M485V 28 V479L/A481G/V482A/M483V/N500S 29 A493K/H494Q/S497L/N500S/N506S 30 A493K/H494Q/S497L/N506S/T487M/K488R/T489A/Y490F consequence, it was technically impossible to determine an accurate Km for LTC4. Login to comment 195 ABCC1 p.Tyr440Phe To confirm whether the increase in Km resulting from the Y440F mutation reflected a decreased affinity for substrate, we examined the effect of the mutation on photolabeling of MRP1 with [3 H]LTC4. Login to comment 200 ABCC1 p.Ile441Leu Photolabeling of both fragments of the I441L mutant protein, which displayed only a 2-fold increase in Km, was essentially indistinguishable from that obtained with the wild-type MRP1 protein. Login to comment 201 ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Ile441Leu However, photolabeling of the NH2-terminal fragments of both the Y440F and double Y440F/I441L mutant proteins was similarly, substantially reduced compared with both the wild-type and the I441L mutant. Login to comment 203 ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Ile441Leu Furthermore, photolabeling of the COOH-terminal fragment of the Y440F and Y440F/I441L mutant proteins was also reduced when compared with wild-type MRP1 or the I441L mutant, despite the fact that this region is identical in all four proteins. Login to comment 209 ABCC1 p.Tyr440Phe All of these mutations had a greater effect on transport than the more conservative Y440F mutation. Login to comment 210 ABCC1 p.Tyr440Gln ABCC1 p.Tyr440Ser Transport by the two polar mutations Y440Q and Y440S was decreased by 75 to 80%, whereas the charged and neutral mutations decreased transport by more than 90% (Fig. 6A). Login to comment 213 ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Trp In addition, unlike the Y440F mutation, which had little effect on E217betaG transport, the Y440W mutation essentially eliminated transport of the conjugated estrogen (Fig. 6B). Login to comment 215 ABCC1 p.Tyr440Phe The Y440F mutation has a major deleterious effect on the transport of LTC4 and the S-methyl GSH-stimulated transport of E13SO4 (Fig. 3, B and D) but not on the other estrogen conjugate tested (E217betaG) or MTX (Fig. 3, C and E). Login to comment 216 ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Phe Because we have shown that the Y440F mutant protein has reduced affinity for LTC4 compared with wild-type MRP1 (Fig. 4B), it is possible that the Y440F mutation also affects the affinity for E13SO4 and/or S-methyl GSH. Login to comment 217 ABCC1 p.Tyr440Phe To test the former hypothesis, we attempted to determine a Km for the transport of E13SO4 by wild-type MRP1 and the Y440F mutant. Login to comment 222 ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Ile441Leu Shown are wild-type MRP1 (f), Y440F (Œ), I441L (), and Y440F/I441L (ࡗ). Login to comment 226 ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Ile441Leu [3 H]LTC4 photolabeling of membrane vesicle isolated from Sf21 cells expressing wild-type and mutant MRP1 proteins. A, immunoblot showing the relative amounts of MRP1 protein in each sample after adjustment for relative MRP1 protein expression; wild-type MRP1 (35 ␮g) and mutants Y440F (75 ␮g), I441L (22 ␮g), and Y440F/I441L (20 ␮g). Login to comment 233 ABCC1 p.Tyr440Phe To test whether the Y440F mutation alters the binding characteristics of S-methyl GSH and thus E13SO4 transport, we examined the binding of a GSH analog, azidophenacyl-GSH, to wild-type and mutant MRP1 expressed in stably transfected HEK cells. Login to comment 235 ABCC1 p.Met443Leu The M443L mutation reduced S-methyl GSH-stimulated E13SO4 transport by 90% in Sf21 cells (Fig. 3D). Login to comment 237 ABCC1 p.Met443Leu Thus, the affinity for azidophenacyl-GSH is severely affected by the M443L mutation. Login to comment 238 ABCC1 p.Ile441Leu ABCC1 p.Ile441Leu In contrast, the I441L mutant protein, which decreased S-methyl GSH-stimulated transport of E13SO4 by 55% compared with wild-type MRP1 (Fig. 3D), bound approximately equivalent levels of azidophenacyl-[35 S]GSH (Fig. 7C), suggesting that, as was determined for LTC4, the affinity for azidophenacyl-GSH is relatively unaffected by the I441L mutation. Login to comment 239 ABCC1 p.Tyr440Phe In contrast, the Y440F mutation, which reduced S-methyl GSH-stimulated transport of E13SO4 by approximately 65% (Fig. 3D), markedly decreased photolabeling with azidophenacyl-[35 S]GSH (Fig. 7C). Login to comment 240 ABCC1 p.Tyr440Phe This suggests that the affinity for this GSH derivative is dramatically affected by the Y440F mutation, as was found for LTC4. Login to comment 241 ABCC1 p.Tyr440Phe The reduced affinity for both azidophenacyl GSH and LTC4 suggests that the Y440F mutation may alter the interaction of MRP1 with the GSH moiety of both compounds and thus may decrease E13SO4 transport by reducing the affinity for GSH and S-methyl GSH. Login to comment 242 ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Ile441Leu ABCC1 p.Met443Leu Effect of the Y440F, I441L, M443L, and Y440F/I441L Mutations on Resistance to Vincristine, Doxorubicin, and VP-16. Login to comment 243 ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Ile441Leu ABCC1 p.Met443Leu Lastly, the drug-resistance profiles of Y440F, I441L, M443L, and Y440F/I441L mutant proteins were examined because unlike MRP1, the profile of resistance to natural product drugs conferred by MRP3 is restricted primarily to epipodophyllotoxins (Deeley et al., 2006). Login to comment 260 ABCC1 p.Tyr440Phe The three single mutations each decreased resistance to vincristine and VP-16, 2-to 3-fold, whereas only the Y440F mutation resulted in a major decrease in resistance to doxorubicin. Login to comment 261 ABCC1 p.Tyr440Phe The effect of the double Y440F/I441I mutation seemed to be additive with respect to both VP-16 and doxorubicin resistance but resulted in no greater decrease in resistance to vincristine than either mutation alone. Login to comment 280 ABCC1 p.Tyr440Phe It is noteworthy that conservative substitution of Tyr440 with Phe as present in MRP3 reduced LTC4 transport by ϳ60% with little effect on transport of E217betaG. Login to comment 282 ABCC1 p.Tyr440Phe The Y440F mutation resulted in a significant decrease in the apparent affinity for LTC4 (4-5-fold increase in Km), whereas Vmax was not affected. Login to comment 283 ABCC1 p.Tyr440Phe In addition, photolabeling of the Y440F protein by [3 H]LTC4 was markedly decreased. Login to comment 284 ABCC1 p.Tyr440Phe Taken together, the data strongly suggested that the primary defect in the Y440F protein was at the level of LTC4 binding. Login to comment 291 ABCC1 p.Tyr440Gln ABCC1 p.Tyr440Glu ABCC1 p.Tyr440Ala Other conservative (W) and nonconservative (A, E, Q, and S) substitutions of Tyr440 caused significant reductions in LTC4 transport ranging from 75% (Y440Q) to 90% (Y440A and Y440E). Login to comment 292 ABCC1 p.Tyr440Phe In particular, the relatively conservative substitution with Trp not only decreased LTC4 transport by ϳ75%, but unlike the Y440F mutation, also essentially eliminated transport of E217betaG. Login to comment 295 ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Phe ABCC1 p.Tyr440Phe ABCC1 p.Ile441Leu ABCC1 p.Met443Leu ABCC1 p.Met443Leu In contrast to the Y440F mutation, the conservatively substituted I441L mutation had no effect on LTC4 or MTX transport but decreased transport of both E217betaG and E13SO4, whereas the M443L mutation decreased transport of all three conjugated substrates but not TABLE 2 Relative Drug Resistance of HEK293 Cells Transfected with Wild-Type and Mutant MRP1 Transfectant Drug (Relative Resistance Factora ) Vincristine VP-16 Doxorubicin HEKMRP1 15.6 Ϯ 2.5 16.2 Ϯ 4.9 4.5 Ϯ 0.3 HEKMRP1-Y440F 2.4 Ϯ 0.5 (5.1) 2.7 Ϯ 0.8 (6.0) 1.3 Ϯ 0.2 (1.9) HEKMRP1-1441L 8.9 Ϯ 2.5 (9.7) 4.1 Ϯ 1.2 (4.4) 3.7 Ϯ 1.4 (4.1) HEKMRP1-M443L 6.5 Ϯ 1.2 (6.0) 5.8 Ϯ 0.5 (5.4) 3.8 Ϯ 0.7 (3.6) HEKMRP1-Y440F/1441L 3.9 Ϯ 1.0 (7.5) 1.3 Ϯ 0.3 (1.7) 1.2 Ϯ 0.2 (1.5) HEKMRP3 1.1 Ϯ 0.1 6.4 Ϯ 1.9 0.87 Ϯ 0.2 a The relative resistance factor was obtained by dividing the IC50 values for the wild-type or mutant MRP1-transfected cells by the IC50 value for cells transfected with the expression vector alone. Login to comment 303 ABCC1 p.Lys332Arg ABCC1 p.Tyr440Phe Most significantly, the conservative substitution of Lys332 by Arg increased the Km for LTC4 ϳ5-fold without affecting the Vmax, as is the case with the Y440F mutation. Login to comment 305 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu In addition, photolabeling with [3 H]LTC4 of K332D and K332L mutant proteins was severely reduced compared with wild-type MRP1. Login to comment 307 ABCC1 p.Tyr440Phe The Y440F mutation had little effect on either E217betaG or MTX transport but markedly decreased S-methyl GSH-stimulated E13SO4 transport, and photolabeling with the GSH analog azidophenacyl- [35 S]GSH was severely reduced. Login to comment 309 ABCC1 p.Tyr440Phe However, it is clear that the Y440F mutation almost entirely eliminates binding of azidophenacyl-GSH, as well as LTC4. Login to comment 311 ABCC1 p.Tyr440Phe Consistent with this suggestion, the Y440F mutation resulted in a major decrease in resistance to all three classes of drugs, transport of, or resistance to which, has been shown to be GSH-dependent (Loe et al., 1996b, 1998; Rappa et al., 1997; Renes et al., 1999). Login to comment