ABCMdb

ABCC4 p.Phe368Trp

Predicted by SNAP2:	A: N (53%), C: N (72%), D: D (75%), E: D (71%), G: D (63%), H: D (53%), I: N (57%), K: D (66%), L: N (53%), M: N (66%), N: D (63%), P: D (80%), Q: D (53%), R: D (71%), S: D (63%), T: D (59%), V: N (53%), W: D (59%), Y: N (72%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: N,

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[hide] Phenylalanine 368 of multidrug resistance-associat... Biochem Pharmacol. 2012 Aug 1;84(3):366-73. Epub 2012 Apr 21. 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., [PMID:22542979]

Abstract [show]
Multidrug resistance-associated protein 4 (MRP4) is a membrane transporter that mediates the cellular efflux of a wide range of anionic drugs and endogenous molecules. MRP4 transport can influence the pharmacokinetics of drugs and their metabolites, therefore more knowledge about the molecular determinants important for its transport function would be of relevance. Here, we substituted amino acids Phe(368), Trp(995), and Arg(998) with conservative or non-conservative residues, and determined the effect on transport of the model substrates estradiol 17-beta-d-glucuronide (E(2)17betaG), cyclic guanosine monophosphate (cGMP), methotrexate (MTX), and folic acid into membrane vesicles isolated from baculovirus transduced HEK293 cells overexpressing the mutant MRP4 proteins. This revealed that all Arg(998) mutations appeared to be deleterious, whereas the effect of a Phe(368) or Trp(995) replacement was dependent on the amino acid introduced and the substrate studied. Substitution of Phe(368) with Trp (F368W) induced a gain-of-function of E(2)17betaG transport and a loss-of-function of MTX transport, which could not be attributed to an altered substrate binding. Moreover, we did not observe any modification in ATP or ADP handling for F368W. These results, in combination with docking of substrates in a homology model of MRP4 in the inward- and outward-facing conformation, suggest that Phe(368) and Trp(995) do not play an important role in the initial binding of substrates. They, however, might interact with the substrates during rearrangement of helixes for substrate translocation, funneling the substrates to the exit site in the outward-facing conformation.

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12 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 Moreover, we did not observe any modification in ATP or ADP handling for F368W. Login to comment
46 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 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 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 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
117 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
119 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 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
136 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 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 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 Fig. 5A shows the ATP-dependency of E217bG transport via MRP4 and F368W at a fixed E217bG concentration (200 mM). Login to comment
142 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 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
147 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
153 Table 1 Kinetic characteristics of transport via MRP4 mutants F368W, F368Y, W995F, and W995Y in comparison to wild type MRP4. Login to comment
154 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
168 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
189 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 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 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 E217bG transport activity of F368W and wild type MRP4 (Fig. 5A) revealed no effect Fig. 5. Login to comment
196 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 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
203 These results indicate that handling of ATP and ADP by mutant F368W is similar to that of wild type MRP4. Login to comment
204 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 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 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 F368W increased E217bG transport and reduced MTX transport, which was not due to an altered substrate affinity. Login to comment
47 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
137 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
144 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
148 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
155 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
202 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
205 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
210 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
219 F368W increased E217bG transport and reduced MTX transport, which was not due to an altered substrate affinity. Login to comment