ABCC8 p.Leu1260Ala
| Predicted by SNAP2: | A: N (72%), C: N (72%), D: D (66%), E: D (71%), F: N (78%), G: N (57%), H: D (66%), I: N (57%), K: D (75%), M: N (66%), N: D (66%), P: D (71%), Q: D (63%), R: D (71%), S: N (57%), T: N (82%), V: N (72%), W: D (63%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] The dileucine motif at the COOH terminus of human ... J Biol Chem. 2005 Jan 28;280(4):2522-8. Epub 2004 Nov 12. Loo TW, Bartlett MC, Clarke DM
The dileucine motif at the COOH terminus of human multidrug resistance P-glycoprotein is important for folding but not activity.
J Biol Chem. 2005 Jan 28;280(4):2522-8. Epub 2004 Nov 12., 2005-01-28 [PMID:15542593]
Abstract [show]
P-glycoprotein (P-gp, ABCB1) actively transports a broad range of cytotoxic compounds out of the cell. The COOH terminus of P-gp contains a dileucine motif (Leu(1260)-Leu(1261)) and a conserved phenylalanine (Phe(1268)). Similar residues in SUR1 (ABCC8) were reported to be important plasma membrane-targeting signals (Sharma, N., Crane, A., Clement, J. P. t., Gonzalez, G., Babenko, A. P., Bryan, J., and Aguilar-Bryan, L. (1999) J. Biol. Chem. 274, 20628-20632). Here, we used alanine-scanning mutagenesis to test whether these residues were essential for trafficking of P-gp to the cell surface. Mutant L1260A expressed a 150-kDa immature protein that did not reach the cell surface and was sensitive to digestion by Endo H(f). By contrast, mutants L1261A, F1268A, and wild-type P-gps expressed the 170-kDa mature proteins at the cell surface. Mutation of Leu(1260) to Gly, Ile, Trp, Lys, or Glu also resulted in the expression of the 150-kDa immature protein. All of the mutants, however, expressed the 170-kDa protein in the presence of the drug substrate/specific chemical chaperone cyclosporin A. Mutant L1260A P-gp exhibited drug-stimulated ATPase activities similar to that of wild-type enzyme after rescue with cyclosporin A. Deletion of the last 22 amino acids (Q(1259)-Q(1280)) also caused misprocessing. The mutant, however, was rescued by expression in the presence of cyclosporin A and conferred resistance to colchicine in transfected cells. These results show that the dileucine motif is not a plasma membrane targeting signal. The COOH terminus is required for proper folding of P-gp but not for activity.
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No. Sentence Comment
106 Accordingly, wild-type P-gp and mutants L1260A, L1261A, K1264A, Y1267A, and F1268A were expressed in HEK 293 cells in the presence or absence of the chemical chaperone/drug substrate cyclosporin A. Whole cell SDS extracts were then subjected to immunoblot analysis. Fig. 2A shows that the cyclosporin A was able to rescue mutants L1260A and Y1267A, because a 170-kDa protein was then detected (Fig. 2A).
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ABCC8 p.Leu1260Ala 15542593:106:40
status: NEWX
ABCC8 p.Leu1260Ala 15542593:106:330
status: NEW5 Mutant L1260A expressed a 150-kDa immature protein that did not reach the cell surface and was sensitive to digestion by Endo Hf.
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ABCC8 p.Leu1260Ala 15542593:5:7
status: NEW9 Mutant L1260A P-gp exhibited drug-stimulated ATPase activities similar to that of wild-type enzyme after rescue with cyclosporin A.
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ABCC8 p.Leu1260Ala 15542593:9:7
status: NEW89 Fig. 2A shows that the major product in mutants L1260A or Y1267A was the 150-kDa protein.
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ABCC8 p.Leu1260Ala 15542593:89:48
status: NEW94 Fig. 2B shows that treatment of wild-type or mutant L1260A P-gps with Endo Hf resulted in the disappearance of the 150-kDa protein and the appearance of a 140-kDa protein (Fig. 2B).
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ABCC8 p.Leu1260Ala 15542593:94:52
status: NEW119 B, wild-type and mutant L1260A P-gps were expressed in HEK 293 cells in the absence of cyclosporin A. Whole cell extracts were then treated with EndoHf (Hf), PNGase F (F), or no endoglycosidase (-).
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ABCC8 p.Leu1260Ala 15542593:119:24
status: NEW127 For cell surface labeling, the A52-epitope-tagged wild-type and mutant L1260A P-gps were used.
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ABCC8 p.Leu1260Ala 15542593:127:71
status: NEW129 Accordingly, HEK293 cells expressing A52-tagged wild-type or mutant L1260A were grown for 24 h in the presence or absence of 10 M cyclosporin A. Whole cell SDS extracts were then subjected to immunoblot analysis with monoclonal antibody A52.
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ABCC8 p.Leu1260Ala 15542593:129:68
status: NEW130 Fig. 3A shows that the major products of A52-tagged mutant L1260A P-gp were the 150and 170-kDa proteins when expression was carried out in the absence or presence of cyclosporin A, respectively.
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ABCC8 p.Leu1260Ala 15542593:130:59
status: NEW136 By contrast, the 170-kDa protein was labeled in mutant L1260A only when grown in the presence of cyclosporin A.
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ABCC8 p.Leu1260Ala 15542593:136:55
status: NEW137 Labeling of the 150-kDa protein was not detected when mutant L1260A was grown without cyclosporin A.
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ABCC8 p.Leu1260Ala 15542593:137:61
status: NEW139 Because residue Leu1260 was very sensitive to replacement with other amino acids, we determined whether mutant L1260A retained drug-stimulated ATPase activity when at the cell surface.
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ABCC8 p.Leu1260Ala 15542593:139:111
status: NEW141 To determine drug-stimulated ATPase activity, we attached a histidine tag at the COOH-end of the wild-type and mutant L1260A P-gps.
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ABCC8 p.Leu1260Ala 15542593:141:118
status: NEW142 The presence of the histidine tag did not affect the maturation characteristics of mutant L1260A.
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ABCC8 p.Leu1260Ala 15542593:142:90
status: NEW143 When histidine-tagged mutant L1260A was expressed in the absence or presence of cyclosporin A, the major products in SDS-PAGE were the 150and 170-kDa proteins, respectively (data not show).
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ABCC8 p.Leu1260Ala 15542593:143:29
status: NEW146 Fig. 4 shows that mutant L1260A retained more than 80% of the verapamil- or vinblastine-stimulated ATPase activity of wild-type P-gp.
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ABCC8 p.Leu1260Ala 15542593:146:25
status: NEW147 Half-maximal activation of wild-type and rescued mutant L1260A occurred at ϳ30 M verapamil.
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ABCC8 p.Leu1260Ala 15542593:147:56
status: NEW148 In the presence of vinblastine, however, both wild-type and rescued mutant L1260A showed half-maximal activity at 6 M vinblastine.
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ABCC8 p.Leu1260Ala 15542593:148:75
status: NEW160 HEK293 cells were transfected with vector alone (Control), A52-tagged wild-type (Wild-type), or mutant L1260A (L1260A) cDNAs.
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ABCC8 p.Leu1260Ala 15542593:160:103
status: NEWX
ABCC8 p.Leu1260Ala 15542593:160:111
status: NEW163 B, HEK293 cells expressing vector alone (Control), A52-tagged wild-type (Wild-type), or mutant L1260A P-gp (L1260A) and grown in the absence (-) or presence (ϩ) of cyclosporin A were surface labeled with biotin-X-hydrazide as described under "Materials and Methods."
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ABCC8 p.Leu1260Ala 15542593:163:95
status: NEWX
ABCC8 p.Leu1260Ala 15542593:163:108
status: NEW166 Drug-stimulated ATPase activity of wild-type and mutant L1260A P-gps.
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ABCC8 p.Leu1260Ala 15542593:166:56
status: NEW167 Histidine-tagged wild-type or mutant L1260A P-gps were expressed in HEK293 cells in the presence of 10 M cyclosporin A for 24 h, and then isolated by nickel-chelate chromatography.
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ABCC8 p.Leu1260Ala 15542593:167:37
status: NEW193 For example, the dileucine motif mutant L1260A and the deletion mutant ⌬22 in which all putative plasma-membrane targeting signals were deleted could be rescued when 2 T. W. Loo, M. Claire Bartlett, and D. M. Clarke, unpublished observations.
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ABCC8 p.Leu1260Ala 15542593:193:40
status: NEW