ABCB1 p.Met68Arg
| Predicted by SNAP2: | A: D (59%), C: D (53%), D: D (80%), E: D (80%), F: D (53%), G: D (66%), H: D (75%), I: N (53%), K: D (85%), L: N (66%), N: D (66%), P: D (85%), Q: D (63%), R: D (80%), S: D (66%), T: D (71%), V: N (72%), W: D (80%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: N, 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] Arginines in the first transmembrane segment promo... J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2. Loo TW, Bartlett MC, Clarke DM
Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11.
J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PMID:18596043]
Abstract [show]
A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.
Comments [show]
None has been submitted yet.
No. Sentence Comment
48 The most efficient rescue was observed with the M68R mutant.
X
ABCB1 p.Met68Arg 18596043:48:48
status: NEW55 For disulfide cross-linking analysis, the cDNA of mutant L339C(TM6)/F728C(TM7) (16) was modified to also encode the G64R, M68R, or V71R mutations.
X
ABCB1 p.Met68Arg 18596043:55:122
status: NEW103 Mutants M68R and L65R showed the highest increase in maturation efficiency (85 and 60% mature 170-kDa mature, respectively) (Fig. 2).
X
ABCB1 p.Met68Arg 18596043:103:8
status: NEW118 Mutants G251V, V53R/G251V (inhibited maturation) and M68R/G251V (enhanced maturation) were subjected endoglycosidase H or F digestion to determine whether the alterations in mobility of the mutant P-gps were indeed due to changes in the glycosylation state of the protein.
X
ABCB1 p.Met68Arg 18596043:118:53
status: NEW125 Because the M68R mutation was most efficient in promoting maturation of the G251V processing mutant, we tested whether it could also promote maturation of a different processing mutant.
X
ABCB1 p.Met68Arg 18596043:125:12
status: NEW126 Accordingly, the M68R mutation was introduced into the ⌬NBD2- P-gp processing mutant (20).
X
ABCB1 p.Met68Arg 18596043:126:17
status: NEW128 Mutants ⌬NBD2- P-gp, M68R/⌬NBD2-P-gp, and ⌬NBD2-P-gp lacking the three glycosylation sites (N91A, N94A, N99A) were expressed in HEK 293 cells, and whole cell SDS extracts were subjected to immunoblot analysis.
X
ABCB1 p.Met68Arg 18596043:128:28
status: NEW129 Fig. 4 shows that the M68R mutant yielded an extra P-gp protein that corresponded in size to the mature protein (Fig. 4).
X
ABCB1 p.Met68Arg 18596043:129:22
status: NEW130 Treatment with endoglycosidases H and F showed that the immature form (100 kDa) was sensitive to endoglycosidases H and F, whereas the slower migrating M68R protein (115 kDa) was sensitive to endoglycosidase F but not endoglycosidase H (data not shown).
X
ABCB1 p.Met68Arg 18596043:130:152
status: NEW138 Whole cell SDS extracts of HEK 293 cells expressing A52-tagged mutants G251V, V53R/G251V, or M68R/G251V were treated with endoglycosidase Hf (H),peptideN-glycosidaseF(F)ornoendoglycosidase(-).Equivalentamountsweresubjectedtoimmunoblot analysis.
X
ABCB1 p.Met68Arg 18596043:138:93
status: NEW149 Mutants L65R and M68R yielded mature 170-kDa P-gp as the major product in the absence of drug substrates.
X
ABCB1 p.Met68Arg 18596043:149:17
status: NEW155 Effect of Suppressor Arginine Mutations on P-gp-Drug Interactions-Because mutant M68R/G251V showed efficient maturation (about 85%) when expressed in the absence of drug substrates (Fig. 2), we tested whether Arg-68 affected P-gp- drug interactions in disulfide protection assays using mutant L339C(TM6)/F728C(TM7) as described previously (9).
X
ABCB1 p.Met68Arg 18596043:155:81
status: NEW159 Effect of the M68R mutation on maturation of the ⌬NBD2 processing mutant.
X
ABCB1 p.Met68Arg 18596043:159:14
status: NEW160 Whole cell extracts of HEK 293 cells expressing mutants ⌬NBD2-P-gp with no changes (None), the M68R mutation, or N91A, N94A and N99A changes to the glycosylation sites (Unglycos) were subjected to immunoblotanalysis.Thepositionsofmature,immature,andunglycosylated(Unglycos) forms of ⌬NBD2 P-gp are indicated.
X
ABCB1 p.Met68Arg 18596043:160:102
status: NEW165 TABLE 1 Effects of drug substrates on the maturation of TM1 arginine mutants containing the G251V mutation Mutation (G251V ؉) No drug Cyclosporin A Verapamil Vinblastine Rhodamine None - 111a,c 111 111 11 V52R -a,b 111 111 111 11 V53R 2 - - - - G64R 2 - - - - T55R 2 - - - - L56R 2 - - - - A57R 2 - - - - A58R 2 - - - - I59R 2 - - - - I60R 2 - - - - H61R - 111 1 1 1 G62R 2 - - - - A63R 2 - - - - G64R 1 111 1 1 11 L65R 11 111 11 11 11 P66R 2 1 - - - L67R - 111 111 111 11 M68R 111 111 111 111 111 M69R - 111 - 11 111 L70R - 111 111 111 11 V71R 1 111 111 111 11 F72R 2 1 1 1 1 a Change in the amount of mature (170 kDa) protein in the presence of drug substrate relative to that in the absence of drug substrate.
X
ABCB1 p.Met68Arg 18596043:165:479
status: NEW173 Membranes were prepared from HEK 293 cells expressing mutants L339C(TM6)/F728C(TM7), G64R/ L339C(TM6)/F728C(TM7), M68R/ L339C(TM6)/F728C(TM7), and V71R/L339C(TM6)/F728C(TM7).
X
ABCB1 p.Met68Arg 18596043:173:114
status: NEW177 A representative example of the effects of various concentrations of vinblastine on cross-linking of mutant M68R/L339C(TM6)/ F728C(TM7) by M14M is shown in Fig. 6.
X
ABCB1 p.Met68Arg 18596043:177:108
status: NEW179 The M68R/ L339C(TM6)/F728C(TM7) mutant, however, required 266 M vinblastine (Ͼ500-fold increase) to inhibit cross-linking by 50%.
X
ABCB1 p.Met68Arg 18596043:179:4
status: NEW183 The G64R, L65R, and M68R mutations preferentially affected P-gp-vinblastine interactions.
X
ABCB1 p.Met68Arg 18596043:183:20
status: NEW188 Effect of the M68R mutation on inhibition of cross-linking by vinblastine.
X
ABCB1 p.Met68Arg 18596043:188:14
status: NEW189 A, membranes were prepared from HEK 293 cells expressing mutants L339C(TM6)/F728C(TM7) or M68R/L339C(TM6)/F728C(TM7).
X
ABCB1 p.Met68Arg 18596043:189:90
status: NEW194 TABLE 2 Concentrations of drug substrates required to inhibit cross-linking by 50% Mutant Vinblastine Cyclosporin A Rhodamine B M M M L339C/F728C 0.5 Ϯ 0.2a 0.8 Ϯ 0.3 52 Ϯ 14 G64R/L339C/F728C 53 Ϯ 15b 1.5 Ϯ 0.4 61 Ϯ 10 M68R/L339C/F728C 266 Ϯ 62b 1.3 Ϯ 0.3 57 Ϯ 7 V71R/L339C/F728C 0.6 Ϯ 0.2 0.8 Ϯ 0.2 57 Ϯ 10 a Each value is the mean Ϯ S.D. (n ϭ 3-4 separate cross-linking experiments).
X
ABCB1 p.Met68Arg 18596043:194:279
status: NEW197 Therefore, we measured drug stimulation of ATPase activity of mutants G64R and M68R using various concentrations of verapamil to test whether the apparent affinity for verapamil of the P-gp mutants was altered.
X
ABCB1 p.Met68Arg 18596043:197:79
status: NEW200 Accordingly, mutations G64R or M68R were introduced into a wild-type P-gp that contained a histidine tag at the COOH-terminal end.
X
ABCB1 p.Met68Arg 18596043:200:31
status: NEW201 Histidine-tagged wild-type and mutants G64R and M68R P-gps were expressed in HEK 293 cells, and the proteins were isolated by nickel-chelate chromatography.
X
ABCB1 p.Met68Arg 18596043:201:48
status: NEW205 Verapamil also stimulated the ATPase activities of mutants G64R and M68R (1.6 and 1.8 mol Pi/min/mg of protein, respectively).
X
ABCB1 p.Met68Arg 18596043:205:68
status: NEW206 Although both mutants G64R and M68R showed relatively high activation of ATPase activity with verapamil, their apparent affinities for verapamil were reduced by about 19-and 4-fold, respectively.
X
ABCB1 p.Met68Arg 18596043:206:31
status: NEW207 Half-maximal activation of the ATPase activities of mutants G64R and M68R required 540 m and 120 M verapamil, respectively, compared with 29 m for wild-type P-gp.
X
ABCB1 p.Met68Arg 18596043:207:69
status: NEW208 Effect of TM11 Mutations on Rescue of Mutant G251V Containing the L65R and M68R Changes-One reason that Arg-68 caused the largest increase in maturation of mutant G251V is that the arginine residue may promote packing of the TM segments.
X
ABCB1 p.Met68Arg 18596043:208:75
status: NEW214 To test if Tyr-950 or Tyr-953 influences the ability of the M68R mutation to promote maturation of the G251V mutant, we introduced the Y950A or Y953A mutations into mutant M68R/ G251V mutant.
X
ABCB1 p.Met68Arg 18596043:214:60
status: NEWX
ABCB1 p.Met68Arg 18596043:214:172
status: NEW216 Fig. 8 shows the maturation efficiencies compared with the M68R/G251V parent.
X
ABCB1 p.Met68Arg 18596043:216:59
status: NEW218 Not all mutations in TM11, however, affected maturation of the M68R/G251V mutant.
X
ABCB1 p.Met68Arg 18596043:218:63
status: NEW219 The presence of mutations Q946A, M948A, M949A, F951A, or S952A did not appear to affect the maturation of the M68R/ G251V mutant (Fig. 8A, lanes 2-5, 7, and 8).
X
ABCB1 p.Met68Arg 18596043:219:110
status: NEW222 In contrast, both Y950A and Y953A affected the maturation efficiency of the M68R/G251V mutant.
X
ABCB1 p.Met68Arg 18596043:222:76
status: NEW229 Mutants L65R/G251V/Y950F, M68R/G251V/ Y950A/Y953A, and M68R/G251V/Y950F/Y953F were constructed, and the cDNAs were expressed in HEK 293 cells.
X
ABCB1 p.Met68Arg 18596043:229:26
status: NEWX
ABCB1 p.Met68Arg 18596043:229:55
status: NEW231 Immunoblot analysis shows the Y950A/Y953A and Y950F/Y953F changes in M68R/G251V (Fig. 8A, lanes 11 and 12) or Y950F change in L65R/G251V (Fig. 8B, lane 5) reduced maturation to levels similar to that observed in the G251V parent.
X
ABCB1 p.Met68Arg 18596043:231:69
status: NEW233 Verapamil-stimulated ATPase activities of wild-type and mutant G64R and M68R P-gps.
X
ABCB1 p.Met68Arg 18596043:233:72
status: NEW264 Effect of TM11 mutations on maturation of M68R/G251V and L65R/G251V mutants.
X
ABCB1 p.Met68Arg 18596043:264:42
status: NEW265 A, HEK 293 cells were transfected with mutants M68R/G251V (lanes 2-12) or G251V (lanes 1, 13-15) containing the indicated TM11 mutations.
X
ABCB1 p.Met68Arg 18596043:265:47
status: NEW276 Mutants H61R/G251V or M69R/G251V showed little drug rescue with verapamil (Fig. 5), and mutants G64R and M68R in a wild-type background showed reductions in apparent affinity for verapamil in ATPase assays (Fig. 7).
X
ABCB1 p.Met68Arg 18596043:276:105
status: NEW280 The observation that the M68R mutation can reduce the apparent affinities for the drug substrates verapamil and vinblastine would be unexpected because it appears that P-gp initially interacts with drug substrates in the inner leaflet of the bilayer (44).
X
ABCB1 p.Met68Arg 18596043:280:25
status: NEW281 One explanation is that the M68R mutation causes an indirect effect on the vinblastine and verapamil binding sites.
X
ABCB1 p.Met68Arg 18596043:281:28
status: NEW282 The structural alterations may be small because the M68R change did not appear to affect P-gp interactions with cyclosporin A or rhodamine B (Table 2).
X
ABCB1 p.Met68Arg 18596043:282:52
status: NEW283 Another explanation is that the M68R mutation has altered the predicted "OFF-sites" of P-gp.
X
ABCB1 p.Met68Arg 18596043:283:32
status: NEW285 Because the OFF-sites would be involved in drug release, they may be located in the outer portions of P-gp and would be sensitive to mutations such as M68R.
X
ABCB1 p.Met68Arg 18596043:285:151
status: NEW298 Some mutations such as G64R, L65R, M68R, and V71R actually promoted maturation of the G251V mutant.
X
ABCB1 p.Met68Arg 18596043:298:35
status: NEW299 The L65R and M68R mutations were particularly effective in promoting maturation as they increased maturation efficiency of the G251V mutant from about 20 to 60-85%.
X
ABCB1 p.Met68Arg 18596043:299:13
status: NEW304 An alternative possibility to explain why the L65R and M68R mutations were particularly effective in promoting maturation of the G251V mutant is that they promoted packing of the TM segments by forming hydrogen bonds with Tyr-950 and/or Tyr-953 located in TM11.
X
ABCB1 p.Met68Arg 18596043:304:55
status: NEW308 This possibility is supported by the observations that the conservative Y950F and Y953F mutations reduced maturation of the M68R/G251V mutant to levels observed with the G251V parent.
X
ABCB1 p.Met68Arg 18596043:308:124
status: NEW314 In P-gp, the introduction of the L65R or M68R mutations appears to promote folding by enhancing TM1-TM11 interactions through hydrogen bond formation with the Tyr-950 and/or Tyr-953 in TM11.
X
ABCB1 p.Met68Arg 18596043:314:41
status: NEW[hide] Mapping the Binding Site of the Inhibitor Tariquid... J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26. Loo TW, Clarke DM
Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.
J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26., [PMID:26507655]
Abstract [show]
ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.
Comments [show]
None has been submitted yet.
No. Sentence Comment
175 In the N-terminal TMD1 domain, the largest number of arginine mutations predicted to line the drug-binding pocket that inhibited tariquidar rescue were located in TM1 (H61R, G64R, L65R, M68R, M69R, and F72R) and TM5 (F303R, I306R, Y307R, S309R, and Y310R) (Fig. 4, A and E).
X
ABCB1 p.Met68Arg 26507655:175:186
status: NEW193 It was found that 16 of the 28 mutants resembled the G251V/I868R mutant as expression in the presence of 5 òe;M cyclosporine A yielded mature P-gp as the major product in TM1 (H61R, G64R, L65R, M68R, and M69R), TM5 (F303R, I306R, and S309R), TM7 (Q725R and F728R), TM10 (I868R and G872R), TM11 (F942R, T945R, and Q946R), and TM12 (V982R) (Fig. 7).
X
ABCB1 p.Met68Arg 26507655:193:198
status: NEW212 Seventeen of the 30 G251V/arginine mutants (M68R, M69R, and F72R in TM1; I306R, Y307R, S309R, and Y310R in TM5; F336R in TM6; F728R and F732R in TM7; I868R and G872R in TM10; F942R, T945R, M949R, and S952R in TM11; and V982R in TM12) that could not be rescued with tariquidar showed little or no stimulation of ATPase activity with tariquidar (Fig. 8A).
X
ABCB1 p.Met68Arg 26507655:212:44
status: NEW283 We identified 13 additional arginine mutations (H61R, G64R, L65R, and M68R in TM1; A129R in TM2; I306R and S309R in TM5; F343R in TM6; F942R, T945R, Q946R, and Y950R in TM11; and L975R in TM12) (Fig. 11A) that were not predicted to lie within 4.5-&#c5; of the predicted site 3 tariquidar-binding site (9).
X
ABCB1 p.Met68Arg 26507655:283:70
status: NEW