ABCMdb

PMID: 17848563

Loo TW, Bartlett MC, Clarke DM
Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites.
J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11., 2007-11-02 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCB1 p.Ile306Arg ABCB1 p.Phe343Arg The folding defect in ⌬F508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (⌬Y490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Login to comment 7 ABCB1 p.Leu65Arg ABCB1 p.Ile306Arg ABCB1 p.Ile306Arg ABCB1 p.Phe343Arg ABCB1 p.Phe343Arg ABCB1 p.Thr199Arg The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. Login to comment 40 ABCB1 p.Ile306Arg ABCB1 p.Phe343Arg Printed in the U.S.A. NOVEMBER 2, 2007•VOLUME 282•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 32043 essing mutants by a direct mechanism because suppressor mutations (arginines) introduced into TM segments 5 (I306R) and 6 (F343R) also promoted maturation of the protein (21). Login to comment 69 ABCB1 p.Leu65Arg ABCB1 p.Ile306Arg ABCB1 p.Phe343Arg ABCB1 p.Thr199Arg For disulfide cross-linking analysis, the cDNA of mutant L339C(TM6)/ F728C(TM7) (34) was modified to also encode the L65R, T199R, I306R, or F343R mutations. Login to comment 86 ABCB1 p.Leu339Cys ABCB1 p.Ile306Arg Disulfide Cross-linking Analysis-The double cysteine mutants L339C(TM6)/F728C(TM7), L65R(TM1)/L339C(TM6)/ F728C(TM7), T199R(TM3)/L339C(TM6)/F728C(TM7), I306R (TM5)/L339C(TM6)/F728C(TM7), or F343R(TM6)/L339C (TM6)/F728C(TM7) were transiently expressed in HEK 293 cells (32). Login to comment 94 ABCB1 p.Gly251Val ABCB1 p.Gly251Val Limited Proteolysis with Trypsin-HEK 293 cells expressing mutants G251V, L65R(TM1)/G251V, or wild-type P-gp were incubated in the presence or absence of 10 ␮M cyclosporin A for 24 h. The membranes were then prepared as described previously (37) and suspended in Tris-buffered saline. Login to comment 99 ABCB1 p.Phe343Tyr ABCB1 p.Ile306Ala Some changes in TM5 (I306A or G) or in TM6 (F343Y) in the ⌬Y490 P-gp mutant also caused the mutant protein to be unstable because cells expressing these mutants contained relatively large amounts of a 130-kDa degradation product (21). Login to comment 101 ABCB1 p.Phe343Tyr In cells expressing mutant F343Y/⌬Y490, the 130-kDa protein was the major product. Login to comment 102 ABCB1 p.Phe343Arg By contrast, mutant F343R/⌬Y490 yielded about equivalent amounts of the mature 170-kDa and immature 150-kDa proteins and very little 130-kDa product. Login to comment 103 ABCB1 p.Ile306Arg ABCB1 p.Phe343Arg Therefore, the presence of arginine mutations in TM5 (I306R) or TM6 (F343R) might have promoted maturation of ⌬Y490 P-gp by influencing distal folding events. Login to comment 112 ABCB1 p.Gly251Val The positions of ⌬Y490 and G251V processing mutations are shown as gray circles. Login to comment 147 ABCB1 p.Thr199Cys Fig. 2A shows that all of the mutants except T199C had activities similar to that of Cys-less P-gp (less than 1.5-fold increase). Login to comment 148 ABCB1 p.Thr199Cys Mutant T199C showed a 6.7-fold increase in activity after treatment with MTS-rhodamine. Login to comment 154 ABCB1 p.Gln195Cys ABCB1 p.Thr199Cys The mutants Q195C and T199C, however, exhibited different properties than Cys-less P-gp after treatment with MTS-rhodamine. Login to comment 155 ABCB1 p.Gln195Cys Whereas mutant Q195C showed a 6.4-fold stimulation of ATPase activity with rhodamine B before treatment with MTS-rhodamine, its activity could only be stimulated 1.3-fold after treatment with MTS-rhodamine. Login to comment 157 ABCB1 p.Thr199Cys By contrast, covalent modification of mutant T199C with MTS-rhodamine fully activated its ATPase activity such that rhodamine B had no further effect on its activity. Login to comment 158 ABCB1 p.Thr199Cys If MTS-rhodamine activates P-gp ATPase activity because it occupies the rhodamine-binding site when it is attached to Cys199 , then labeling of mutant T199C should be protectable with rhodamine B. Login to comment 160 ABCB1 p.Thr199Cys Histidine-tagged mutant T199C was expressed in HEK 293 cells, solubilized with detergent, and reacted with various concentrations of MTS-rhodamine. Login to comment 162 ABCB1 p.Thr199Cys To test whether rhodamine B protected mutant T199C from labeling, HEK 293 cells expressing the histidine-tagged mutant were solubilized with detergent and treated with 1 mM MTS-rhodamine in the presence or absence of 5 mM rhodamine B for 10 min at 20 °C. Login to comment 163 ABCB1 p.Thr199Cys The P-gps were isolated by nickel-chelate chromatography and mixed with lipids, and ATPase activities were determined. Fig. 3B shows that ATP hydrolysis was reduced by 78% when mutant T199C was reacted with MTS-rhodamine in the presence of 5 mM rhodamine B. Login to comment 164 ABCB1 p.Leu65Arg ABCB1 p.Thr199Arg Effect of L65R and T199R Mutations on Maturation of a P-gp Processing Mutant-We then tested whether arginines introduced into TM1 (Leu65 ) (27) or TM3 (Thr199 ) promoted maturation of a P-gp processing mutant. Login to comment 165 ABCB1 p.Ile306Arg ABCB1 p.Phe343Arg ABCB1 p.Gly251Val Processing mutant G251V was chosen because it yields very low levels of mature 170-kDa protein and could be rescued by the presence of arginine in TM5 (I306R) or in TM6 (F343R) (21). Login to comment 166 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val Accordingly, mutants L65R(TM1)/G251V, T199R(TM3)/G251V, I306R(TM5)/G251V (positive control), and A342R(TM6)/G251V (negative control) were constructed and expressed in HEK 293 cells. Whole cell SDS extracts were then subjected to immunoblot analysis. Fig. 4A shows that the presence of an arginine residue at positions 65(TM1), 199(TM3), and 306(TM5) promoted maturation of mutant G251V. Login to comment 168 ABCB1 p.Phe194Cys ABCB1 p.Met192Cys ABCB1 p.Gln195Cys ABCB1 p.Gln195Cys ABCB1 p.Phe193Cys ABCB1 p.Gly191Cys ABCB1 p.Ile190Cys ABCB1 p.Thr199Cys ABCB1 p.Thr199Cys ABCB1 p.Met197Cys ABCB1 p.Gly203Cys ABCB1 p.Phe200Cys ABCB1 p.Thr202Cys ABCB1 p.Thr209Cys ABCB1 p.Ala198Cys ABCB1 p.Phe201Cys ABCB1 p.Ser196Cys ABCB1 p.Ile205Cys ABCB1 p.Phe208Cys ABCB1 p.Gly207Cys Fold-stimulation 1 2 3 4 5 6 Cys-less I190C G191C M192C F193C F194C Q195C S196C M197C A198C T199C F200C F201C T202C G203C F204C I205C V206C G207C F208C T209C A 7 Fold-stimulation 1 2 3 4 5 6 Cys-less B _ + MTS-rhod Rhod B+ _ + Q195C + + _ + T199C + + _ + _ _+ + + 7 FIGURE 2. Login to comment 173 ABCB1 p.Gln195Cys ABCB1 p.Thr199Cys B, Cys-less, Q195C and T199C P-gp mutants were treated with (ϩ) or without (-) 2 mM MTS-rhodamine and histidine-tagged P-gp isolated by nickel-chelate chromatography. Equivalent amounts of P-gp were mixed with lipid, and ATPase activity was determined in the presence (ϩ) or absence (-) of 2 mM rhodamine B (Rhod B). Login to comment 177 ABCB1 p.Thr199Cys Labeling of mutant T199C with increasing concentrations of MTS-rhodamine and protection by rhodamine B. Login to comment 178 ABCB1 p.Thr199Cys A, HEK 293 cells expressing histidine-tagged mutant T199C were solubilized with n-dodecyl-beta-D-maltoside, and insoluble material was removed by centrifugation. Login to comment 185 ABCB1 p.Gly251Val Arginines Disrupt Subset of P-gp Drug-binding Sites NOVEMBER 2, 2007•VOLUME 282•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 32047 By contrast, the presence of an arginine at position 342(TM6) did not rescue mutant G251V. Login to comment 193 ABCB1 p.Leu339Cys Accordingly, mutants L65R(TM1)/L339C(TM6)/F728C(TM7), T199R(TM3)/L339C (TM6)/F728C(TM7), I306R(TM5)/L339C(TM6)/F728C(TM7), and F343R(TM6)/L339C(TM6)/F728C(TM7) were constructed and expressed in HEK 293 cells. Login to comment 198 ABCB1 p.Ile306Arg The presence of the I306R mutation, however, caused a large reduction in the apparent affinity for vinblastine (42 Ϯ 9 ␮M; 60-fold) and for cyclosporin A (15 Ϯ 3 ␮M; 25-fold) and had little effect on the apparent affinity for rhodamine B (96 Ϯ 11 ␮M; 1.1-fold). Login to comment 204 ABCB1 p.Leu65Arg Effect of L65R Mutation on Protease Sensitivity of P-gp-In a previous study (37), we observed that expression of P-gp processing mutants in the presence of drug substrates appeared to convert the protein from a loosely folded conformation to a more compact structure because they became about 100-fold more resistant to trypsin. Login to comment 207 ABCB1 p.Gly251Val Effect of arginine mutations on maturation of the G251V and ⌬Y490 processing mutants. Login to comment 208 ABCB1 p.Gly251Val Wild-type, G251V (A), or ⌬Y490 (B) P-gps containing mutations L65R(TM1), T199R(TM3), I306R(TM5), or A342R(TM6) were expressed in HEK 293 cells. Whole cell SDS extracts were subjected to SDS-PAGE on 5.5% gels and immunoblot analysis. Login to comment 215 ABCB1 p.Gly251Val ABCB1 p.Gly251Val The positions of mature (170 kDa) and cross-linked (X-link) P-gps are indicated. Arginines Disrupt Subset of P-gp Drug-binding Sites 32048 prepared from cells expressing mutant G251V that were grown in the absence or presence of cyclosporin A, mutant L65R(TM1)/G251V, or wild-type P-gp. Login to comment 217 ABCB1 p.Ile306Arg ABCB1 p.Ile306Arg ABCB1 p.Ile306Arg ABCB1 p.Ile306Arg ABCB1 p.Ile306Arg ABCB1 p.Ile306Arg The reactions were stopped by the addition of trypsin inhibitor, and the samples were subjected to SDP-PAGE on 6% gels followed A B I306 I306R X-link 170 kDa X-link 170 kDa 0 0.08 0.7 2.2 6.6 19.8 59 178 533 1600 0.24 [Vin] µM 0.1 1 10 100 [Vinblastine] (µM) PercentCross-linked 20 40 60 80 100 0 I306R I306 1000 C D I306 I306R X-link 170 kDa X-link 170 kDa 0 0.007 0.06 0.18 0.54 1.6 4.9 14.6 44 132 0.02 [Cyclo] µM 0.01 0.1 1 10 [Cyclosporin] (µM) PercentCross-linked 20 40 60 80 100 0 I306R I306 100 E F I306 I306R X-link 170 kDa X-link 170 kDa 0 0.6 5.5 16.5 49 148 444 1333 4000 1.8 [Rhod B] µM 1 10 100 1000 [Rhodamine B] (µM) PercentCross-linked 20 40 60 80 100 0 I306R I306 10000 FIGURE 6. Login to comment 227 ABCB1 p.Gly251Val Arginines Disrupt Subset of P-gp Drug-binding Sites NOVEMBER 2, 2007•VOLUME 282•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 32049 by immunoblot analysis. Fig. 7 shows that the mature 170-kDa protein in mutant G251V expressed in the presence of cyclosporin A was 100-fold more resistant to trypsin compared with the mutant expressed in the absence of drug substrate. Login to comment 228 ABCB1 p.Gly251Val ABCB1 p.Gly251Val Similarly, the L65R(TM1)/G251V mutant and wild-type mature proteins were 100-fold more resistant to trypsin when compared with the 150-kDa protein of mutant G251V. Login to comment 229 ABCB1 p.Leu65Arg ABCB1 p.Gly251Val These results indicate that the L65R mutation converts mutant G251V into a more protease-resistant conformation. Login to comment 234 ABCB1 p.Leu65Arg For example, the L65R mutation only caused a large reduction (57-fold) in the apparent affinity for vinblastine and little change in affinities for cyclosporin A or rhodamine B. Login to comment 236 ABCB1 p.Phe343Arg The F343R mutation exhibited decreased apparent affinity for rhodamine B (3.6-fold) and cyclosporin A (18-fold) but not for vinblastine (Ͻ2-fold). Login to comment 240 ABCB1 p.Gly251Val ABCB1 p.Gly251Val Protease sensitivity of mutants G251V and L65R(TM1)/ G251V. Login to comment 241 ABCB1 p.Gly251Val ABCB1 p.Gly251Val Membranes prepared from HEK 293 cells expressing mutant G251V that were grown in the absence or presence (ϩCyclo A) of cyclosporin A, mutant L65R(TM1)/G251V or wild-type P-gps were treated with various concentrations of TPCK-trypsin. Login to comment 269 ABCB1 p.Thr199Cys The labeling of Cys199 also appeared to be specific for MTS-rhodamine because the ATPase activity of mutant T199C was not activated by MTS-verapamil. Login to comment 270 ABCB1 p.Leu65Arg By contrast, the ATPase activity of mutant L65R was activated by treatment with MTS-verapamil (27) but not by MTS-rhodamine (Ͻ2-fold activation after treatment with 2 mM MTS-rhodamine) (data not shown). Login to comment 271 ABCB1 p.Thr199Arg It is possible, however, that residue 199 lies some distance away from the actual rhodamine B binding site because the T199R mutation did not reduce the apparent affinity for rhodamine B in the cross-linking protection assay (Table 1). Login to comment 273 ABCB1 p.Thr199Arg The residue at position 199 in TM3, however, does appear to lie close to the vinblastine-binding site because the T199R mutation reduced the apparent affinity for vinblastine by 6.4-fold (Table 1). Login to comment 276 ABCB1 p.Ile306Arg For example, it appeared that vinblastine interactions with mutant I306R were completely abolished because it did not stimulate ATPase activity at vinblastine concentrations of up to 200 ␮M (45). Login to comment 278 ABCB1 p.Ile306Arg The results from the cross-linking protection assay, however, showed that mutant I306R could still interact with vinblastine but with reduced affinity (Fig. 6A). Login to comment