ABCMdb

PMID: 9819232

Hafkemeyer P, Dey S, Ambudkar SV, Hrycyna CA, Pastan I, Gottesman MM
Contribution to substrate specificity and transport of nonconserved residues in transmembrane domain 12 of human P-glycoprotein.
Biochemistry. 1998 Nov 17;37(46):16400-9., 1998-11-17 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCB1 p.Leu975Ala Substitutions including L975A in combination with any one of the other two replacements had the least effect on Pgp function. Login to comment 8 ABCB1 p.Val981Ala ABCB1 p.Phe983Ala The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. Login to comment 19 ABCB1 p.Phe978Ala Replacement of the conserved residues (for example, F978A) within the TM regions has profound effects on the substrate specificity and transport function of Pgp (11). Login to comment 50 ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala Moreover, double mutants based on the triple mutant were cloned (L975A-V981A-F983, L975A-V981-F983A, L975- V981A-F983A). Login to comment 98 ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala Alanine mutants were arranged in such a way that three alanine mutations were putatively located in the outer plasma membrane leaflet (L975A, V981A, and F983A) and four in the inner leaflet (M986A, V988A, Q990A, and V991A), assuming an R-helical structure to TM 12 (Figure 1b). Login to comment 121 ABCB1 p.Gln990Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala Substitution of the four nonconserved amino acid residues (M986A, V988A, Q990A, and V991A) in the carboxy-terminal half of TM 12 had no effect on transport of calcein-AM, rhodamine 123, and bodipy-taxol. Login to comment 124 ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala Double mutants combining pairwise L975A, V981A, and F983A were constructed in order to determine the critical nonconserved residues that were responsible for mediating drug transport in the amino-proximal half of TM 12. Login to comment 125 ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala Those double mutants were L975A-V981A, L975A-F983A, and V981A-F983A. Login to comment 126 ABCB1 p.Gln990Ala ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala The V981-F983 double mutant was Table 1: Properties of Wild-Type and Mutant P-glycoproteinsa drug transporta cell surface expressionc rhodamine daunomycin bodipy-verapamil calcein-AM bodipy-taxol wild-type ++++ ++++ ++++ ++++ ++++ ++++ L975A ++++ ++++ ++++ ++++ ++++ ++++ V981A ++++ ++++ ++++ ++++ ++++ ++++ F983A ++++ ++++ ++++ ++++ ++++ ++++ M986A ++++ ++++ n.d. ++++ ++++ n.d. V988A ++++ ++++ n.d. ++++ ++++ n.d. Q990A ++++ ++++ n.d. ++++ ++++ n.d. V991A ++++ ++++ n.d. ++++ ++++ n.d. L975A, V981A, F983A ++++ no transport no transport ++ ++ ++ M986A, V988A, Q990A, V991A ++++ ++++ +++ ++ ++++ ++++ V981A, F983A ++++ + no transport ++ ++ +++ L975A, F983A ++++ + ++ ++++ +++ ++++ L975A, V981A ++++ ++ no transport ++++ +++ ++++ a Symbols are noted as follows: ++++, wild-type activity; ++, impaired activity; +, residual activity; and n.d., not determined. b Drug transport was determined by FACS. Login to comment 130 ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala Mutant Pgp`s are the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A, - -), the quadruple mutant in the carboxy-terminal half of TM 12 (M986A-V988A-Q990A-V991A, ‚‚‚), the double mutants L975A-V981A (- - -), L975A-F983A (-‚‚-), and V981A-F983A (-‚-). Login to comment 134 ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala The double mutants involving L975A and either V981A and/or F983A were still capable of transporting calcein-AM, bodipy-taxol, and bodipy-verapamil but rhodamine 123 and daunorubicin transport was significantly reduced compared to wild-type Pgp (Figure 4), indicating a significant contribution of L975 to drug specificity. Login to comment 141 ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala Mutant Pgp`s are the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A) and the quadruple mutant (M986A-V988A-Q990A-V991A) (s). Login to comment 144 ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala Photoaffinity labeling demonstrated that the triple mutant L975A-V981A-F983A displayed a significant reduction in binding of IAAP compared to wild-type Pgp which is consistent with its reduced drug transport ability (Figure 5b). Login to comment 146 ABCB1 p.Gln990Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala The quadruple mutant, M986A-V988A-Q990A-V991A, in the carboxy-terminal half still retained a substantial ability to bind IAAP. Login to comment 147 ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala The three double mutants L975A-V981A, L975A-F983A, and V981A-F983A were able to bind IAAP but somewhat less than wild-type Pgp as was also true for the single mutants investigated in this study (Figure 5b,c). Login to comment 153 ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala Mutant MDR1s are double mutants L975A-V981A, L975A-F983A, and V981A-F983A (s). Login to comment 156 ABCB1 p.Val981Ala ABCB1 p.Leu975Ala The double and single mutants displayed near wild-type levels of verapamil-stimulated ATPase activity except for L975A-V981A (Figure 6b). Login to comment 159 ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ATP binding was equivalent for wild-type MDR1 and the MDR1 mutants L975A-V981A-F983A as well as for M986A-V988A-Q990A-V991A (Figure 7). Login to comment 162 ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala Vanadate-trapped [R-32P]-8-azido-ADP was significantly reduced in the triple mutant L975A-V981A-F983A (Figure 8b,c), consistent with the decrease a b c FIGURE 5: Photoaffinity labeling of wild-type and mutant Pgp`s. Login to comment 174 ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala (a) pTM: cells infected with vTF 7-3 virus and transfected with the expression vector containing no MDR1 (pTM1) (negative control), WT (wild-type human MDR1), and mutant Pgp`s are the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A) and the quadruple mutant in the carboxy-terminal half of TM 12 (M986A- V988A-Q990A-V991A). Login to comment 176 ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala Mutant MDR1s are the three double mutants L975A-V981A, L975A-F983A, and V981A-F983A. Login to comment 188 ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala pTM: cells infected with vTF 7-3 virus and transfected with the expression vector containing no MDR1 (pTM1) (negative control), WT (wild-type human MDR1), the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A), the quadruple mutant in the carboxy-terminal half of TM 12 (M986A- V988A-Q990A-V991A), WT (wild-type human MDR1) in the presence of 250 µM EDTA and without MgCl2 to assess the specificity of the 170 kDa band representing Pgp as indicated with the arrow. Login to comment 193 ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala (Middle panel) Vanadate-induced [R-32P]-8-azido-ADP labeling was performed at 37 °C. pTM: cells infected with vTF 7-3 virus and transfected with the expression vector containing no MDR1 (pTM1) (negative control), WT (wild-type human MDR1), the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A), and the quadruple mutant in the carboxy-terminal half of TM 12 (M986A- V988A-Q990A-V991A). Login to comment 215 ABCB1 p.Gln990Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala Changing all four amino acid residues M986A, V988A, Q990A, or V991A in the carboxy-terminal part of TM 12 still allows almost normal transport function of Pgp. Login to comment 227 ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala To determine which combinations of these three residues of TM 12 predicted to lie in the outer leaflet were responsible for the loss of transport activity, we constructed double mutants (L975A-V981A, L975A-F983A, and V981A-F983A). Login to comment 228 ABCB1 p.Gln990Ala ABCB1 p.Gln990Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val981Ala ABCB1 p.Val988Ala ABCB1 p.Val988Ala ABCB1 p.Met986Ala ABCB1 p.Met986Ala ABCB1 p.Val991Ala ABCB1 p.Val991Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala ABCB1 p.Leu975Ala The double mutants showed reduced drug transport suggesting that a change in any two of these three amino Table 2: Properties of Wild-Type and Mutant P-Glycoproteinsa photoaffinity labelinga ATP bindingb ATPase activityc wild-type ++++ ++++ ++++ L975A +++ n.d. +++ V981A +++ n.d. +++ F983A +++ n.d. +++ M986A n.d. n.d. n.d. V988A n.d. n.d. n.d. Q990A n.d. n.d. n.d. V991A n.d. n.d. n.d. L975A,V981A, F983A + ++++ + M986A, V988A, Q990A, V991A ++ ++++ ++ V981A, F983A +++ n.d. +++ L975A, F983A +++ n.d. +++ L975A, V981A +++ n.d. +++ a Symbols are noted as follows: ++++, wild-type activity; ++, impaired activity; +, residual activity; and n.d. not determined. b Photoaffinity labeling was done in HeLa crude membrane preparations using [125 I]iodoarylazidoprazosin. Login to comment 236 ABCB1 p.Val981Ala ABCB1 p.Phe983Ala While no single residue is crucial, combinations of residues, especially V981A and F983A, affect binding of specific substrates such as daunomycin and rhodamine 123. Login to comment 248 ABCB1 p.Phe983Ala F983A in combination with the other substitutions was also found to have a major effect on fluorescent drug transport. Login to comment