ABCMdb

PMID: 22542979

Wittgen HG, van den Heuvel JJ, Krieger E, Schaftenaar G, Russel FG, Koenderink JB
Phenylalanine 368 of multidrug resistance-associated protein 4 (MRP4/ABCC4) plays a crucial role in substrate-specific transport activity.
Biochem Pharmacol. 2012 Aug 1;84(3):366-73. Epub 2012 Apr 21., [PubMed]

Sentences

No. Mutations Sentence Comment

12 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp Substitution of Phe368 with Trp (F368W) induced a gain-of-function of E217bG transport and a loss-of-function of MTX transport, which could not be attributed to an altered substrate binding. Login to comment 13 ABCC4 p.Phe368Trp Moreover, we did not observe any modification in ATP or ADP handling for F368W. Login to comment 46 ABCC4 p.Arg998Lys ABCC4 p.Phe368Trp ABCC4 p.Trp995Phe ABCC4 p.Trp995Ala ABCC4 p.Phe368Tyr ABCC4 p.Arg998Leu ABCC4 p.Phe368Ala ABCC4 p.Arg998Ser ABCC4 p.Arg998Tyr ABCC4 p.Trp995Tyr Site-directed mutagenesis of MRP4 and generation of expression vectors and baculovirus The previously described Gateway entry vector containing the human MRP4 coding sequence [8] was used as a template for site-directed mutagenesis of the following amino acids: F368W, F368Y, F368A, W995F, W995Y, W995A, R998S, R998K, R998Y, and R998L. Login to comment 47 ABCC4 p.Arg998Lys ABCC4 p.Phe368Trp ABCC4 p.Trp995Phe ABCC4 p.Trp995Ala ABCC4 p.Phe368Tyr ABCC4 p.Arg998Leu ABCC4 p.Phe368Ala ABCC4 p.Arg998Ser ABCC4 p.Arg998Tyr ABCC4 p.Trp995Tyr Site-directed mutagenesis of MRP4 and generation of expression vectors and baculovirus The previously described Gateway entry vector containing the human MRP4 coding sequence [8] was used as a template for site-directed mutagenesis of the following amino acids: F368W, F368Y, F368A, W995F, W995Y, W995A, R998S, R998K, R998Y, and R998L. Login to comment 82 ABCC4 p.Phe368Trp ABCC4 p.Trp995Phe ABCC4 p.Phe368Tyr ABCC4 p.Trp995Tyr Kinetic analysis of MRP4 mutant proteins To determine the apparent Km and Vmax values of wild type and mutant MRP4 proteins F368W, F368Y, W995F, and W995Y, concentration curves were made for the different substrates. Login to comment 86 ABCC4 p.Phe368Trp For mutant F368W the Km value of ATP was determined by measuring the uptake rate of 200 mM E217bG in presence of different concentrations of ATP (up to 4 mM). Login to comment 89 ABCC4 p.Phe368Trp Trapping of ADP during E217bG transport using vanadate To test the effect of ADP trapping on E217bG transport via MRP4 and mutant F368W, the vesicular transport assay was performed as described in Section 2.6.1 for E217bG transport, using 100 mM E217bG in presence of different concentrations of vanadate. Login to comment 115 ABCC4 p.Trp995Ala ABCC4 p.Phe368Ala Substitution of Phe368 and Trp995 with Ala, and all amino acid substitutions of Arg998 almost completely disrupted transport activity for all substrates tested. Login to comment 116 ABCC4 p.Trp995Phe ABCC4 p.Trp995Tyr Transport via W995F and W995Y was not completely abolished, but the aromatic substitutions significantly decreased transport of all substrates compared to wild type MRP4. Login to comment 117 ABCC4 p.Phe368Trp ABCC4 p.Phe368Tyr Whereas amino acid substitutions of Trp995 and Arg998 diminished or completely abolished transport of all substrates, substitutions F368W and F368Y had dual effects on the transport function, which appeared to be substrate- and mutation-dependent. Login to comment 118 ABCC4 p.Phe368Tyr F368Y-mediated cGMP transport was increased to 160 Æ 15% (Fig. 3B), while transport of the other substrates was decreased by 20-50% compared to wild type MRP4 activity (Fig. 3A, C and D). Login to comment 119 ABCC4 p.Phe368Trp Moreover, F368W-mediated E217bG transport was increased to 250 Æ 11% of wild type MRP4 (Fig. 3A), whereas cGMP transport was drastically decreased to 24 Æ 2% (Fig. 3B), and MTX and folic acid transport was also reduced to 55-80% of wild type transport activity (Fig. 3C and D). Login to comment 121 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp ABCC4 p.Trp995Phe ABCC4 p.Trp995Phe ABCC4 p.Phe368Tyr ABCC4 p.Phe368Tyr ABCC4 p.Trp995Tyr ABCC4 p.Trp995Tyr Kinetic properties of MRP4 wild type and F368W, F368Y, W995F, and W995Y mutants To further explore the mechanism by which the amino acid substitutions affected MRP4 transport activity, we determined the apparent affinity (Km) and maximum transport capacity (Vmax) of wild type MRP4 and mutants F368W, F368Y, W995F and W995Y for E217bG, cGMP, MTX, and folic acid. Login to comment 122 ABCC4 p.Trp995Ala ABCC4 p.Phe368Ala As F368A, W995A, and all mutations of Arg998 completely disrupted the transport activity of MRP4, it was not possible to include these mutant proteins into the kinetic analysis. Login to comment 136 ABCC4 p.Phe368Trp Surprisingly, the significant gain of E217bG transport function by mutant F368W appeared to be completely due to a 3-fold increase in Vmax, whereas the Km was exactly the same as for wild type MRP4. Login to comment 137 ABCC4 p.Phe368Trp Surprisingly, the significant gain of E217bG transport function by mutant F368W appeared to be completely due to a 3-fold increase in Vmax, whereas the Km was exactly the same as for wild type MRP4. Login to comment 139 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp ATP and ADP handling of MRP4 wild type and F368W mutant We explored the possibility of a changed handling of ATP or ADP by F368W during E217bG transport. Login to comment 140 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp For this purpose, we investigated E217bG transport by mutant F368W and wild type MRP4 in presence of different concentrations of ATP, and the inhibitory effect of vanadate, which traps ADP to the NBDs. Login to comment 141 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp Fig. 5A shows the ATP-dependency of E217bG transport via MRP4 and F368W at a fixed E217bG concentration (200 mM). Login to comment 142 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp The Km values of the wild type and F368W mutant were similar (1.5 Æ; 0.4 mM and 1.7 Æ 0.7 mM, respectively). Login to comment 143 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp In addition, Fig. 5B shows that vanadate inhibited E217bG transport by MRP4 and F368W with comparable IC50 values (160 Æ 40 mM and 180 Æ 30 mM, respectively). Login to comment 144 ABCC4 p.Phe368Trp In addition, Fig. 5B shows that vanadate inhibited E217bG transport by MRP4 and F368W with comparable IC50 values (160 40 mM and 180 30 mM, respectively). Login to comment 147 ABCC4 p.Phe368Trp ABCC4 p.Trp995Phe ABCC4 p.Phe368Tyr ABCC4 p.Trp995Tyr Kinetics of ATP-dependent transport of different substrates into membrane vesicles from HEK293 cells containing wild type MRP4 (&) or MRP4 mutants F368W (*), F368Y (Â), W995F (5), and W995Y (~). Login to comment 148 ABCC4 p.Phe368Trp ABCC4 p.Trp995Phe ABCC4 p.Phe368Tyr ABCC4 p.Trp995Tyr Kinetics of ATP-dependent transport of different substrates into membrane vesicles from HEK293 cells containing wild type MRP4 (&) or MRP4 mutants F368W (*), F368Y (), W995F (5), and W995Y (~). Login to comment 153 ABCC4 p.Phe368Trp ABCC4 p.Trp995Phe ABCC4 p.Phe368Tyr ABCC4 p.Trp995Tyr Table 1 Kinetic characteristics of transport via MRP4 mutants F368W, F368Y, W995F, and W995Y in comparison to wild type MRP4. Login to comment 154 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp ABCC4 p.Trp995Phe ABCC4 p.Trp995Phe ABCC4 p.Phe368Tyr ABCC4 p.Phe368Tyr ABCC4 p.Trp995Tyr ABCC4 p.Trp995Tyr E217bG cGMP MTX Folic acid Km (mM) Vmax (%) Km (mM) Vmax (%) Km (mM) Vmax (%) Km (mM) Vmax (%) MRP4 17 Æ 2 99 Æ 3 630 Æ 67 99 Æ 5 170 Æ 30 97 Æ 6 250 Æ 73 100 Æ 13 F368W 17 Æ 3 310 Æ 14*** >2000 n.d. 160 Æ 36 53 Æ 4* 240 Æ 100 85 Æ 15 F368Y 31 Æ 3 130 Æ 5*** 480 Æ 63 110 Æ 7 260 Æ 89 130 Æ 17 300 Æ 182 42 Æ 12*** W995F 54 Æ 24** 42 Æ 7*** 360 Æ 46 21 Æ 1** 150 Æ 81 19 Æ 3*** 350 Æ 275 31 Æ 12*** W995Y 13 Æ 4 26 Æ 2*** 1800 Æ 1020 94 Æ 36 130 Æ 48 16 Æ 2*** 390 Æ 116 61 Æ 9** n.d. not determined: concentration of cGMP in the experiment was not high enough to accurately determine Vmax. Login to comment 155 ABCC4 p.Phe368Trp ABCC4 p.Trp995Phe ABCC4 p.Phe368Tyr ABCC4 p.Trp995Tyr E217bG cGMP MTX Folic acid Km (mM) Vmax (%) Km (mM) Vmax (%) Km (mM) Vmax (%) Km (mM) Vmax (%) MRP4 17 2 99 3 630 67 99 5 170 30 97 6 250 73 100 13 F368W 17 3 310 14*** >2000 n.d. 160 36 53 4* 240 100 85 15 F368Y 31 3 130 5*** 480 63 110 7 260 89 130 17 300 182 42 12*** W995F 54 24** 42 7*** 360 46 21 1** 150 81 19 3*** 350 275 31 12*** W995Y 13 4 26 2*** 1800 1020 94 36 130 48 16 2*** 390 116 61 9** n.d. not determined: concentration of cGMP in the experiment was not high enough to accurately determine Vmax. Login to comment 168 ABCC4 p.Phe368Trp We found substrate-dependent effects for the transport activity of mutant F368W that were not related to a change in apparent Km but due to an altered Vmax. Login to comment 171 ABCC4 p.Arg998Ala In a previous study we already showed that R998A disrupted transport of MTX and cGMP [8]. Login to comment 179 ABCC4 p.Trp995Phe ABCC4 p.Trp995Tyr The decreased transport of W995F and W995Y appeared to be mainly caused by a decreased Vmax, indicating a decreased catalytic turnover number of these MRP4 mutants. Login to comment 180 ABCC4 p.Trp995Tyr In addition, the increased apparent Km for cGMP (W995Y) indicates that binding of this substrate might have been affected (Fig. 4 and Table 1). Login to comment 186 ABCC2 p.Phe368Ala Mutation of Phe368 to Ala decreased transport of all substrates tested, whereas conservative substitutions with the aromatic Trp and Tyr residues caused substrate-dependent effects. Login to comment 189 ABCC4 p.Phe368Trp In the present study, we observed that the F368W mutation induced an increased transport rate of E217bG, whereas transport rates of cGMP and MTX were reduced. Login to comment 191 ABCC4 p.Phe368Trp However, F368W did not affect the Km values for MTX and E217bG transport, implicating it is not involved in initial binding of these substrates. Login to comment 193 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp Therefore, the effects of F368W on E217bG and MTX transport appeared to be caused by an oppositely directed change in Vmax, indicating that the transport rate of F368W is influenced in a substrate-dependent fashion. Login to comment 195 ABCC4 p.Phe368Trp E217bG transport activity of F368W and wild type MRP4 (Fig. 5A) revealed no effect Fig. 5. Login to comment 196 ABCC4 p.Phe368Trp Effect of different concentrations of ATP (A) and vanadate (B) on ATP-dependent E217bG uptake into membrane vesicles containing wild type (&) and mutant F368W (*) MRP4. Login to comment 201 ABCC4 p.Phe368Trp Vmax was set at 100%, and mean Æ S.E.M. of four to five independent experiments are shown. H.G.M. Wittgen et al. / Biochemical Pharmacology 84 (2012) 366-373 371 of F368W substitution on the apparent affinity for ATP, indicating that it does not affect the rate of ATP binding and release. Login to comment 202 ABCC4 p.Phe368Trp of F368W substitution on the apparent affinity for ATP, indicating that it does not affect the rate of ATP binding and release. Login to comment 203 ABCC4 p.Phe368Trp These results indicate that handling of ATP and ADP by mutant F368W is similar to that of wild type MRP4. Login to comment 204 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp Because neither a change in substrate nor in ATP or ADP binding of F368W appeared to explain its opposite effects on rate of transport of E217bG and MTX, we made homology models of MRP4 to visualize the localization of this amino acid (Fig. 6). Login to comment 205 ABCC4 p.Phe368Trp Because neither a change in substrate nor in ATP or ADP binding of F368W appeared to explain its opposite effects on rate of transport of E217bG and MTX, we made homology models of MRP4 to visualize the localization of this amino acid (Fig. 6). Login to comment 208 ABCC4 p.Phe368Trp This could provide an explanation for the finding that the MRP4 substitution F368W increased the catalytic turnover rate of E217bG transport, whereas it decreased that of MTX, implicating that Phe368 plays a crucial role in the substrate-dependent conformational changes that are involved in the transport of MTX and E217bG to the extracellular side. Login to comment 209 ABCC4 p.Phe368Trp ABCC4 p.Phe368Trp Another explanation for these opposite effects of the F368W mutant could be that Phe368 plays a role in the dissociation of these substrates in the outward-facing conformation. Login to comment 210 ABCC4 p.Phe368Trp Another explanation for these opposite effects of the F368W mutant could be that Phe368 plays a role in the dissociation of these substrates in the outward-facing conformation. Login to comment 218 ABCC4 p.Phe368Trp F368W increased E217bG transport and reduced MTX transport, which was not due to an altered substrate affinity. Login to comment 219 ABCC4 p.Phe368Trp F368W increased E217bG transport and reduced MTX transport, which was not due to an altered substrate affinity. Login to comment