ABCB1 p.Phe978Cys

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PMID: 19456124 [PubMed] Crowley E et al: "Transmembrane helix 12 modulates progression of the ATP catalytic cycle in ABCB1."
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6 Two of the mutations (L976C and F978C) were found to reduce the binding of [γ-32 P]-azido-ATP to ABCB1.
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ABCB1 p.Phe978Cys 19456124:6:32
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67 This necessitated the centrifugation (100000g for 30 min) of 500 μL Table 1: Mutagenic Oligonucleotide Primers Used To Generate TM12 Mutationsa mutation primer sequence (50 -30 ) diagnostic restriction digest L976C GAGGATGTTCTAtgtGTATTTTCAGCTGTTG -SpeI F978C GTTCTACTAGTATgTTCtGCaGTTGTCTTTGGTG +PstI A980C CTACTAGTATTTTCAtgcGTTGTCTTTGGTGCCATGGCC -PvuII V982C CTAGTATTTTCAGCgGTTtgCTTTGGTGCCATGGCC -PvuII G984C GCTGTTGTCTTTtGTGCtATGGCCGTGG -NcoI M986C GTATTTGGTGCttgtGCtGTGGGGCAAGTC -NcoI V988C GGTGCCATGGCCtgtGGGCAAGTCAGTTC -BstXI G989C CTTTGGTGCCATGGCCGTGtGcCAAGTCAGTTCATTTGC +BstXI Q990C GGCCGTGGGGtgtGTCtcTTCATTTGCTCC +EarI a Primer sequences contain an introduced cysteine residue (bold) and additional silent mutations (lower case), with respect to the coding sequence that generates or removes the indicated restriction site.
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ABCB1 p.Phe978Cys 19456124:67:259
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118 The most striking alterations were the approximately 5-fold reductions in basal ATPase activity for the L976C (Vmax=26 ( 4 nmol min-1 mg-1 )and F978C (Vmax=27 ( 9 nmol min-1 mg-1 ) mutations compared to the control cysteine-less isoform of ABCB1.
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ABCB1 p.Phe978Cys 19456124:118:144
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141 L976C (Vmax = 231 ( 80 nmol min-1 mg-1 ), F978C (Vmax = 142 ( 40 nmol min-1 mg-1 ), V988C, G989C, and Q990C all caused statisticallysignificant (p<0.05) reductionsinthe Vmax valuesfor nicardipine-stimulated ATPase activities (Figure 4B).
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ABCB1 p.Phe978Cys 19456124:141:42
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146 However, the F978C isoform did display an altered nicardipine-ABCB1 interaction; that is, the potency of nicardipine was reduced from 4.1 ( 1.1 to 24.1 ( 2.3 μM. Vinblastine also stimulates the ATPase activity of ABCB1.
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ABCB1 p.Phe978Cys 19456124:146:13
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149 For example, mutations L976C, F978C, V988C, and Q990C all displayed statistically significant reductions in maximal ATPase activity in the presence of vinblastine compared to the control cysteine-less isoform.
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ABCB1 p.Phe978Cys 19456124:149:30
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150 However, of these, only the F978C isoform again displayed a reduction in the potency for stimulation of its ATPase activity by vinblastine, although the degree of stimulation remained unaffected.
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ABCB1 p.Phe978Cys 19456124:150:28
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155 Table 2: Potency and Degree of Drug Stimulation of ATP Hydrolysis by ABCB1a nicardipine vinblastine EC50 (μM) fold stimulation EC50 (μM) fold stimulation Cys-less 4.1 ( 1.1 4.0 ( 0.6 5.91 ( 2.9 2.2 ( 0.2 L976C 5.2 ( 0.2 7.4 ( 1.4 10.0 ( 0.0 3.5 ( 0.6 F978C 24.1 ( 2.3b 9.5 ( 1.4 42.9 ( 4.3b 2.3 ( 0.5 A980C 3.4 ( 0.3 5.1 ( 0.9 12.3 ( 1.8 3.2 ( 0.8 V982C 5.8 ( 0.9 4.2 ( 0.5 2.0 ( 0.7 1.8 ( 0.2 G984C 37.6 ( 11.2b 16.2 ( 6.6b 6.7 ( 1.7 6.2 ( 2.3 M986C 9.2 ( 0.8 4.7 ( 1.1 15.0 ( 2.0b 2.8 ( 0.7 V988C 3.9 ( 0.6 3.1 ( 0.1 7.3 ( 2.3 1.9 ( 0.2 G989C 13.6 ( 1.5 5.1 ( 1.6 4.9 ( 0.9 2.4 ( 0.3 Q990C 6.9 ( 1.1 3.7 ( 1.0 NDc NDc S992C 4.9 ( 0.5 4.2 ( 0.6 7.1 ( 2.6 2.3 ( 0.4 F994C 1.7 ( 0.4 3.2 ( 0.8 5.9 ( 2.5 1.6 ( 0.3 a ATPase activity was plotted as a function of the drug concentration and potency (EC50) and degree of stimulation obtained by nonlinear regression of the dose-response relationship equation.
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ABCB1 p.Phe978Cys 19456124:155:263
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172 Figure 5A presents a representative autoradiogram of [γ-32 P]-azido-ATP binding to the mutants L976C, F978C, A980C, V988C, G989C, and Q990C.
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ABCB1 p.Phe978Cys 19456124:172:108
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174 The results indicate that at a concentration of 10 μM [γ-32 P]-azido-ATP there was a discernible difference between the binding of the ATP analogue to L976C, F978C, and A980C.
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ABCB1 p.Phe978Cys 19456124:174:170
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175 Mutant isoforms L976C and F978C displayed a 4and 15-fold decrease in binding, whereas A980C showed a 1.8-fold increase in binding at the concentration of [γ-32 P]-azido-ATP used.
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ABCB1 p.Phe978Cys 19456124:175:26
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179 Figure 5C shows the densitometric analysis of the dose-response curve for the cysteine-less, A980C, L976C, and F978C isoforms.
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ABCB1 p.Phe978Cys 19456124:179:111
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181 In contrast, the mutant isoforms L976C (Bmax = 0.24, KD = 17 μM) and F978C (Bmax = 0.19, KD =17 μM) displayed 4-5-fold reductions in the amount of [γ-32 P]-azido-ATP binding compared to the cysteine-less control.
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ABCB1 p.Phe978Cys 19456124:181:75
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182 Therefore, the decrease in basal and drug-stimulated ATPase activity observed for the L976C and F978C isoforms can be explained at least in part by impaired ATP binding at the NBDs.
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ABCB1 p.Phe978Cys 19456124:182:96
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187 To investigate whether such differences are a reflection of a physical asymmetry between the helices and their surroundings, we attempted to rationalize three of the functionally perturbed TM12 single cysteine mutants (i.e., F978C, A980C, and V988C) byreference toamolecularmodel ofABCB1.
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ABCB1 p.Phe978Cys 19456124:187:225
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193 (C) Data obtained for the cysteine-less, L976C, F978C, and A980C isoforms were analyzed by densitometry, and the amount bound was plotted as a function of the [γ-32 P]-azido-ATP concentration.
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ABCB1 p.Phe978Cys 19456124:193:48
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203 The F978C mutation, located near the extracellular protein-lipid interface in the membrane-embedded region of TM12, is part of a conserved sequence motif present in TM12 and TM6.
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ABCB1 p.Phe978Cys 19456124:203:4
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207 However, unlike the TM12 F978C mutation, the TM6 F335C mutation does not cause a significant reduction in ATPase activity (15).
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ABCB1 p.Phe978Cys 19456124:207:25
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208 In TM12, mutation to the smaller 978C increases the distance between F978C and F72, preventing hydrophobic interactions (Figure 6D) and decreasing the number of TM12-TM1 contact points.
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ABCB1 p.Phe978Cys 19456124:208:69
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210 Interestingly, the modeled mutation also alters the orientation of the F978C side chain, resulting in the complete exposure of the cysteine side chain into a pore below L975, which has been implicated in dibromobimane binding (47).
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ABCB1 p.Phe978Cys 19456124:210:71
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220 The most dramatic effects in TM12 were observed in residues at the extracellular Table 3: Nucleotide Binding to ABCB1a ABCB1 isoform [32 P]-N3-ATP [32 P]-N3-ATP+ 1 mM ATP [32 P]-N3-ATP+ 1 mM ADP Cys-less 1.00 0.21 ( 0.05 0.23 ( 0.06 L976C 0.21 ( 0.05 0.10 ( 0.02 0.05 ( 0.03 F978C 0.07 ( 0.01 ND ND A980C 1.81 ( 0.71 0.45 ( 0.10 0.15 ( 0.08 V988C 0.53 ( 0.20 ND ND G989C 0.83 ( 0.04 0.10 ( 0.05 0.13 ( 0.06 Q990C 1.05 ( 0.30 0.19 ( 0.11 0.01 ( 0.01 a The ABCB1 isoforms were incubated with 10 μM [γ32 P]-azido-ATP in the presence or absence of excess unlabeled nucleotides (1 mM).
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ABCB1 p.Phe978Cys 19456124:220:275
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231 (D) Mutation to F978C (CPK) prevents hydrogen bonding to F72.
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ABCB1 p.Phe978Cys 19456124:231:16
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232 (L976C and F978C) and intracellular (V988C and Q990C) ends of the helix.
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ABCB1 p.Phe978Cys 19456124:232:11
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234 In contrast, the reduced basal activity observed with mutants L976C and F978C appeared to be a consequence of reduced nucleotide binding.
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ABCB1 p.Phe978Cys 19456124:234:72
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PMID: 8910331 [PubMed] Loo TW et al: "Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates."
No. Sentence Comment
66 Accordingly, site-directed mutagenesis was used to change the codon for each residue surrounding Phe-335 and Phe-978 to cysteine (Fig. 1B).
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ABCB1 p.Phe978Cys 8910331:66:109
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156 A, membranes prepared from cells transfected with vector alone (control) or cotransfected with cDNA for mutant L332C in the Cys-less NH2-terminal half-molecule A52 and the cDNA for mutant L975C in the Cys-less COOH-terminal half-molecule A52 or with cDNA for mutant F335C in the NH2-terminal half-molecule A52 and the cDNA for mutant F978C in the COOH-terminal half-molecule A52 were treated with oxidant for various intervals and at different temperatures.
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ABCB1 p.Phe978Cys 8910331:156:334
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PMID: 9405384 [PubMed] Loo TW et al: "Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate."
No. Sentence Comment
81 One mutant, F335C (TM6) showed enhanced activity (280%), whereas the equivalent residue in TM12 (F978C) showed decreased activity (31%).
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ABCB1 p.Phe978Cys 9405384:81:97
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PMID: 24349290 [PubMed] Chufan EE et al: "Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1)."
No. Sentence Comment
55 For biochemical studies, crude membranes were prepared from High-Five insect cells infected with baculovirus coding for cysless WT, single mutants Y307C, F343C, Q725C, F728C, F978C, V982C, double mutants Y307C/V982C, F343C/V982C, Q725C/V982C, F728C/V982C, and a triple mutant Y307C/Q725C/V982C.
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ABCB1 p.Phe978Cys 24349290:55:175
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69 One mutant, F978C, behaves in a manner similar to that of cysless WT Pgp, with only a change in the binding affinity for CsA, IC50 = 0.54 &#b5;M, compared to 0.05 &#b5;M for cysless WT Pgp (Table 1).
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ABCB1 p.Phe978Cys 24349290:69:12
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71 Similarly, tariquidar inhibits IAAP labeling of F978C to the Figure 1.
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ABCB1 p.Phe978Cys 24349290:71:48
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82 However a majority of the mutants show low basal activity; F728C/V982C shows the lowest (4 &#b1;1.6 nmol Pi/min/mg protein) while V982C and F978C shows fairly low activity (11 &#b1; 2.2 and 13 &#b1; 0.6, respectively).
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ABCB1 p.Phe978Cys 24349290:82:140
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90 Mutation(s) CsA Tariquidar Max Inhibition (%) IC50 (&#b5;M) Max Inhibition (%) IC50 (&#b5;M) Cysless WT 86 &#b1; 3 0.05 &#b1; 0.01 97 &#b1; 4 0.14 &#b1; 0.03 Q725C 24 &#b1; 4 -- 37 -- Q725C/V982C 11 -- 22 -- Y307C 35 &#b1; 2 -- ND -- Y307C/V982C 46 -- ND -- F728C 48 -- 40 -- F728C/V982C 49 -- ND -- V982C 56 0.40 64 0.70 Y307C/Q725C/ V982C 12 -- 23 -- F978C 86 0.54 73 3.6 Mean values with standard errors are reported when more than two experiments were carried out; otherwise only average values are reported.
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ABCB1 p.Phe978Cys 24349290:90:353
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175 Mutation(s) Cell surface expression Transport function CalAM BD-PRA NBD-CsA Rh123 Dauno BD-PAC Y307C 100 90-100 80-90 90-100 90-100 90-100 90-100 Q725C 100 90-100 90-100 90-100 90-100 90-100 90-100 F728C 100 90-100 80-90 90-100 90-100 90-100 90-100 V982C 100 90-100 80-100 <10 90-100 90-100 90-100 F343C 50-60 90-100 80-100 90-100 90-100 90-100 90-100 F978C 100 90-100 90-100 90-100 90-100 90-100 90-100 Y307C/ V982C 100 90-100 50-60 50-60 90-100 90-100 90-100 Q725C/ V982C 100 90-100 80-90 <20 90-100 90-100 90-100 F728C/ V982C 30-40 55-65 30-40 <20 70-80 50-60 90-100 F343C/ V982C 70-80 90-100 80-90 <20 90-100 90-100 90-100 Y307/ Q725C/ V982C 100 90-100 30-40 50-60 70-80 60-70 90-100 For cell surface expression, the cells were incubated with MRK-16 antibody for 30 min followed by FITC-labeled anti-mouse secondary antibody for 30 min.
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ABCB1 p.Phe978Cys 24349290:175:352
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