ABCMdb

ABCB1 p.His1195Tyr

Predicted by SNAP2:	A: N (61%), C: N (61%), D: N (53%), E: N (53%), F: N (53%), G: N (57%), I: N (57%), K: N (97%), L: N (53%), M: N (72%), N: N (72%), P: N (82%), Q: N (93%), R: N (87%), S: N (78%), T: N (72%), V: N (61%), W: D (53%), Y: N (53%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: N,

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Publications
[hide] A T3587G germ-line mutation of the MDR1 gene encod... Mol Cancer Ther. 2006 Apr;5(4):877-84. Mutoh K, Mitsuhashi J, Kimura Y, Tsukahara S, Ishikawa E, Sai K, Ozawa S, Sawada J, Ueda K, Katayama K, Sugimoto Y
A T3587G germ-line mutation of the MDR1 gene encodes a nonfunctional P-glycoprotein.
Mol Cancer Ther. 2006 Apr;5(4):877-84., [PMID:16648557]

Abstract [show]
The human multidrug resistance gene 1 (MDR1) encodes a plasma membrane P-glycoprotein (P-gp) that functions as an efflux pump for various structurally unrelated anticancer agents. We have identified two nonsynonymous germ-line mutations of the MDR1 gene, C3583T MDR1 and T3587G MDR1, in peripheral blood cell samples from Japanese cancer patients. Two patients carried the C3583T MDR1 allele that encodes H1195Y P-gp, whereas a further two carried T3587G MDR1 that encodes I1196S P-gp. Murine NIH3T3 cells were transfected with pCAL-MDR-IRES-ZEO constructs carrying either wild-type (WT), C3583T, or T3587G MDR1 cDNA and selected with zeocin. The resulting zeocin-resistant mixed populations of transfected cells were designated as 3T3/WT, 3T3/H1195Y, and 3T3/I1196S, respectively. The cell surface expression of I1196S P-gp in 3T3/I1196S cells could not be detected by fluorescence-activated cell sorting, although low expression of I1196S P-gp was found by Western blotting. H1195Y P-gp expression levels in 3T3/H1195Y cells were slightly lower than the corresponding WT P-gp levels in 3T3/WT cells. By immunoblotting analysis, both WT P-gp and H1195Y P-gp were detectable as a 145-kDa protein, whereas I1196S P-gp was visualized as a 140-kDa protein. 3T3/I1196S cells did not show any drug resistance unlike 3T3/H1195Y cells. Moreover, a vanadate-trap assay showed that the I1196S P-gp species lacks ATP-binding activity. Taken together, we conclude from these data that T3587G MDR1 expresses a nonfunctional P-gp and this is therefore the first description of such a germ-line mutation. We contend that the T3587G MDR1 mutation may affect the pharmacokinetics of MDR1-related anticancer agents in patients carrying this allele.

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No. Sentence Comment
3 Two patients carried the C3583T MDR1 allele that encodes H1195Y P-gp, whereas a further two carried T3587G MDR1 that encodes I1196S P-gp. Login to comment
5 The resulting zeocin-resistant mixed populations of transfected cells were designated as 3T3/WT, 3T3/H1195Y, and 3T3/I1196S, respectively. The cell surface expression of I1196S P-gp in 3T3/I1196S cells could not be detected by fluorescence-activated cell sorting, although low expression of I1196S P-gp was found by Western blotting. Login to comment
6 H1195Y P-gp expression levels in 3T3/H1195Y cells were slightly lower than the corresponding WT P-gp levels in 3T3/WT cells. Login to comment
7 By immunoblotting analysis, both WT P-gp and H1195Y P-gp were detectable as a 145-kDa protein, whereas I1196S P-gp was visualized as a 140-kDa protein. Login to comment
8 3T3/I1196S cells did not show any drug resistance unlike 3T3/H1195Y cells. Login to comment
36 We were subsequently able to identify a novel germ-line mutation in the MDR1 gene, C3583T MDR1, which causes a substitution of tyrosine for His1195 in the P-gp (H1195Y P-gp). Login to comment
37 In our current study, we have established T3587G MDR1 and C3583T MDR1 cDNA transfectants and examined both expression levels and functional properties of I1196S P-gp and H1195Y P-gp. Login to comment
48 The zeocin-resistant mixed populations of the transfected cells were designated as 3T3/WT, 3T3/H1195Y, and 3T3/I1196S, respectively. Login to comment
79 The C3583T MDR1 and T3587G MDR1 alleles encode H1195Y P-gp and I1196S P-gp, respectively, and both of the His1195 and Ile1196 residues are located in the Walker B region of the second ATP-binding site of P-gp (Fig. 1A). Login to comment
87 Arrows, location of the H1195Y and I1196S substitutions. Login to comment
91 April 2006 American Association for Cancer ResearchCopyright (c) 2006 on September 29, P-gp Expression Levels in the MDR1 Transfectants To investigate the molecular functions of the H1195Y mutant P-gp and I1196S mutant P-gp, we generated 3T3/ WT, 3T3/H1195Y, and 3T3/I1196S cells, which were stably transfected with WT MDR1, C3583T MDR1, and T3587G MDR1 cDNA, respectively. The P-gp expression levels on the cell surfaces of these transfectants were subsequently examined by FACS analysis using the MRK16 antibody, which recognizes a cell surface epitope of human P-gp. Login to comment
92 Both 3T3/WT and 3T3/H1195Y cells express P-gp on their cell surface, although these expression levels in 3T3/ H1195Y cells (mean channel, 510) were slightly lower than in 3T3/WT cells (mean channel, 980; Fig. 2A). Login to comment
96 Moreover, both WT P-gp and H1195Y P-gp were detectable as a 145-kDa protein, whereas I1196S P-gp was observed as a 140-kDa protein (Fig. 2B). Login to comment
106 As shown in Fig. 3, P-gp expression was observed in NIH3T3 cells transduced with both WT and H1195Y MDR1 retroviruses but not in cells transduced with I1196S MDR1 retrovirus. Login to comment
107 Transduction efficiencies were 70% and 60% for WT and H1195Y MDR1 retroviruses, respectively. Login to comment
108 P-gp expression in cells transduced with H1195Y MDR1 retrovirus was again found to be at a slightly lower levels than in cells transduced with WT MDR1 retrovirus (Fig. 3B and C). Login to comment
111 3T3/ H1195Y cells also showed higher levels of resistance to these drugs compared with the parental cells, but these were at slightly lower levels than 3T3/WT cells (Fig. 4). Login to comment
125 Loss of ATP-Binding Ability in I1196S P-gp Because H1195Y P-gp and I1196S P-gp have amino acid substitutions in the second ATP-binding site of P-gp, we examined the ATP-binding activities of these variants. Login to comment
128 Because 3T3/I1196S clone 23 expressed f25% of the WT P-gp levels, and 3T3/H1195Y cells expressed f50% of the WT levels, we normalized these amounts in the relevant experiments (Fig. 5B and C). Login to comment
130 However, verapamil stimulated the nucleotide trapping of both WT P-gp and H1195Y P-gp, both of which showed similar levels of ATP-binding activity (Fig. 5C and D). Login to comment
150 C, NIH3T3 cells transduced with H1195Y MDR1 retrovirus. Login to comment
155 ), 3T3/WT (o), 3T3/H1195Y (4), and 3T3/I1196S (w ) cells were cultured for 5 d with various concentrations of vincristine or doxorubicin. Cell numbers were determined using a cell counter. Login to comment
174 As shown in Fig. 2A, most of the 3T3/WT and 3T3/H1195Y cells expressed cell surface P-gp. Login to comment
176 To confirm our finding of a lower expression level of H1195Y P-gp, we did retrovirus-mediated gene transfer. Login to comment
177 Cells transduced with H1195Y MDR1 retrovirus showed slightly lower P-gp expression levels than those transduced with WT MDR1 retrovirus (Fig. 3). Login to comment
178 We therefore speculate that the difference in P-gp expression between 3T3/WT and 3T3/H1195Y cells is genuine and can be attributed to post-transcriptional events, such as protein maturation and/or stability. Login to comment
185 C, ATP-binding activity of H1195Y P-gp. Plasma membrane protein extracts of NIH3T3 (20 Ag), 3T3/WT (10 Ag), and 3T3/H1195Y (20 Ag) cells were analyzed as in B. Top, autoradiography using a radioimaging analyzer; bottom, Western blotting analysis of the same blot with the anti-P-gp antibody C219. Arrows, P-gps. Login to comment
200 Our present study also shows that substitution of serine for Ile1196 results in the loss of ATP-binding activity but that the substitution of tyrosine for His1195 does not affect P-gp function. Login to comment