ABCMdb

PMID: 21182301

Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PubMed]

Sentences

No. Mutations Sentence Comment

42 ABCB1 p.Gly251Val In this study, the G251V processing mutant was used as a reporter molecule because it exhibits partial maturation (about 15% mature). Login to comment 48 ABCB1 p.Trp232Arg The W232R mutation may stabilize TMD1 as it promotes maturation of a mutant lacking the critical NBD2-TMD1 interface (ΔNBD2- P-gp) (42). Login to comment 49 ABCB1 p.Trp232Arg Whether the W232R mutation could promote maturation of a TMD1 þ 2 mutant lacking both NBDs is unknown. Login to comment 50 ABCB1 p.Ile306Arg Previously identified suppressor mutations in TMD1 (I306R- (TM5) or F343R(TM6)) (40) did not promote maturation of TMD1 þ 2 (unpublished data). Login to comment 54 ABCB1 p.Trp232Arg FIGURE 1: Rescue of P-gp mutants containing processing mutations in different domains by W232R. Login to comment 57 ABCC7 p.Leu1260Ala ABCB1 p.Gly251Val ABCB1 p.Pro709Ala ABCB1 p.Gly722Ala The red balls show the locations of Trp232 and the processing mutations at positions 251 (G251V), 490 (ΔY490), 709 (P709A), 722 (G722A), and 1260 (L1260A). Login to comment 58 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Trp232Ala (B) HEK 293 cells were transfected with A52-tagged wild-type P-gp, mutants G251V, G251V/W232R, and G251V/ W232A,orplasmidvector(control).Wholecellextractsweresubjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment 59 ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg (C) HEK 293 cells were transfected with A52-tagged mutants containing processing mutations in different domains and with or without the W232R mutation ((ΔY490(NBD1) ( W232R, P709A(linker region) ( W232R, G722A(TMD2) ( W232R, or L1260A(NBD2) ( W232R). Login to comment 67 ABCC7 p.Leu1260Ala ABCB1 p.Gly251Val ABCB1 p.Pro709Ala ABCB1 p.Gly722Ala Mutations were introduced into wild-type P-gp or processing mutants containing processing mutations in different domains (G251V in TMD1, ΔY490 in NBD1, P709A in the linker region, G722A in TMD2, or L1260A in NBD2) as described previously (28). Login to comment 84 ABCB1 p.Thr945Ala ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg ABCB1 p.Trp232Ala ABCB1 p.Asn296Ala ABCB1 p.Glu875Ala Baby hamster kidney (BHK) cells expressing A52-tagged wild-type P-gp or mutants G251V, G251V/W232R, W232R, W232A, N296A, E875A, or T945A were generated as described previously (25). Login to comment 91 ABCC7 p.Leu1260Ala ABCB1 p.Gly251Val ABCB1 p.Trp232Arg The G251V or L1260A processing mutations were introduced into Cys-less P-gp containing pairs of cysteines in different domains (L443C(NBD1)/ S909C(TMD2), L531C(NBD1)/C1074(NBD2), or C137(TMD1)/A935C(TMD2)) with or without the W232R mutation. Login to comment 92 ABCC7 p.Leu1260Ala ABCC7 p.Leu1260Ala ABCB1 p.Trp232Arg The L1260A and L1260A/W232R mutations were also introduced into a Cys-less P-gp containing the A266C(TMD1)/ F1086C(NBD2) mutations. Login to comment 99 ABCB1 p.Trp232Arg Membranes were prepared from HEK 293 cells expressing A52-tagged TMD1 þ 2 or TMD1 þ 2(W232R) and suspended in TBS, pH 7.4, at a protein concentration of 5 mg/mL. Login to comment 111 ABCB1 p.Trp232Arg RESULTS W232R Promotes Maturation of Mutants with Processing Mutations in Any Domain. Login to comment 112 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg The W232R suppressor mutation that promotes maturation of the G251V processing mutant is located in TM4 of TMD1 (Figure 1A). Login to comment 114 ABCB1 p.Gly251Val ABCB1 p.Trp232Ala We tested whether a W232A change would also promote maturation of the G251V mutant. Login to comment 116 ABCB1 p.Trp232Arg It was found that only the W232R change promoted maturation of the mutant (Figure 1B, lanes 1-3). Login to comment 117 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg The amount of mature P-gp relative to total (mature plus immature) was 70 ( 6% for mutant W232R/G251V and 92 ( 5% for wild-type P-gp. Login to comment 118 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Ala By contrast, immature protein was the major product in mutants G251V (10 ( 4% mature) and G251V/W232A (13 ( 5% mature). Login to comment 119 ABCB1 p.Gly251Val The results indicate that introduction of the arginine at 232 rather than removal of the tryptophan was responsible for the increase in maturation in mutant G251V. Login to comment 121 ABCC7 p.Arg555Lys ABCC7 p.Arg516Lys ABCC7 p.Arg29Lys ABCC7 p.Arg766Lys ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg Suppressor mutations can rescueΔF508-CFTRbya variety ofmechanisms.Examplesinclude removal of the ER retention signals (arginine-framed trafficking motif mutations; R29K, R516K, R555K, and R766K) (61, 62), introduction of a combination of CFTR suppressor mutations (F949/Q637R or F29S/F494N/Q637R) that increase solubility of NBD1(63),orintroductionofsuppressormutationssuchasV510D (TMD1) (64) and R1070W(TMD2) (65) that restore NBD1-TMD2 interactions. Login to comment 122 ABCC7 p.Asn1303Lys ABCC7 p.Leu1065Pro ABCC7 p.Arg1066Cys ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys In a recent study of four of the CFTR suppressor mutations located in NBD1 (I539T, G550E, R553M, and R555K), it was found that they only restored maturation of mutants that had processing mutations in NBD1 but not those that had processing mutations in other domains such as NBD2 (N1303K) or TMD2 (L1065P or R1066C) (66). Login to comment 123 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg We previously observed that W232R could rescue the ΔY490 (NBD1) and G251V (TMD1) P-gp processing mutants (42). Login to comment 124 ABCB1 p.Trp232Arg We then tested whether W232R could rescue mutants containing processing mutations in other domains. Login to comment 125 ABCC7 p.Leu1260Ala ABCB1 p.Trp232Arg ABCB1 p.Pro709Ala ABCB1 p.Gly722Ala The W232R mutation was introduced into mutants P709A (linker region), G722A (TMD2), or L1260A (NBD2). Login to comment 127 ABCB1 p.Trp232Arg The previously reported ΔY490 and ΔY490/W232R mutants (42) were included for comparison. Login to comment 128 ABCB1 p.Trp232Arg In all cases, introduction of the W232R mutation rescued the processing mutants such that the 170 kDa mature form of P-gp was the major product (Figure 1C). Login to comment 129 ABCB1 p.Trp232Arg The results show that the W232R mutation could rescue mutants with processing mutations in any domain. Login to comment 135 ABCB1 p.Trp232Arg It is possible that the W232R mutation promotes maturation because the charged arginine helps TM4 to insert stably into the membrane. Login to comment 136 ABCB1 p.Gly251Val Therefore, some of the other Trp to Arg changes to flanking tryptophans could also promote maturation of G251V P-gp by helping to anchor other TM segments in the membrane. Login to comment 137 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp315Arg Accordingly, mutants G251V/W212R(TM3), G251V/W315R- (TM5), G251V/W708R(TM7), and G251V/W855R(TM10) were constructed. Login to comment 138 ABCB1 p.Gly251Val ABCB1 p.Trp136Arg The W136R mutation inhibited maturation of G251V as shown previously (42), and the mutant was included in this study as a control (Figure 2B, lanes 8 and 9). Login to comment 141 ABCB1 p.Gly251Val Mutant G251V/T55R(TM1) (Figure 2B, lanes 3 and 4) was included as a negative control because its maturation was unaffected by the presence of cyclosporin A (41). Login to comment 143 ABCB1 p.Gly251Val It was found that none of the other Trp to Arg mutations promoted maturation of the G251V processing mutant in the absence of cyclosporin A (Figure 2B, lanes 8, 10, 14, 16, and 18). Login to comment 144 ABCB1 p.Trp232Arg Indeed, all of the mutations except W232R (Figure 2B, lane 12) inhibited maturation when expression was carried out in the absence of drug substrates (Figure 2C). Login to comment 146 ABCB1 p.Trp232Arg The results show that only the W232R mutation promoted maturation in the absence of cyclosporin A. Login to comment 147 ABCB1 p.Trp315Arg ABCB1 p.Trp136Arg ABCB1 p.Trp212Arg ABCB1 p.Trp708Arg ABCB1 p.Trp855Arg The effect of the W136R, W212R, W315R, W708R, and W855R mutations on maturation of wild-type P-gp was then tested. Login to comment 149 ABCB1 p.Trp708Arg All of the mutants except W708R (Figure 2D, lane 9) expressed the mature 170 kDa protein as the major product (Figure 2D, lanes 1, 3, 5, 7, and 11), and the amount of mature protein increased in the presence of cyclosporin A (Figure 2D, lanes 2, 4, 6, 8, and 12). Login to comment 150 ABCB1 p.Trp708Arg The major product in mutant W708R was the immature 150 kDa protein (Figure 2D, lane 9). Login to comment 152 ABCB1 p.Trp232Arg Effect of W232R on Domain-Domain Interactions. Login to comment 155 ABCC7 p.Leu1260Ala ABCB1 p.Gly251Val ABCB1 p.Trp232Arg To test if the W232R mutation restored domain-domain contacts, it was introduced into the G251V or L1260A processing mutants that also contained pairs of cysteines at the TMD1-TMD2 (C137(TMD1)/A935C(TMD2), NBD1-NBD2 (L531C(NBD1)/C1074(NBD2), or NBD1-TMD2 (L443C(NBD1)/S909C(TMD2) interfaces. Login to comment 156 ABCC7 p.Leu1260Ala ABCC7 p.Leu1260Ala ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Arg The G251V and L1260A parents were used because the G251V mutation is in the same domain as W232R (TMD1) whereas L1260A is in another domain (NBD2). Login to comment 158 ABCC7 p.Leu1260Ala ABCB1 p.Gly251Val ABCB1 p.Trp232Arg Introduction of the W232R mutation into all of the G251V (Figure 3A, lanes 3, 7, and 11) or L1260A (Figure 3B, lanes 3, 7, and 11) double-cysteine processing mutants restored maturation. Login to comment 159 ABCB1 p.Trp232Arg In the absence of the W232R mutation, all of the double cysteine mutants yielded immature protein that did not show cross-linking when treated with the copper phenanthroline (CuP) oxidant (lanes 2, 6, and 10 in Figure 3A,B). Login to comment 160 ABCB1 p.Trp232Arg The mature W232R mutants, however, showed cross-linking between the cysteines when they were treated with oxidant (lanes 4, 8, and 12 in Figure 3A,B). Login to comment 161 ABCB1 p.Trp232Arg The results suggest that the W232R mutation promotes maturation by inducing long-range conformation changes to promote TMD1-TMD2, NBD1-NBD2, and NBD1-TMD2 interactions in P-gp. Login to comment 162 ABCC7 p.Leu1260Ala ABCB1 p.Trp232Arg Since the L1260A mutation is located in the NBD2 (Figure 1A), we also examined the effects of W232R on NBD2-TMD1 interactions. Login to comment 164 ABCC7 p.Leu1260Ala ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys ABCB1 p.Trp232Arg Accordingly, we introduced the W232R mutation into mutant A266C/F1086C/L1260A. Login to comment 166 ABCC7 p.Leu1260Ala ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys ABCB1 p.Trp232Arg Figure 3C shows that the W232R mutation promoted maturation of the mutant A266C/F1086C/L1260A (lane 3) and that only the mature protein was cross-linked (lane 4). Login to comment 167 ABCB1 p.Trp232Arg ABCB1 p.Asn296Ala The N296A Mutation Abolishes W232R Rescue. Login to comment 170 ABCB1 p.Gly251Val (B) Cells transfected with vector (control), A52-tagged wild-type P-gp (WT), mutant G251V cDNA containingT55R, or the indicated Trp to Arg mutation were expressed in the absence (-) or presence (þ) of 10 μM cyclosporin A (cyclo A). Login to comment 177 ABCB1 p.Trp232Arg The model in Figure 1A, however, suggests that W232R must enhance matura- tionbya different mechanismsince itdoes not lie closetoany TM segments in TMD2. Login to comment 179 ABCB1 p.Trp232Arg Therefore, W232R may promote maturation through hydrogen bond interactions with residues in TM5 or TM6. Login to comment 181 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg To test for evidence of hydrogen bond interactions of W232R with residues in TM segments 5, 6, or 12, potential hydrogen bond partners (Thr294(TM5), Asn296(TM5), Ser298(TM5), Ser344(TM6), Gln347(TM6), Ser349(TM6), Ser351(TM6)), (Gln990(TM12), Ser992(TM12), or Ser993(TM12)) (see Figure 4A,B) were mutated to alanine in a G251V/W232R background. Login to comment 182 ABCB1 p.Trp232Arg The rationale was that removal of a hydrogen-bonding partner would abolish the ability of W232R to promote maturation. Login to comment 184 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Asn296Ala It was observed that only one mutation in TM5 (N296A) inhibited maturation of the G251V/W232R mutant when it was expressed in the absence of cyclosporin A (Figure 4C, lane 7). Login to comment 185 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg By contrast, mutation of potential hydrogen-bonding residues in TM6 (Ser344, Q347, S349, or Q351) or TM12 (Gln990, Ser992, or S993) to alanine did not affect maturation of G251V/W232R (Figure 4D). Login to comment 186 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Asn296Ala Introduction of the N296A mutation into G251V/W232R(TM4) caused the mutant to behave in a fashion similar to the original FIGURE 3: Effect of W232R on cross-linking between domains of processing mutants. Login to comment 187 ABCC7 p.Leu1260Ala ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg Membranes were prepared from cells expressing P-gp processing mutants G251V ( W232R (A) or L1260A ( W232R (B) that also contained pairs of cysteines in various domains (L443C(NBD1)/S909C(TMD2), L531C(NBD1)/C1074(NBD2), C137(TMD1)/A935C(TMD2)). Login to comment 188 ABCC7 p.Leu1260Ala ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys ABCB1 p.Trp232Arg Membranes were also prepared from cells expressing the L1260A processing mutant ( W232R containing cysteines in TMD1 and NBD2 (A266C/F1086C) (C). Login to comment 192 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg FIGURE 4: Effect of mutating residues capable of forming hydrogen bonds with W232R on maturation of G251V. Login to comment 194 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Asn296Ala ABCB1 p.Asn296Ala ABCB1 p.Ser298Ala ABCB1 p.Thr294Ala (C) HEK 293 cells transfected with A52-tagged mutant G251V or G251V/ W232R containing alanine mutations in potential hydrogen-bonding residues in TM5 (T294A, N296A, S298A) or G251V/N296A were expressed in the absence (-) or presence (þ) of cyclosporin A (cyclo A). Login to comment 196 ABCB1 p.Gln990Ala ABCB1 p.Gly251Val ABCB1 p.Gln347Ala ABCB1 p.Ser344Ala ABCB1 p.Ser992Ala ABCB1 p.Ser351Ala ABCB1 p.Ser993Ala ABCB1 p.Ser349Ala (D) HEK 293 cells transfected with A52-tagged mutant G251V/W232R(TM4) containing mutations in potential hydrogen-bonding residues in TM6 (S344A, Q347A, S349A, S351A) or TM12 (Q990A, S992A, S993A) were cultured in the absence (-) or presence (þ) of cyclosporin A (cyclo A). Login to comment 199 ABCB1 p.Gly251Val G251V parent. Login to comment 200 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Asn296Ala Like the G251V parent (Figure 4C, lanes 1 and 2), the G251V/W232R/N296A mutant (Figure 4C, lane 7) showed about 15% maturation efficiency when expressed in the absence of drug substrates, and the mature 170 kDa P-gp was the major product when expressed in the presence of cyclosporin A (Figure 4C, lane 8). Login to comment 201 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Asn296Ala Therefore, the presence of the N296A mutation prevented the ability of W232R to promote maturation of the G251V mutant. Login to comment 202 ABCB1 p.Gly251Val ABCB1 p.Asn296Ala Figure 4A (lanes 11 and 12) shows that the N296A change alone had little effect on the maturation characteristics of the G251V parent because it could still be rescued with cyclosporin A. Login to comment 203 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Asn296Ala These results suggest that Arg232(TM4) promotes maturation of the G251V mutant through hydrogen bond interactions with Asn296(TM5) because the N296A mutation inhibited the ability of W232R to promote maturation of G251V (Figure 4C, lane 7). Login to comment 205 ABCB1 p.Trp232Arg If W232R rescues by forming a hydrogen bond with Asn296, then we infer from the models (Figure 4A,B) that it would require a slight rotation of TM5 to bring the Asn side chain closer to the side chainof arginine at position 232. Login to comment 207 ABCB1 p.Gly251Val To test if other mutations to Trp232 would promote maturation, it was changed to other residues capable (Asp, Asn, Ser, Tyr) or incapable (Ala, Gly, Ile, Phe) of forming hydrogen bonds in a G251V background. Login to comment 210 ABCB1 p.Gly251Val No significant increase in maturation efficiency of G251V was observed when Trp232 was changed to amino acids that do not form hydrogen bonds (Ala, Gly, Ile, or Phe). Login to comment 211 ABCB1 p.Gly251Val ABCB1 p.Trp232* The pattern of rescue of the G251V/W232X mutants was consistent with the prediction that hydrogen bond interactions were responsible for the increase in maturation efficiency. Login to comment 212 ABCB1 p.Trp232Arg Rescue by W232R Does Not Require the NBDs. Login to comment 215 ABCB1 p.Trp232Arg Alanine-scanning mutagenesis of potential hydrogen bond partners suggested that W232R rescue involved Asn296 in TM5 (Figure 4C, lane 7). Login to comment 216 ABCB1 p.Trp232Arg Since residues W232R and Asn296 were located in the TMDs, it was possible that the NBDs would not be required for rescue. Login to comment 217 ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg To test if the NBDs were required for rescue, the W232R mutation was introduced into a truncation mutant lacking both NBDs (TMD1 þ 2(W232R)). Login to comment 218 ABCB1 p.Trp232Arg When TMD1 þ 2(W232R) was expressed in HEK 293 cells, it was found that about 50% of the protein was present as mature protein (Figure 6A). Login to comment 219 ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg ABCB1 p.Asn296Ala No detectable mature protein was observed in cells expressing TMD1 þ 2 lacking W232R or with mutant TMD1 þ 2(W232R/N296A) (Figure 6A). Login to comment 222 ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg To test if W232R also converted TMD1 þ 2 into a protease-resistant conformation, membranes prepared from cells expressing TMD1 þ 2 with or without W232R were treated with various concentrations of trypsin. Login to comment 223 ABCB1 p.Gly251Val It was found that the FIGURE 5: Effect of different W232 mutations on maturation of G251V. Login to comment 224 ABCB1 p.Gly251Val (A) HEK 293 cells expressing A52-tagged G251V containing mutations in W232 (-, no change) capable (Arg(R), Asp(D), Asn- (N), Ser(S), Tyr(Y)) or incapable (Ala(A), Gly(G), Ile(I), Phe(F)) of forming hydrogen bonds were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment 226 ABCB1 p.Gly251Val ABCB1 p.Trp232* (B) The amount of mature P-gp (percent mature) relative to total P-gp (170 plus 150 kDa) in mutant G251V/W232X (X = W, R, D, N, S, Y, A, G, I, or F) is shown. Login to comment 227 ABCB1 p.Gly251Val The results are the average values from three separate transfections ( SD. An asterisk indicates significant difference (P < 0.05) from parent (G251V). Login to comment 228 ABCB1 p.Trp232Arg FIGURE 6: Effect of the W232R mutation on maturation and protease sensitivity of a P-gp truncation mutant lacking the NBDs (TMD1 þ 2). Login to comment 229 ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg ABCB1 p.Asn296Ala (A) Whole cell extracts of cells transfected with A52-tagged wild-type, W232R, or W232R/N296A forms of TMD1 þ 2 were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment 230 ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg (B) Membranes prepared from cells expressing A52-tagged wild-type (-W232R) or mutant W232R (þW232R) forms of TMD1 þ 2 were treated with the indicated concentrations of trypsin and samples subjected to immunoblot analysis. Login to comment 232 ABCB1 p.Trp232Arg mature form of TMD1 þ 2(W232R) was 15.5 times more resistant totrypsinrelativetotheimmatureformsoftheprotein(Figure6B). Login to comment 233 ABCB1 p.Trp232Arg Rescue by W232R resembled drug rescue as both converted TMD1 þ 2 to a mature protease-resistant conformation. Login to comment 235 ABCB1 p.Thr945Arg To test if intradomain interactions in TMD2 could also act as a rescue mechanism, we examined the T945R suppressor mutation. Login to comment 236 ABCB1 p.Thr945Arg The T945R mutation was selected as it was one of the most effective suppressor mutations identified in TMD2 (42). Login to comment 238 ABCB1 p.Gly251Val ABCB1 p.Glu875Ala ABCB1 p.Thr945Arg A G251V/T945R/E875A mutant was constructed to test if removal of the negative charge would affect maturation. Login to comment 239 ABCB1 p.Glu875Ala ABCB1 p.Thr945Arg It was found that T945R no longer promoted maturation when the E875A mutation was present (Figure 7C, lane 4). Login to comment 241 ABCB1 p.Gln990Ala ABCB1 p.Ser992Ala ABCB1 p.Ser993Ala Maturation was not reduced by the Q990A, S992A, or S993A TM12 mutations (Figure 7C, lanes 7, 8, and 9). Login to comment 243 ABCB1 p.Gly251Val ABCB1 p.Thr945Glu ABCB1 p.Glu875Arg Therefore, we tested the effect of reversing the charges by constructing mutant G251V/T945E/ E875R. Login to comment 244 ABCB1 p.Gly251Val ABCB1 p.Thr945Glu ABCB1 p.Thr945Glu ABCB1 p.Glu875Arg ABCB1 p.Glu875Arg It was found that the T945E/E875R combination but not individual T945E or E875R mutations promoted maturation of G251V (Figure 7D). Login to comment 245 ABCB1 p.Thr945Arg Since both Glu875 and Thr945 are located in TMD2, we tested if the T945R mutation would promote maturation of the truncation mutant lacking the NBDs (TMD1 þ 2). Login to comment 246 ABCB1 p.Thr945Arg Immunoblot analysis of whole cell extracts of the transfected cells showed that the T945R mutation promoted maturation of TMD1 þ 2 (Figure 7E). Login to comment 251 ABCB1 p.Thr945Arg To test if T945R would promote or inhibit the alternative topology, it was introduced into C-half P-gp. Login to comment 254 ABCB1 p.Thr945Arg Expression of C-half/T945R yielded a single protein product corresponding to unglycosylated P-gp (Figure 8C). Login to comment 255 ABCB1 p.Thr945Arg The C-half/ T945R protein was not glycosylated as it was not sensitive to endoglycosidases (data not shown). Login to comment 256 ABCB1 p.Gly251Val FIGURE 7: Effect of mutations in Thr945 or Glu875 on the rescue of G251V P-gp or mutant TMD1 þ 2. Login to comment 258 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Thr945Arg ABCB1 p.Thr945Arg (C) Whole cell extracts from cells expressing A52-tagged mutant G251V (none) or G251V/T945R (T945R) with the indicated changes topotential hydrogen bondpartnersinTM10(lane4) orTM12(lanes 7-9) were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment 260 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Thr945Glu ABCB1 p.Glu875Arg (D) Whole cell extracts of cells expressing A52-tagged G251V (none) or G251V mutants containing opposite charges at positions 945 and 875 (T945E/E875, T954E, or E875R) were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment 262 ABCB1 p.Thr945Arg (E) Whole cell extracts of cells transfected with A52-tagged wild-type or T945R forms of TMD1 þ 2 were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment 264 ABCB1 p.Thr945Arg FIGURE 8: Effect of T945R mutation on expression of C-half P-gp. Login to comment 267 ABCB1 p.Thr945Arg (C) Whole cell extracts of cells expressing A52-tagged wild-type (None) or T945R forms of C-half P-gp were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment 270 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Arg To test if rescue by an arginine suppressor mutation yielded an active transporter, we examined the ability of mutants G251V and G251V/W232R to confer resistance to cytotoxic compounds colchicine, paclitaxel, and vinblastine. Login to comment 272 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg Stable BHK cell lines expressing wild-type P-gp or mutants G251V, W232R, or G251V/W232R were generated by cotransfecting P-gp cDNAs with pWL-neo vector followed by selection with G418. Login to comment 277 ABCB1 p.Trp232Arg The W232R mutation altered the drug resistance profile as it conferred only a 4-fold increase in resistance to vinblastine but showed relatively high levels of resistance to colchicine (16-fold; P < 0.05) and paclitaxel (19-fold; P < 0.05) (Figure 9A). Login to comment 278 ABCB1 p.Trp232Arg The decreased resistance to vinblastine and increased resistance to colchicine were consistent with the reported effects of W232R on drug-stimulatedATPaseactivity (42).MutantW232Rshowed an increased S50 (concentration required for 50% stimulation of activity) for vinblastine and increased maximum stimulation in the presence of colchicine. Login to comment 279 ABCB1 p.Gly251Val Cells expressing mutant G251V showed almost no increase (P<0.05) in drug resistance compared to control cells. Login to comment 280 ABCB1 p.Gly251Val This is to be expected since mutant G251V is a processing mutant in which the majority of P-gp is misfolded in the cell. Login to comment 281 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg Introduction of the W232R mutation into G251V, however, yielded a functional P-gp. Login to comment 282 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg Cells expressing mutant G251V/W232R were more resistant to colchicine (12-fold; P < 0.05) compared to cells expressing wild-type P-gp (9-fold) but were less resistant to vinblastine (3-fold (P < 0.05) versus 17-fold) and paclitaxel (18-fold (P < 0.05) versus 29-fold). Login to comment 283 ABCB1 p.Gly251Val ABCB1 p.Trp232Arg These results show that the W232R mutation restored maturation of mutant G251V P-gp to yield a functional transporter at the cell surface. Login to comment 285 ABCB1 p.Thr945Ala ABCB1 p.Trp232Ala ABCB1 p.Asn296Ala ABCB1 p.Glu875Ala Stable BHK cell lines expressing mutants W232A, N296A, E875A, or T945A were generated. Login to comment 289 ABCB1 p.Trp232Ala ABCB1 p.Asn296Ala For example, cells expressing mutants W232A and N296A were more resistant to colchicine (P < 0.05) than cells expressing wild-type P-gp, but their resistance to paclitaxel was reduced (P < 0.05). Login to comment 290 ABCB1 p.Thr945Ala Cells expressing mutant T945A were as resistant to vinblastine as cells with wild-type P-gp but showed decreased resistance to colchicine (P < 0.05). Login to comment 297 ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Trp232Arg ABCB1 p.Trp232Arg (A) Stable BHK cell lines expressing no P-gp (control), equivalent levels of wild type, mutants G251V, W232R, or G251V/W232R P-gp were incubated for 6 days in the presence of various concentrations of drug substrates vinblastine (gray bars), colchicine (white bars), or paclitaxel (black bars). Login to comment 299 ABCB1 p.Thr945Ala ABCB1 p.Trp232Ala ABCB1 p.Asn296Ala ABCB1 p.Glu875Ala Each value is average of triplicate assays ( SD. (B) Whole cell extracts of BHK cells expressing vector (control), A52-tagged wild-type, mutant W232A, N296A, E875A, or T945A P-gp were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment 303 ABCB1 p.Thr945Ala ABCB1 p.Glu875Ala Higher concentrations of verapamil were required for half-maximal activation (S50) of ATPase activity for mutants T945A (370 μM) and E875A (>1 mM) compared to wild-type P-gp (48 μM). Login to comment 304 ABCB1 p.Trp232Ala ABCB1 p.Asn296Ala The verapamil-stimulated ATPase activities of mutants W232A and N296A, however, resembled that of wild-type enzyme (S50 of 30 and 42 μM, respectively). Login to comment 306 ABCB1 p.Asn296Ala The maximum activity was increased by 50% in mutant N296A when compared to wild-type P-gp. Login to comment 307 ABCB1 p.Thr945Ala ABCB1 p.Trp232Ala ABCB1 p.Glu875Ala By contrast, the maximum activity of mutants W232A, E875A, and T945A were reduced relative to wild-type P-gp. Login to comment 308 ABCB1 p.Thr945Ala ABCB1 p.Trp232Ala ABCB1 p.Asn296Ala ABCB1 p.Glu875Ala The S50 of mutants W232A (45 μM) and T945A (200 μM) differed from wild-type P-gp (102 μM) while those of mutants N296A (78 μM) and E875A (86 μM) were similar. Login to comment 309 ABCB1 p.Glu875Cys ABCB1 p.Thr945Cys ABCB1 p.Trp232Cys ABCB1 p.Asn296Cys It was not surprising that the mutations affected rhodamine B-stimulated ATPase activity because it was previously shown that treatment of mutants W232C, N296C, and T945C (E875C was not tested) with a thiol-reactive analogue of rhodamine inhibited activity (15). Login to comment 310 ABCB1 p.Thr945Ala ABCB1 p.Trp232Ala ABCB1 p.Trp232Ala ABCB1 p.Asn296Ala ABCB1 p.Asn296Ala ABCB1 p.Glu875Ala In summary, the E875A and T945A mutations altered both verapamiland rhodamine B-stimulated ATPase activity while the W232A and N296A mutations only affected rhodamine B-stimulated ATPase activity. Login to comment 311 ABCB1 p.Thr945Ala ABCB1 p.Trp232Arg ABCB1 p.Thr945Arg ABCB1 p.Thr945Arg The large effect of the T945A mutation on ATPase activity is consistent with the observation that wild-type P-gp containing W232R exhibits verapamiland rhodamine-stimulated ATPase activity whereas wild-type P-gp containing T945R does not (42). Login to comment 313 ABCB1 p.Trp232Arg First, the W232R suppressor mutation located inthe first domain (TMD1) can repair defects in maturation caused by processing mutations located in any other domain. Login to comment 314 ABCB1 p.Gly251Val It also restored maturation of the G251V processing mutant to yield a functional transporter at the cell surface (Figure 9A). Login to comment 316 ABCB1 p.Trp232Arg ABCB1 p.Thr945Arg Second, the NBDs are not required for rescue by W232R or T945R. Login to comment 329 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys It appears that the ΔF508 mutation inhibits folding of NBD1 and its ability to stably associate with other domains resulting in altered CFTR-chaperone interactions, ER retention,andenhanceddegradation(65).Second-sitesuppressor mutations in NBD1 (such as I539T/G550E/R553M/R555K) can restore interdomain assembly (65, 66) to yield a more stable ΔF508-CFTR molecule (64, 66). Login to comment 331 ABCC7 p.Arg1070Trp For example, the R1070W mutation was postulated to promote folding through hydrophobic interactions with NBD1 in ΔF508-CFTR (65). Login to comment 332 ABCC7 p.Val510Asp Similarly, it was suggested that the V510D suppressor mutation in NBD1 promoted folding of ΔF508-CFTR by forming a salt bridge with Arg1070 (64). Login to comment 333 ABCC7 p.Gly550Glu Other suppressor mutations in NBD1 such as G550E appear to rescue ΔF508-CFTR by altering the conformation of NBD1 (62). Login to comment 336 ABCB1 p.Thr945Ala ABCB1 p.Trp232Ala ABCB1 p.Asn296Ala ABCB1 p.Glu875Ala Histidine-tagged wild-type or mutant W232A, N296A, E875A, or T945A P-gp was expressed in HEK 293 cells and isolated by nickel-chelate chromatography. Login to comment 350 ABCB1 p.Trp232Arg P-gp TMD1 þ 2 could be rescued to yield mature protein in a protease-resistant conformation by the W232R mutation. Login to comment 352 ABCB1 p.Trp232Arg Therefore, one possible explanation for the ability of the W232R- (TM4) mutation to promote maturation of the P-gp processing mutants was that the positively charged side chain of arginine could serve as a better anchor for TM4. Login to comment 357 ABCB1 p.Thr945Arg Strong support for the prediction that intradomain interactions between TM segments can modulate topologywas theobservation that T945R inhibited the formation of an alternative topology in C-half P-gp when it was expressed alone (Figure 8C). Login to comment 362 ABCB1 p.Trp232Arg Multiple topologies have been detected (34-37), and drug substrates (8) or suppressor mutations like W232R (this study) can convert the domains from protease-sensitive to protease- resistantconformations.TheplasticityoftheTMDsmaycontribute to alterations in the substrate specificity of P-gp since they contain the drug-binding sites. Login to comment 366 ABCB1 p.Glu875Cys It was previously reported that residue Glu875 could contribute to folding of TM segments because the E875C mutation altered the topology of C-half Pgp (45). Login to comment 368 ABCB1 p.Thr945Cys ABCB1 p.Trp232Cys ABCB1 p.Asn296Cys These results are consistent with those from cysteine mutagenesis studies where modification of mutant T945C by thiol-reactive analogues of verapamil or rhodamine or mutants W232C and N296C by a thiol reactive analogue of rhodamine inhibited their activity (13, 15). Login to comment 386 ABCB1 p.Gly251Val Evidence for mobility of the TM segments is suggested by the large number of cross-links observed between TM segments in a cysteine mutagenesis study (51), that drug binding occurs through an induced-fit mechanism (93), that ATP hydrolysis can cause rotation of one or more helices (94), and that other residues at position 232 whose side chains are of different sizes and capable of forming hydrogen bonds (Asp, Ser, Tyr) could rescue G251V (Figure 5). Login to comment