ABCMdb

PMID: 12924948

Zhang DW, Gu HM, Vasa M, Muredda M, Cole SP, Deeley RG
Characterization of the role of polar amino acid residues within predicted transmembrane helix 17 in determining the substrate specificity of multidrug resistance protein 3.
Biochemistry. 2003 Aug 26;42(33):9989-10000., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC3 p.Ser1229Ala Mutation S1229A reduced only methotrexate transport. Login to comment 6 ABCC3 p.Ser1231Ala ABCC3 p.Asn1241Ala Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E217βG and methotrexate but not taurocholate. Login to comment 7 ABCC3 p.Gln1235Ala Mutation Q1235A also reduced resistance to VP-16 and transport of E217βG but increased taurocholate transport without affecting transport of methotrexate. Login to comment 8 ABCC3 p.Tyr1232Phe ABCC3 p.Ser1233Ala Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. Login to comment 9 ABCC3 p.Thr1237Ala In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. Login to comment 75 ABCC3 p.Ser1229Ala ABCC3 p.Ser1231Ala ABCC3 p.Asn1241Ala ABCC3 p.Gln1235Ala ABCC3 p.Tyr1232Phe ABCC3 p.Ser1233Ala ABCC3 p.Thr1237Ala ABCC3 p.Thr1237Ser ABCC3 p.Thr1237Leu ABCC3 p.Thr1237Gly They are as follows: S1229A (5'-GGG CTG GTG GGG CTA GCT GTG TCC TAC TCC-3'), S1231A (5'-GC CTT TCT GTG GCC TAC TCC CTG CAG GTG ACA-3'), Y1232F (5'-T TCT GTG TCC TTC TCC TTA CAG GTG ACA TTT G-3'), S1233A (5'-CT GTG TCC TAC GCC CTG CAG GTG ACA TTT G-3'), Q1235A (5'-G TCC TAC TCC TTG GCG GTG ACA TTT GCT C-3'), T1237A (5'-CC TTG CAG GTG GCA TTC GCT CTG AAC TGG-3'), T1237S (5'-CC TTG CAG GTG TCC TTC GCT CTG AAC TGG-3'), T1237G (5'-CC TTG CAG GTG GGA TTC GCT CTG AAC TGG-3'), T1237L (5'-CC TTG CAG GTG CTA TTC GCT CTG AAC TGG-3'), and N1241A (5'-GTG ACA TTT GCG CTA GCC TGG ATG ATA C-3'). Login to comment 133 ABCC3 p.Asn1241Ala ABCC3 p.Tyr1232Phe To examine the functional importance of all remaining polar residues in TM17 of MRP3, we generated a series of seven mutant proteins in which Ser1229 , Ser1231 , Ser1233 , Gln1235 , Thr1237 , or Asn1241 was replaced with Ala and Tyr1232 was substituted with Phe (Figure 3). Login to comment 138 ABCC3 p.Ser1229Ala ABCC3 p.Ser1231Ala ABCC3 p.Asn1241Ala ABCC3 p.Gln1235Ala ABCC3 p.Tyr1232Phe ABCC3 p.Ser1233Ala Replacement of Ser1229 with Ala had no significant effect on MRP3-mediated E217βG uptake, whereas mutations S1231A, Y1232F, S1233A, Q1235A, and N1241A all decreased the transport activity. Login to comment 139 ABCC3 p.Thr1237Ala In contrast, conversion of Thr1237 to Ala resulted in a significant (~2.0-fold) increase in transport activity. Login to comment 141 ABCC3 p.Gln1235Ala Replacement of Gln1235 by Ala had no FIGURE 1: Resistance of stably transfected HEK293 cells to cisplatin (B), doxorubicin (C), vincristine (D), and VP-16 (E). Login to comment 146 ABCC3 p.Ser1229Ala ABCC3 p.Ser1231Ala ABCC3 p.Asn1241Ala ABCC3 p.Tyr1232Phe ABCC3 p.Ser1233Ala However, mutations S1229A, S1231A, Y1232F, S1233A, and N1241A all reduced methotrexate transport activity. Login to comment 147 ABCC3 p.Thr1237Ala Similar to the results obtained with E217βG as a substrate, substitution of Thr1237 with Ala resulted in a mutant protein that displayed an enhanced transport activity of the drug. Login to comment 149 ABCC3 p.Ser1229Ala ABCC3 p.Ser1231Ala ABCC3 p.Asn1241Ala Mutations S1229A, S1231A, and N1241A did not significantly influence transport activity. Login to comment 150 ABCC3 p.Tyr1232Phe ABCC3 p.Ser1233Ala However, mutations Y1232F and S1233A both decreased taurocholate transport activity by approximately 30-50%. Login to comment 151 ABCC3 p.Gln1235Ala ABCC3 p.Thr1237Ala In contrast, mutations Q1235A and T1237A increased the ability of MRP3 to transport the bile salt approximately 1.5-and 3-fold, respectively. Login to comment 154 ABCC3 p.Thr1237Ala We have shown that mutation T1237A increased the ability of MRP3 to transport both E217βG and taurocholate. Login to comment 155 ABCC3 p.Gln1235Ala ABCC3 p.Thr1237Ala Like mutation T1237A, replacement of Gln1235 with Ala also increased taurocholate uptake. Login to comment 160 ABCC3 p.Gln1235Ala ABCC3 p.Thr1237Ala The results suggest that mutations Q1235A and T1237A affect a step in the transport process after initial binding of this substrate. Login to comment 161 ABCC3 p.Gln1235Ala ABCC3 p.Thr1237Ala The effects of mutations Q1235A and T1237A on kinetic parameters of E217βG transport were also examined (Figure 7B, Table 1). Login to comment 162 ABCC3 p.Gln1235Ala Mutation Q1235A had no significant effect on the Km values (29 and 32 µM for wild-type MRP3 and mutant MRP1Q1235A , respectively) but decreased the Vmax FIGURE 2: Transport of taurocholate by MRP3. Panel A: Time course of ATP-dependent [3H]taurocholate uptake by membrane vesicles prepared from HEK293 stable transfectant expressing wild-type MRP3. Membrane vesicles were incubated at 37 °C with 4 µM [3H]taurocholate in transport buffer for the time indicated, as described. Login to comment 179 ABCC3 p.Gln1235Ala Thus, similar to the results obtained with taurocholate as a substrate, mutation Q1235A appeared not to influence the initial binding of the conjugated estrogen to the protein. Login to comment 180 ABCC3 p.Thr1237Ala Interestingly, converting Thr1237 to Ala decreased Km values for E217βG transport by approximately 5-fold (Km ) 6 µM for MRP1T1237A ). Login to comment 182 ABCC3 p.Thr1237Ala Thus, in contrast to the results obtained with taurocholate, these findings demonstrated that the increase in E217βG transport observed with the T1237A mutation at subsaturating concentrations was attributable to increased affinity of the protein for this substrate, which is associated with a decrease in Vmax. Login to comment 185 ABCC3 p.Ser1229Ala As observed with the effects of the mutations on E217βG transport, substitution of Ser1229 with Ala did not influence the ability of MRP3 to confer VP-16 resistance. Login to comment 186 ABCC3 p.Ser1231Ala ABCC3 p.Asn1241Ala ABCC3 p.Gln1235Ala ABCC3 p.Tyr1232Phe ABCC3 p.Ser1233Ala However, mutation of five hydrophilic amino acid residues (S1231A, Y1232F, S1233A, Q1235A, and N1241A) caused approximately a 2-3-fold reduction of resistance to VP-16. Login to comment 187 ABCC3 p.Thr1237Ala Interestingly, conversion of Thr1237 to Ala resulted in a mutant protein with enhanced resistance to VP-16 (3-fold). Login to comment 188 ABCC3 p.Ser1229Ala ABCC3 p.Ser1231Ala ABCC3 p.Asn1241Ala ABCC3 p.Gln1235Ala ABCC3 p.Tyr1232Phe ABCC3 p.Ser1233Ala ABCC3 p.Thr1237Ala Thus, on the basis of the substrates tested in this study, mutations S1229A, S1231A, Q1235A, and N1241A affected substrate specificity of MRP3, whereas mutations Y1232F, S1233A, and T1237A influenced the overall activity of the protein. Login to comment 189 ABCC3 p.Thr1237Ser ABCC3 p.Thr1237Leu ABCC3 p.Thr1237Gly Effect of Mutations T1237G, T1237S, and T1237L on VP-16 Resistance. Login to comment 190 ABCC3 p.Thr1237Ala Since replacement of Thr1237 with Ala resulted in a mutant protein with an enhanced capacity to confer VP-16 resistance, we also mutated this residue to Gly, Ser, and Leu. Login to comment 193 ABCC3 p.Thr1237Ala ABCC3 p.Thr1237Gly As shown in Table 2, mutation T1237G, like T1237A, significantly FIGURE 4: ATP-dependent [3H]E217βG uptake by membrane vesicles prepared from HEK293 cells stably transfected with wild-type or mutant MRP3. Panel A: Relative protein expression levels of wild-type and mutant MRP3 proteins in stably transfected HEK293 cells. Login to comment 201 ABCC3 p.Thr1237Leu Substitution of Thr1237 with Leu only moderately enhanced resistance to VP-16. Login to comment 210 ABCC3 p.Thr1237Ser ABCC3 p.Thr1237Leu ABCC3 p.Thr1237Gly Effects of Mutations T1237G, T1237S, and T1237L on the Transport Profile of MRP3. Login to comment 211 ABCC3 p.Thr1237Ala In addition to the effect on VP-16 resistance, substitution of Thr1237 with Ala increased the capacity of MRP3 to transport methotrexate, E217βG, and taurocholate. Login to comment 212 ABCC3 p.Thr1237Ser ABCC3 p.Thr1237Leu ABCC3 p.Thr1237Gly Thus, the effects of the mutations T1237G, T1237S, and T1237L on the ability of MRP3 to transport these three substrates were also examined by in vitro transport assays (Figure 8B-D). Login to comment 213 ABCC3 p.Thr1237Ala ABCC3 p.Thr1237Gly Replacement of Thr1237 with Gly, like mutation T1237A, dramatically increased the ability of MRP3 to transport methotrexate (Figure 8B), E217βG (Figure 8C), and taurocholate (Figure 8D). Login to comment 248 ABCC3 p.Thr1237Ala In MRP3, elimination of the hydrogen-bonding capability of Tyr1232 and Ser1233 negatively affected all functions, while mutation of Thr1237 to Ala had a positive effect. Login to comment 277 ABCC3 p.Gln1235Ala Consistent with this possibility, mutation Q1235A increased and decreased the Vmax for transport of taurocholate and E217βG, respectively, without affecting the Km for either substrate. Login to comment 278 ABCC3 p.Thr1237Ser ABCC3 p.Thr1237Leu ABCC3 p.Thr1237Gly Although the majority of mutations that eliminated hydrogen-bonding potential had a negative effect on the activity FIGURE 8: Effect of mutations T1237S, T1237L, and T1237G on ATP-dependent [3H]methotrexate (panel B), [3H]E217βG (panel C), and [3H]taurocholate (panel D) uptake by wild-type MRP3. Login to comment 285 ABCC3 p.Gln1235Ala Mutation Q1235A caused a decrease in VP-16 resistance and E217βG transport, consistent with the involvement of hydrogen bonding in the interaction with these substrates, but the mutation increased taurocholate transport. Login to comment 288 ABCC3 p.Thr1237Ala ABCC3 p.Thr1237Gly In addition, conversion of polar residue Thr1237 to either Ala or Gly markedly increased the ability to confer VP-16 resistance and to transport E217βG, taurocholate, and methotrexate, while mutation to a bulkier and more hydrophobic Leu residue resulted in only a moderate increase in transport of all three substrates. Login to comment 289 ABCC3 p.Thr1237Ser The effect of replacement of Thr1237 with Ser was more complex. Login to comment 292 ABCC3 p.Thr1237Ala Interestingly, although mutation T1237A increased the ability of MRP3 to transport both taurocholate and E217βG, the mutation resulted in a major increase in Vmax for taurocholate uptake, without significantly affecting Km values. Login to comment 295 ABCC3 p.Thr1237Ala If so, the fact that the T1237A mutations increase the affinity of the protein for E217βG suggests that a single amino acid may contribute both to the high-affinity binding of one substrate and the low-affinity binding of another. Login to comment