ABCMdb

ABCC1 p.Asp1084Glu

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Publications

PMID: 12954620 [PubMed] Zhang DW et al: "Functional importance of polar and charged amino acid residues in transmembrane helix 14 of multidrug resistance protein 1 (MRP1/ABCC1): identification of an aspartate residue critical for conversion from a high to low affinity substrate binding state."

No. Sentence Comment

51 They are as follows: T1082A (5Ј-C TCC AAG GAG CTC GAC GCA GTG GAC TCC-3Ј), D1084N (5Ј-CTG GAC ACA GTG AAT TCC ATG ATC CCG-3Ј), D1084A (5Ј-CTG GAC ACA GTG AAA TCG ATG ATC CCG-3Ј), D1084E (5Ј-CTG GAC ACA GTG GAC TCG ATG ATC CCG-3Ј), D1084V (5Ј-CTG GAC ACA GTG GTA TCG ATG ATC CCG-3Ј), S1085A (5Ј-CTG GAC ACA GTC GAC GCC ATG ATC CCG G-3Ј), K1092M (5Ј-C CCG GAG GTC ATC ATG ATG TTC ATG GGC-3Ј), K1092A (5Ј-C CCG GAG GTC ATC GCG ATG TTC ATG GGC-3Ј), K1092E (5Ј-C CCG GAG GTC ATC GAG ATG TTC ATG GGC-3Ј), K1092R (5Ј-C CCG GAG GTC ATC AGG ATG TTC ATG GGC-3Ј), S1097A (5Ј-G ATG TTC ATG GGC GCC CTG TTC AAC-3Ј), N1100A (5Ј-TTC ATG GGC TCG CTC TTC GCC GTC ATT GGT G-3Ј), N1100S (5Ј-TTC ATG GGC TCG CTC TTC AGT GTC ATT GGT G-3Ј). Login to comment
218 Effects of Mutations D1084A, D1084E, D1084V, D1084R, K1092A, K1092E, K1092R, and N1100S on Transport of [3 H]LTC4 and [3 H]E217betaG by Wild Type MRP1-Because mutation D1084N dramatically affected all of the MRP1 functions tested, we also mutated Asp1084 to Ala, Glu, Arg, and Val. Login to comment
242 We also examined the ability of the mutants D1084E and D1084R to bind [3 H]LTC4 by photolabeling studies. Login to comment
243 As shown in Fig. 8E, the relative normalized densitometry values of photolabeling of D1084E and D1084R with [3 H]LTC4 were 120 and 100, respectively. Login to comment
244 Thus, similar to the results obtained with photolabeling of mutant D1084N with LTC4, substitution of Asp1084 by Arg or Glu had no significant effect on the photolabeling. Login to comment
273 In contrast, conservative substitution of Asp1084 with Glu resulted in a mutant protein which retained ϳ50% of the ability of the wild type protein to transport LTC4 and E217betaG, and to confer vincristine and doxorubicin resistance, while having no effect on VP-16 resistance. Login to comment
322 Photolabeling studies with [3 H]LTC4 also showed that the replacement of Asp1084 by Asn and Glu had no effect on binding of the substrate to the protein. Login to comment

PMID: 15208328 [PubMed] Situ D et al: "Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter."

No. Sentence Comment

93 A, MRP1 expression in membrane vesicles prepared from HEK293T cells transfected with empty vector [pcDNA3.1] and vector containing wild-type (WT-MRP1) and mutant (R1046D, D1084R, D1084E, and R1131E) cDNAs was determined by immunoblotting with mAb QCRL-1. Shown is a representative immunoblot of membrane vesicles (1 and 2 ␮g of protein) from a single transfection. Login to comment
95 B-F, levels of 3 H-labeled organic anion uptake by the membrane vesicles shown in A were determined and corrected to take into account any differences in MRP1 protein expression (empty pcDNA3.1 vector control (open bars), wild-type MRP1 (black bars), and mutants R1046D, D1084R, D1084E, and R1131E (gray bars)). Login to comment
100 Because the overall transport activity of the oppositely charged Asp1084 mutant was greatly diminished (Fig. 2, B-E), a like-charge substituted D1084E mutant was created to clarify whether it was the loss of the acidic character or a change in size of the amino acid side chain that was responsible for the diminished activity. Login to comment
101 Like the D1084R mutant, the D1084E mutant showed substantially reduced LTC4 uptake levels (Ͻ20% of wild-type MRP1) (27) (Fig. 2C). Login to comment
102 In contrast to D1084R, however, the D1084E mutant exhibited E217betaG (Fig. 2B), E1SO4 (Fig. 2D), and MTX (Fig. 2E) uptake levels that were similar or only moderately reduced compared with those of wild-type MRP1. Login to comment
103 Because of the apparent selectively greater loss of LTC4 transport activity by the D1084E mutant, GSH uptake by this mutant was also examined and compared with that of D1084R. Login to comment
104 As shown in Fig. 2F, apigenin-stimulated GSH uptake by the D1084R and D1084E mutants was reduced by Ͼ90 and 80%, respectively. Login to comment
118 Immunoblots of membrane vesicle proteins prepared from cells expressing the Glu1204 mutants E1204L and E1204D were carried out as described in A. TABLE I Summary of organic anion transport activity of MRP1 mutants with substitutions of ionizable amino acids in and proximal to TM13 to TM17 of MSD3 Mutation % Wild-type MRP1 transport activitya E217betaG LTC4 E1SO4 MTX GSH TM13 R1046D 115 70 80 120 NDb TM14 D1084R Ͻ10 Ͻ10 15 25 Ͻ10 D1084E 80 20 65 90 20 TM15 R1131E 70 50 80 60 ND TM16 R1197E Ͻ10 Ͻ10 Ͻ15 Ͻ10 ND R1197K 20 Ͻ25 Ͻ20 Ͻ10 ND R1202G 115 115 75 70 ND R1202L 115 120 50 110 ND E1204L Ͻ10 50 10 110 Ͻ25 E1204D 100 115 100 115 Ͻ25 TM17 R1249D Ͻ10 Ͻ15 Ͻ10 Ͻ10 ND R1249K Ͻ10 10 Ͻ15 Ͻ10 ND a The values shown are means of duplicate or triplicate determinations and are derived from Fig. 2, 4, and 5 (see figure legends for details). Login to comment
182 We reported previously (27) that nonconservative substitutions of Asp1084 proximal to TM14 caused a substantial reduction in E217betaG, LTC4, and GSH transport and drug resistance, and we have now shown that these mutations also reduce MTX and E1SO4 transport activity, demonstrating a global disruption of MRP1 activity. We have also found that the same-charge mutant, D1084E, has significant transport activity with respect to E217betaG, MTX, and E1SO4 whereas transport of GSH and the glutathione conjugate LTC4 remains quite low. Login to comment
184 However, neither physical property of Asp1084 appears necessary for substrate binding, because GSH is still able to stimulate E1SO4 transport indicating that GSH binding to D1084E is intact. Login to comment
185 Similarly, D1084E and D1084R can still be photolabeled with LTC4 as well as wild-type MRP1. Login to comment