ABCB3 p.Ala192Thr
| Predicted by SNAP2: | C: N (72%), D: D (53%), E: D (53%), F: N (57%), G: N (57%), H: D (53%), I: N (61%), K: D (59%), L: N (53%), M: N (61%), N: N (66%), P: N (53%), Q: N (61%), R: D (53%), S: N (78%), T: N (78%), V: N (61%), W: D (66%), Y: N (61%), |
| Predicted by PROVEAN: | C: D, D: 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, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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.
Comments [show]
None has been submitted yet.
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.
X
ABCB3 p.Ala192Thr 9521654:191:143
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).
X
ABCB3 p.Ala192Thr 9521654:194:173
status: NEW50 Mutants Q128R, Q139H, F147L, E155L, T169I, T172P, G181R, A192T, W208G, L210I, and S224P were introduced into pHILD2mdr3 by the direct replacement of an internal 1.6 kb Afl II/Sma I mdr3 cDNA subfragment (pst 170 to 1764) by the corresponding mutated mdr3 cDNA segment in pVTM3IC1.
X
ABCB3 p.Ala192Thr 9521654:50:57
status: NEW197 Other mutants in this group including A192T and L210I retained robust activity against FK506 and VAL, but showed a severe reduction in mating activity.
X
ABCB3 p.Ala192Thr 9521654:197:38
status: NEW223 The P. pastoris expression system was used to express and functionally characterize a representative subset of 11 loss of function mutants mapping to the various structural domains targeted for random mutagenesis: Q128R, Q139H, F147L, E155K, T169I, T172P, G181R, A192T, W208G, L210I, and S224P.
X
ABCB3 p.Ala192Thr 9521654:223:263
status: NEW252 Two other mutants showed VRP and VAL-stimulated specific activities that were intermediate: T172P (53%, 26% of WT) and A192T (45%, 29% of WT).
X
ABCB3 p.Ala192Thr 9521654:252:119
status: NEW256 The Km of the VRP stimulated ATPase activity was found to be in the same range for the mutants F147L, T172P, A192T, and W208G, which was between 0.6 and 1.0 mM and similar to the WT isoform.
X
ABCB3 p.Ala192Thr 9521654:256:109
status: NEW270 Table 2: ATPase Activities of P-gp Mutantsa no drug, Vmax Vmax VRP stimulation KM pHILD2 0.037 0.031 0.8 ND WT 0.028 0.078 2.8 0.6 Q128R 0.022 0.034 1.6 ND Q139H 0.023 0.028 1.3 ND F147L 0.039 0.081 2.0 0.8 E155K 0.033 0.034 1.0 ND T169I 0.014 0.017 1.2 ND T172P 0.032 0.056 1.7 0.9 G181R 0.032 0.033 1.1 ND A192T 0.035 0.052 1.5 0.6 W208G 0.046 0.100 2.2 0.9 L210I 0.042 0.046 1.1 ND S224P 0.032 0.032 1.0 ND a Summary of kinetic analysis performed on a representative sample of mutants.
X
ABCB3 p.Ala192Thr 9521654:270:308
status: NEW312 The most extreme examples of such mutations were Q139H, Q139R, F147L, and A192T that retained close to wild-type activity against one or more substrates but showed complete loss of function toward another one.
X
ABCB3 p.Ala192Thr 9521654:312:74
status: NEW328 This group consisted of mutants in TM2 (Q128R), IC1 (Q139H, F147L, E155K, T169I, T172P, G181R), TM3 (A192T), EC2 (W208G), and TM4 (L210I, S224P).
X
ABCB3 p.Ala192Thr 9521654:328:101
status: NEW