ABCMdb

ABCB1 p.Asp1200Asn

Predicted by SNAP2:	A: D (91%), C: D (91%), E: D (91%), F: D (91%), G: D (91%), H: D (91%), I: D (91%), K: D (95%), L: D (95%), M: D (91%), N: D (91%), P: D (95%), Q: D (91%), R: D (95%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, E: D, F: 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: D,

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[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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165 But Hrycyna et al. [9] reported that the mutations D555N and D1200N (D555 and D1200 are presumably involved in Mg2+ binding) resulted in impaired nucleotide photoaffinity labeling only for D555N. Login to comment

[hide] Both ATP sites of human P-glycoprotein are essenti... Biochemistry. 1999 Oct 19;38(42):13887-99. Hrycyna CA, Ramachandra M, Germann UA, Cheng PW, Pastan I, Gottesman MM
Both ATP sites of human P-glycoprotein are essential but not symmetric.
Biochemistry. 1999 Oct 19;38(42):13887-99., 1999-10-19 [PMID:10529234]

Abstract [show]
Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.

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71 The D1200N mutant DNA was constructed similarly with two internal complementary primers, each containing the mutation of interest (G f A at position 3598 for D1200N). Login to comment
72 The coding sequence for the D1200N mutant primer was 5'-ATATTTTGCTTTTGAATGAAG-3'. Login to comment
78 The resulting plasmid was named pTM1-MDR1-D1200N. Login to comment
79 The D555N/D1200N double mutant was constructed exactly like pTM1-MDR1-D1200N except that pTM1-MDR1-D555N was used as the template for the first PCR reaction. Login to comment
80 The resulting expression plasmid was named pTM1-MDR1-D555N/ D1200N. Login to comment
106 Recombinant vaccinia viruses vvT7MDR1(WT) (wild-type P-gp), vvMDR1-CM (P-gp-D1200N), vvMDR1-Mg5 (P-gp-D555N), and vvMDR1-DM (P-gp-D555N/D1200N) were constructed as previously described (28) from the corresponding pTM1-MDR1 vectors described above. Login to comment
163 The two mutants, designated P-gp-D555N and P-gp-D1200N, were constructed by site-directed mutagenesis, cloned into pTM1-MDR1, and transiently expressed in HeLa cells coinfected with the vaccinia virus vTF7-3 as described under Experimental Procedures. Login to comment
165 Total extracts from HeLa cells expressing wild-type P-gp, P-gp-D555N, P-gp-D1200N, and the double mutant P-gp-D555N/ D1200N were subjected to immunoblot analysis with the monoclonal antibody C219 (32). Login to comment
166 While P-gp-D1200N was found to be expressed at a level comparable to wild-type P-gp, in this preparation of membranes it appears that less P-gp-D555N and P-gp D555N/D1200N were expressed compared to wild-type. Login to comment
169 Fluorescent substrate accumulation assays were performed on intact HeLa cells expressing wild-type P-gp, P-gp-D555N, and P-gp-D1200N with the P-gp substrate calcein-AM. Login to comment
170 HeLa cells were transiently infected with vTF7-3 and transfected with pTM1-MDR1 (wild-type), pTM1-MDR1-D555N, and pTM1-MDR1-D1200N, with pTM1 as the negative control, and allowed to incubate for approximately 24 h. After incubation with 0.5 µM calcein-AM for 10 min, cells expressing wild-type P-gp accumulated less substrate than the pTM1 control as measured by a decrease in fluorescence intensity (Figure 2). Login to comment
172 Strikingly, the two Walker B mutants P-gp-D555N and P-gp-D1200N were completely devoid of transport function (Figure 2). Login to comment
178 The ability of Walker B mutants P-gp-D555N, P-gp-D1200N, and P-gp-D555N/D1200N to hydrolyze ATP either in the absence (basal) or presence (drug-stimulated) of 25 µM verapamil was measured as the vanadate-sensitive release of inorganic phosphate from Mg2+ ‚ATP as described under Experimental Procedures, with membrane preparations from vTF7-3-infected HeLa cells alone or vTF7-3-infected cells that were coinfected with recombinant vaccinia viruses encoding wild-type P-gp, P-gp-D555N, P-gp-D1200N, and P-gp-D555N/D1200N. Login to comment
184 (A) vTF7-3-infected-transfected HeLa cells (500 000) expressing wild-type P-gp (bold line), P-gp-D555N (---), and P-gp-D1200N (-‚-) were subjected to FACS analysis after incubation with 5 µg of MRK-16 for 30 min at 37 °C, washing (200g, 5 min), and staining with FITC-conjugated anti-mouse IgG secondary antibody (1 µg) for 30 min at 37 °C. Login to comment
188 HeLa cells were infected with vTF7-3 recombinant vaccinia viruses expressing wild-type P-gp, P-gp-D555N, P-gp-D1200N, and P-gp-D555N/D12000N and harvested after 48 h. Membranes (1 µg/lane) prepared from these cells were subjected to SDS-PAGE and immunoblotting with monoclonal antibody C219 (1:1500). Login to comment
191 HeLa cells were infected with vTF7-3 and transfected with pTM1-MDR1 (wild type), pTM1-MDR1-D555N, and pTM1-MDR1-D1200N for 24 h. Calcein (0.5 µM) accumulation was determined in these cells by FACS after a 10 min incubation at 37 °C in the presence (thin line) and absence (bold line) of 2 µM cyclosporin A. vTF7-3-infected cells transfected with pTM1 vector DNA were included as a negative control. Login to comment
195 Crude membrane preparations derived from HeLa cells infected with vTF7-3 alone and from cells coinfected with recombinant vaccinia viruses encoding wild-type P-gp, P-gp-D555N, P-gp-D1200N, and P-gp-D555N/D1200N were labeled with [125I]IAAP at a concentration of 7.5 nM, immunoprecipitated with anti-P-gp polyclonal antibody PEPG-2 (33), and subjected to SDS-PAGE and autoradiography (Figure 4A). Login to comment
202 To further demonstrate that ATP hydrolysis is essential and to evaluate the roles of the two NBDs for inhibition of [125I]IAAP labeling as a result of vanadate trapping, we analyzed the effects of the Walker B consensus motif mutations D555N, D1200N, and D555N/D1200N. Login to comment
207 Human P-gp-D555N and P-gp-D1200N Differ Dramatically in Their Ability To Be Labeled with [R-32P]-8-Azido-ATP. Login to comment
210 Membrane preparations from HeLa cells infected with vTF7-3 and coinfected with vvT7MDR1 (WT) (wild-type P-gp), vvMDR1-CM (P-gp-D1200N), vvMDR1-Mg5 (P-gp-D555N), or vvMDR1-DM (P-gp-D555N/D1200N). Login to comment
216 (A) Wild type and the mutant P-gps D555N, D1200N, and D555N/1200N were expressed in HeLa cells by use of recombinant vaccinia viruses, and crude membranes (45 µg) were used for [125I]IAAP photoaffinity labeling. Login to comment
220 Wild-type (b) and mutant Pgps, D555N ([), D1200N (4), and D555N/D1200N (O) were expressed in HeLa cells by use of recombinant vaccinia viruses and crude membranes (15 µg) were used for [125I]IAAP labeling in the presence of varying concentrations of vanadate, 5 mM MgCl2, and 2.5 mM ATP. Login to comment
225 Membranes expressing wild-type P-gp and P-gp-D1200N were labeled with [R-32 P]-8-azido-ATP. Login to comment
227 Strikingly, the [R-32P]-8-azido-ATP labeling observed in membranes expressing P-gp-D555N and P-gp-D555N/D1200N was severely impaired. Login to comment
238 As can be seen, the N-half of P-gp is labeled predominantly in the wild-type and D1200N proteins (Figure 6A). Login to comment
251 Crude membrane preparations (50 µg) from HeLa cells infected with vTF7-3 and recombinant vaccinia viruses expressing wild-type P-gp, P-gp-D555N, P-gp-D1200N, or P-gp-D555N/ D1200N were photoaffinity-labeled on ice (0 °C) with either (A) 2.5 µM or (B) 77.5 µM [R-32P]-8-azido-ATP. Login to comment
255 Crude membrane preparations (50-60 µg) from HeLa cells infected with vTF7-3 and recombinant vaccinia viruses expressing wild-type P-gp, P-gp-D555N, P-gp-D1200N, or P-gp-D555N/D1200N were photoaffinity-labeled with either (A) [R-32P]- 8-azido-ATP (2.5 µM) or (B) [125I]IAAP (15 nM) as described in the legend to Figures 4 and 5. Login to comment
270 There was no apparent increase in the amount of label associated with the C-half of P-gp-D1200N under hydrolysis conditions, suggesting that the mutant is incapable of catalyzing even one ATP hydrolysis reaction (Figure 7B, lane 8). Login to comment
271 However, the N-half of P-gp-D1200N retained its ability to be labeled with [R-32P]-8-azido-ATP (Figure 7B, lanes 6 and 8) under both binding and hydrolysis conditions. Login to comment
272 Similar to P-gp-D1200N, P-gp-D555N and P-gp-D555N/D1200N were not capable of being vanadate-trapped, confirming their lack of ATPase activity (data not shown). Login to comment
277 Wild-Type and D1200N Human P-gp Proteins Do Not Differ Significantly in Their Apparent Affinity for ATP. Login to comment
279 The specific P-gp bands were quantified by phosphorimaging and the data were analyzed as described under Experimental FIGURE 7: [R-32P]-8-Azido-ATP photoaffinity labeling and vanadate trapping of wild-type and P-gp-D1200N from HeLa cell membranes. Login to comment
280 Crude membrane preparations (100 µg) from HeLa cells infected with vTF7-3 and the recombinant vaccinia virus expressing wild-type or P-gp-D1200N were photoaffinity-labeled with 2.5 µM [R-32P]-8-azido-ATP for 10 min. Login to comment
286 (B) Examination of wild-type P-gp (lanes 1-4) and D1200N P-gp (lanes 5-8) under [R-32P]-8-azido-ATP hydrolysis/binding conditions: lanes 1 and 5, 0 °C, (-) trypsin, (-) 300 µM vanadate; lanes 2 and 6, 0 °C, (+) trypsin, (-) 300 µM vanadate; lanes 3 and 7, 37 °C, (+) 300 µM vanadate, (+) 25 µM verapamil; lanes 4 and 8, 37 °C, (+) 300 µM vanadate, (+) 25 µM verapamil, (+) trypsin. Login to comment
293 These data suggest that the wild-type and P-gp D1200N mutant proteins have similar apparent affinities for ATP. Login to comment
297 To determine if conformational differences exist between active wild-type Pgp and inactive P-gp-D555N and P-gp-D1200N, the reactivity of these constructs expressed transiently in HeLa cells was assessed by use of the conformation-sensitive monoclonal anti-human P-gp antibody UIC2 (5, 45). Login to comment
300 Furthermore, the UIC2 reactivity of cells expressing either P-gp-D555N and P-gp-D1200N is indistinguishable from each other, in either the presence or absence of cyclosporin A, and is equivalent to that of the cyclosporin A-treated wild-type-expressing cells. Login to comment
301 These UIC2 reactivity data, taken together with the ATPase data that demonstrated complete abrogation of ATPase activity for the D555N and D1200N P-gp proteins (Figure 3) and the ATP labeling data that demonstrated marked differences in the ability of these molecules to bind ATP (Figure 5), strongly suggest that the intrinsic ability of the P-gp molecule to hydrolyze ATP or associate with hydrolysis products accounts for the difference in conformation detected by UIC2. Login to comment
304 Crude membrane preparations from HeLa cells expressing either wild-type P-gp or P-gp-D1200N were incubated and photoaffinity-labeled in the presence of increasing MgCl2 (0-10 mM) followed by immunoprecipitation with PEPG-2, SDS-PAGE, and autoradiography. Login to comment
306 Figure 9 demonstrates that for both the wild-type P-gp (Figure 9A) and P-gp-D1200N (Figure 9B) the labeling with [R-32P]-8-azido-ATP increased in a magnesium concentration-dependent manner with half-maximum incorporation at 152.9 ( 29.9 µM for wild-type P-gp and 161.5 ( 39.7 µM for P-gp-D1200N. Login to comment
310 Taken together, these data demonstrate that the P-gp-D1200N and wild-type P-gp proteins have similar requirements for Mg2+ and that the requirement is 50-60 times greater than the [R-32 P]-8-azido-ATP concentration (2.5 µM) used in the assay, suggesting another role for magnesium in addition to complexing with ATP in the active site. Login to comment
315 FIGURE 8: UIC2 reactivity shift induced by incubation with cyclosporin A. vTF7-3-infected-transfected HeLa cells expressing wild-type P-gp, P-gp-D555N, or P-gp-D1200N were subjected to FACS analysis after staining with UIC2 in the presence (thin line) and absence (bold line) of 5 µM cyclosporin A. Login to comment
335 As shown in Figure 11, both the wild-type (WT) P-gp and the P-gp-1N/1N molecules were labeled FIGURE 9: Effect of increasing concentrations of MgCl2 on [R-32P]- 8-azido-ATP photoaffinity labeling of wild-type P-gp and P-gp- D1200N in membrane preparations from HeLa cells. Login to comment
336 Crude membrane preparations (75 µg) from HeLa cells expressing (A) wild-type P-gp and (B) P-gp-D1200N were photoaffinity-labeled with 2.5 µM [R-32P]-8-azido-ATP on ice (0 °C) in the presence of increasing concentrations of MgCl2 (0-10 mM). Login to comment
354 These mutations at positions D555N and D1200N, made separately or in concert, did not affect the ability of the transporter to bind substrate but completely abrogated transport function and both basal and drug-stimulated ATPase activity. Login to comment
365 Whereas P-gp-D1200N, the mutant transporter with a C-terminal amino acid substitution, labels comparably to the wild-type protein, P-gp-D555N, containing a homologous N-terminal mutation, is severely impaired in its ability to be labeled with [R-32P]-8-azido-ATP. Login to comment
368 After mild trypsinization, it was apparent that the N-half is predominantly labeled in the D1200N and wild-type proteins, whereas little to no labeling of the N-half of the P-gp-D555N was observed. Login to comment
393 This model is also consistent with the data from the nonfunctional Walker B motif mutants, P-gp-D555N and P-gp-D1200N. Login to comment
397 In the case of P-gp-D1200N, although the N-half can bind ATP, the C-half is disabled by the mutation and the molecule remains hydrolysis-incompetent. Login to comment

[hide] Nucleotide-induced conformational changes in P-gly... Biochemistry. 2000 Apr 18;39(15):4559-68. Julien M, Gros P
Nucleotide-induced conformational changes in P-glycoprotein and in nucleotide binding site mutants monitored by trypsin sensitivity.
Biochemistry. 2000 Apr 18;39(15):4559-68., 2000-04-18 [PMID:10758006]

Abstract [show]
Limited trypsin digestion was used to monitor nucleotide-induced conformational changes in wild-type P-glycoprotein (Pgp) as well as in nucleotide binding domain (NBD) Pgp mutants. Purified and reconstituted wild-type or mutant mouse Mdr3 Pgps were preincubated with different hydrolyzable or nonhydrolyzable nucleotides, followed by limited proteolytic cleavage at different trypsin:protein ratios. The Pgp tryptic digestion products were separated by SDS-PAGE followed by immunodetection with the mouse monoclonal anti-Pgp antibody C219, which recognizes a conserved epitope (VVQE/AALD) in each half of the protein. Different trypsin digestion patterns were observed for wild-type Pgp incubated with MgCl(2) alone, MgADP, MgAMP.PNP, MgATP, and MgATP + vanadate. A unique trypsin digestion profile suggestive of enhanced resistance to trypsin was observed under conditions of vanadate-induced trapping of nucleotides (MgATP + vanadate). The trypsin sensitivity profiles of Pgp mutants bearing either single or double mutations in Walker A (K429R, K1072R) and Walker B (D551N, D1196N) sequence signatures of NBD1 and NBD2 were analyzed under conditions of vanadate-induced trapping of nucleotides. The proteolytic cleavage pattern observed for the double mutants K429R/K1072R and D551N/D1196N, and for the single mutants K429R, K1072R, and D1196N were similar and clearly distinct from wild-type Pgp under the same conditions. This is consistent with the absence of ATP hydrolysis and of vanadate-induced trapping of 8-azido-ADP previously reported for these mutants [Urbatsch et al. (1998) Biochemistry 37, 4592-4602]. Interestingly, the trypsin digestion profiles observed under vanadate-induced trapping for the D551N and D1196N mutants were quite different, with the D551N mutant showing a profile resembling that seen for wild-type Pgp. The different sensitivity profiles of Pgp mutants bearing mutations at the homologous residue in NBD1 (D551N) and NBD2 (D1196N) suggest possible structural and functional differences between the two sites.

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232 Additional photolabeling experiments using the Walker B mutants D555N and D1200N showed that in contrast to results from Urbatsch et al. (15), only the D1200N mutant can be photolabeled under binding conditions with all the label being present at the NBD1 (16). Login to comment

[hide] Functionally similar vanadate-induced 8-azidoadeno... J Biol Chem. 2001 Jun 15;276(24):21199-208. Epub 2001 Apr 3. Sauna ZE, Smith MM, Muller M, Ambudkar SV
Functionally similar vanadate-induced 8-azidoadenosine 5'-[alpha-(32)P]Diphosphate-trapped transition state intermediates of human P-glycoprotin are generated in the absence and presence of ATP hydrolysis.
J Biol Chem. 2001 Jun 15;276(24):21199-208. Epub 2001 Apr 3., 2001-06-15 [PMID:11287418]

Abstract [show]
P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpression confers multidrug resistance to cancer cells. Pgp exhibits a robust drug substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activity effectively by trapping Pgp nucleotide in a non-covalent stable transition state conformation. In this study we compare Vi-induced [alpha-(32)P]8-azido-ADP trapping into Pgp in the presence of [alpha-(32)P]8-azido-ATP (with ATP hydrolysis) or [alpha-(32)P]8-azido-ADP (without ATP hydrolysis). Vi mimics P(i) to trap the nucleotide tenaciously in the Pgp.[alpha-(32)P]8-azido-ADP.Vi conformation in either condition. Thus, by using [alpha-(32)P]8-azido-ADP we show that the Vi-induced transition state of Pgp can be generated even in the absence of ATP hydrolysis. Furthermore, half-maximal trapping of nucleotide into Pgp in the presence of Vi occurs at similar concentrations of [alpha-(32)P]8-azido-ATP or [alpha-(32)P]8-azido-ADP. The trapped [alpha-(32)P]8-azido-ADP is almost equally distributed between the N- and the C-terminal ATP sites of Pgp in both conditions. Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogation of [alpha-(32)P]8-azido-ADP trapping into Pgp in the absence of hydrolysis. These data suggest that both ATP sites are dependent on each other for function and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP). Similarly, Pgp in the transition state conformation generated with either ADP or ATP exhibits drastically reduced affinity for the binding of analogues of drug substrate ([(125)I]iodoarylazidoprazosin) as well as nucleotide (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate). Analyses of Arrhenius plots show that trapping of Pgp with [alpha-(32)P]8-azido-ADP (in the absence of hydrolysis) displays an approximately 2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of [alpha-(32)P]8-azido-ATP (62 kJ/mol). In aggregate, these results demonstrate that the Pgp.[alpha-(32)P]8-azido-ADP (or ADP).Vi transition state complexes generated either in the absence of or accompanying [alpha-(32)P]8-azido-ATP hydrolysis are functionally indistinguishable.

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55 Preparation of Crude Membranes from HeLa Cells Expressing Mutant and Wild-type Pgp-A 70-80% confluent monolayer of HeLa cells was infected with vTF7-3 and transfected with pTM1-MDR1 (wild type) or pTM1-MDR1 bearing the homologous mutations at positions 555 (D555N) and 1200 (D1200N) as described previously (9). Login to comment
56 The vaccinia virus expression vectors pTM1-MDR1 (wild type) and pTM1-MDR1(D555N) and pTM1-MDR1(D1200N) were provided by C. A. Hrycyna and M. M. Gottesman, Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda. Login to comment
224 The Pgp mutants D555N and D1200N in Walker B domain of ATP sites do not show Vi-induced trapping of [␣-32 P]8-azido-ADP using either [␣-32 P]8-azido-ADP or [␣-32 P]8-azido-ATP. Login to comment
225 Crude membranes were prepared from HeLa cells transiently expressing the pTM1-MDR1 (designated wild type) and pTM1-MDR1 bearing the homologous mutations in Walker B region of ATP sites at positions 555 and 1200 (designated D555N and D1200N), respectively. Login to comment
230 Lane 1, wild-type Pgp membranes labeled with [␣-32 P]8-azido-ATP; lane 2, D555N membranes labeled with [␣-32 P]8-azido-ATP; lane 3, D1200N membranes labeled with [␣-32 P]8-azido-ATP; lane 4, wild-type membranes labeled with [␣-32 P]8-azido-ADP; lane 5, D555N membranes labeled with [␣-32 P]8-azido-ADP; and lane 6, D1200N membranes labeled with [␣-32 P]8-azido-ADP. Login to comment
290 The mutations in either the N-terminal ATP site (D555N) or the C-terminal site (D1200N) abolish Vi-induced trapping of [␣-32 P]8-azido-ADP by both the hydrolysis and non-hydrolysis routes (Fig. 6). Login to comment

[hide] P-glycoprotein plays an insignificant role in the ... Cancer Res. 1998 Oct 15;58(20):4688-93. Russ G, Ramachandra M, Hrycyna CA, Gottesman MM, Pastan I, Bennink JR, Yewdell JW
P-glycoprotein plays an insignificant role in the presentation of antigenic peptides to CD8+ T cells.
Cancer Res. 1998 Oct 15;58(20):4688-93., [PMID:9788623]

Abstract [show]
Most antigenic peptides presented to CD8+ T cells are generated from cytosolic precursors and are translocated by TAP into the endoplasmic reticulum, where they associate with MHC class I molecules. TAP-deficient cells exhibit a limited capacity to deliver peptides from cytosolic proteins to class I molecules. One candidate for an alternative peptide transporter is P-glycoprotein, which transports numerous substances, including peptides, across membranes. Elevation of P-glycoprotein expression is partially responsible for the resistance developed by neoplasias to chemotherapeutic drugs. Overexpression of P-glycoprotein has been reported to enhance the expression of class I molecules. Here, we investigated the role of P-glycoprotein in the generation of peptide-MHC complexes. We were unable to detect P-glycoprotein-mediated transport of synthetic peptides into the endoplasmic reticulum of either T2 cells (TAP-deficient) infected with a recombinant vaccinia virus (rVV) expressing P-glycoprotein or drug-resistant cells in which TAP is inactivated by a peptide from the herpes simplex virus ICP47 protein. Expression of rVV-encoded P-glycoprotein in T2 cells was unable to enhance cell surface expression of any of three MHC class I allomorphs tested. rVV-mediated expression of P-glycoprotein enabled T2 cells to produce limited amounts of class I-peptide complexes from cytosolic antigens, but this was not blocked by a drug that inhibits its transporter function, and a similar degree of presentation was mediated by functionally inactive mutated forms of P-glycoprotein. Thus, this was a nonspecific effect that we attributed to diminished membrane integrity resulting from P-glycoprotein overexpression. Taken together, our findings cast serious doubts that P-glycoprotein is a biologically significant transporter of cytosolic peptides.

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67 Further rVVs were used to express P-glycoprotein |MDRI(T7)] and P-glycoprotein mutants (NM- D555N, CM-D1200N, and DM-D555N + D1200N), under the control of the T7 promoter (14). Login to comment
109 The introduced mutations alter one or both of highly conserved Asp residues located, respectively, within Walker B regions of either NH2-terminal (D555N) or COOH-terminal (D1200N) ATP binding/utilization sites that are believed to be involved in binding to Mg2+. Login to comment
64 Further rVVs were used to express P-glycoprotein |MDRI(T7)] and P-glycoprotein mutants (NM- D555N, CM-D1200N, and DM-D555N + D1200N), under the control of the T7 promoter (14). Login to comment
105 To determine the requirement for a functional P-glycoprotein, we used rVVs expressing three mutated forms of the protein under the control of the T7 promoter, termed CM, NM, and DM. The introduced mutations alter one or both of highly conserved Asp residues located, respectively, within Walker B regions of either NH2-terminal (D555N) or COOH-terminal (D1200N) ATP binding/utilization sites that are believed to be involved in binding to Mg2+. Login to comment