ABCMdb

ABCC4 p.Phe368Tyr

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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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
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
118 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
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
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
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
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