ABCMdb

ABCC8 p.Leu1260Ala

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Publications

PMID: 15542593 [PubMed] Loo TW et al: "The dileucine motif at the COOH terminus of human multidrug resistance P-glycoprotein is important for folding but not activity."

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). Login to comment
5 Mutant L1260A expressed a 150-kDa immature protein that did not reach the cell surface and was sensitive to digestion by Endo Hf. Login to comment
9 Mutant L1260A P-gp exhibited drug-stimulated ATPase activities similar to that of wild-type enzyme after rescue with cyclosporin A. Login to comment
89 Fig. 2A shows that the major product in mutants L1260A or Y1267A was the 150-kDa protein. Login to comment
94 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). Login to comment
119 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 (-). Login to comment
127 For cell surface labeling, the A52-epitope-tagged wild-type and mutant L1260A P-gps were used. Login to comment
129 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. Login to comment
130 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. Login to comment
136 By contrast, the 170-kDa protein was labeled in mutant L1260A only when grown in the presence of cyclosporin A. Login to comment
137 Labeling of the 150-kDa protein was not detected when mutant L1260A was grown without cyclosporin A. Login to comment
139 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. Login to comment
141 To determine drug-stimulated ATPase activity, we attached a histidine tag at the COOH-end of the wild-type and mutant L1260A P-gps. Login to comment
142 The presence of the histidine tag did not affect the maturation characteristics of mutant L1260A. Login to comment
143 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). Login to comment
146 Fig. 4 shows that mutant L1260A retained more than 80% of the verapamil- or vinblastine-stimulated ATPase activity of wild-type P-gp. Login to comment
147 Half-maximal activation of wild-type and rescued mutant L1260A occurred at ϳ30 ␮M verapamil. Login to comment
148 In the presence of vinblastine, however, both wild-type and rescued mutant L1260A showed half-maximal activity at 6 ␮M vinblastine. Login to comment
160 HEK293 cells were transfected with vector alone (Control), A52-tagged wild-type (Wild-type), or mutant L1260A (L1260A) cDNAs. Login to comment
163 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." Login to comment
166 Drug-stimulated ATPase activity of wild-type and mutant L1260A P-gps. Login to comment
167 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. Login to comment
193 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. Login to comment