ABCB2 p.Phe200Leu
| Predicted by SNAP2: | A: N (57%), C: D (53%), D: D (75%), E: D (71%), G: N (57%), H: N (57%), I: N (66%), K: D (75%), L: N (82%), M: D (53%), N: D (53%), P: D (80%), Q: D (66%), R: N (61%), S: N (66%), T: N (72%), V: N (66%), W: N (61%), Y: N (82%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: D, G: D, H: D, I: N, K: N, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: N, V: N, W: N, Y: N, |
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[hide] Mutational analysis of the P-glycoprotein first in... Biochemistry. 1998 Mar 10;37(10):3337-50. Kwan T, Gros P
Mutational analysis of the P-glycoprotein first intracellular loop and flanking transmembrane domains.
Biochemistry. 1998 Mar 10;37(10):3337-50., 1998-03-10 [PMID:9521654]
Abstract [show]
The role of individual intracellular (IC) loops linking transmembrane (TM) domains in P-glycoprotein (P-gp) function remains largely unknown. The high degree of sequence conservation of these regions in the P-gp family and other ABC transporters suggests an important role in a common mechanism of action of these proteins. To gain insight into this problem, we have randomly mutagenized a portion of TM2, the entire IC1 loop, TM3, the entire extracellular loop (EC2), and part of TM4, and analyzed the effect of such mutations on P-gp function. Random mutagenesis was carried out using Taq DNA polymerase and dITP under conditions of low polymerase fidelity, and the mutagenized segments were reintroduced in the full length mdr3 cDNA by homologous recombination in the yeast Saccharomyces cerevisiae strain JPY201. The biological activity of mutant P-gp variants was analyzed in yeast by their ability to confer cellular resistance to the antifungal drug FK506 and the peptide ionophore valinomycin, and by their ability to complement the yeast Ste6 gene and restore mating in a yeast strain bearing a null mutation [Raymond, M., et al. (1992) Science 256, 232-4] at this locus. The analysis of 782 independent yeast transformants allowed the identification of 49 independent mutants bearing single amino acid substitutions in the mutagenized segment resulting in an altered P-gp function. The mutants could be phenotypically classified into two major groups, those that resulted in partial or complete overall loss of function and those that seemed to affect substrate specificity. Several of the mutants affecting overall activity mapped in IC1; in particular we identified a segment of four consecutive mutation sensitive residues (TRLT, positions 169-172) with such a phenotype. On the other hand, we identified a cluster of mutants affecting substrate specificity within the short EC2 segment and in the adjacent portion of the neighboring TM4 domain. Expression and partial purification of a representative subset of these mutants showed that in all but two cases, loss of function was associated with loss of drug-induced ATPase activity of P-gp. Therefore, it appears that TM domains, IC and EC loops, are structurally and functionally tightly coupled in the process of drug stimulatable ATPase characteristic of P-gp.
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No. Sentence Comment
191 Group 2 comprised 19 mutants with intermediate phenotypes: Q128H, A136V, Q139H, Q139P, Q139R, E155G, E155K, S176P, K177I, E180G, G183D, D184N, A192T, F200L, F204S, R206L, L210I, T211P, and V213A; these were found throughout the mutagenized segment, with a cluster at the C-terminal portion of IC1.
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ABCB2 p.Phe200Leu 9521654:191:150
status: NEW194 Such mutants showed near wild-type transport Table 1: Summary of Mutations Identifieda TM2 IC1 TM3 EC2 TM4 Q128H R138H F159I S176P G187E R206L L210I Q128R Q139H V161E K177I A192T W208G T211P L134P Q139P H162R N179S F200L K209E V213A A136V Q139R T169I E180G F204S I214L Q145H R170L G181R I214T F147L L171P G183D S224P F148S T172P D184N E155G D174G E155K S176F a Summary of the mutations identified in the current screen together with their position within the predicted secondary structure of P-glycoprotein, with respect to the second (TM2), third (TM3), and fourth (TM4) predicted transmembrane domains together with the first intracellular (IC1) and second extracellular loop (EC2).
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ABCB2 p.Phe200Leu 9521654:194:215
status: NEW