ABCMdb

PMID: 9287132

Taguchi Y, Morishima M, Komano T, Ueda K
Amino acid substitutions in the first transmembrane domain (TM1) of P-glycoprotein that alter substrate specificity.
FEBS Lett. 1997 Aug 11;413(1):142-6., [PubMed]

Sentences

No. Mutations Sentence Comment

42 ABCB1 p.Ala63Arg Cells transfected by He60 -> Arg, or Ala63 -> Arg cDNA were selected with stepwise increasing concentrations (10, 20, 40 ng/ml) of vinblastine and finally maintained in 30 ng/ml Vbl. Login to comment 43 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg Cells transfected with Gly64 -> Arg, or Leu65 -> Arg cDNAs were selected with stepwise increasing concentrations (5, 10, 20 ng/ml) of vinblastine and finally maintained in 20 ng/ ml vinblastine. Login to comment 50 ABCB1 p.His61Arg In the previous study, we showed that the replacements of His61 by Arg changed the substrate specificity of P-glycoprotein drastically [13]. Login to comment 55 ABCB1 p.His61Arg ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg ABCB1 p.Ala63Arg From cells transfected with the wild type and He60 -> Arg, His61 -> Arg, Ala63 -> Arg, Gly64 -> Arg, or Leu65 -> Arg mutant cDNA, both Vbl- and Col-resistant colonies were obtained (Fig. 1). Login to comment 57 ABCB1 p.His61Arg In contrast, cells transfected with mutants His61 -> Arg slightly more resistant colonies in the presence of Col than in the presence of Vbl. Login to comment 58 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg Noticeably, cells transfected with mutant Gly64 -> Arg or Leu65 -> Arg yielded comparable number of Col-resistant colonies, but much less Vbl-resistant colonies, suggesting that the substrate specificities of these two mutants were altered from that of the wild type. Login to comment 68 ABCB1 p.Leu65Arg Membrane proteins were obtained from stable transformants maintained in 20 ng/ml vinblastine for mutants Gly64 -»Arg and Leu65 -> Arg, or 30 ng/ml vinblastine for wild type and the other mutants. Login to comment 72 ABCB1 p.Ala57Arg ABCB1 p.Ala58Arg We also failed to detect the mature form of P-glycoprotein, when Ala57 -> Arg, Ala58 -> Arg, Ile59 ->Arg, and Gly62 -> Arg mutant cDNAs were transiently expressed in HEK 293 cells (data not shown). Login to comment 74 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg The drug-resistance profiles of the cells expressing He60 -> Arg, His61 -»Arg, Ala63 -»Arg, Gly64 -> Arg, and Leu65 -> Arg mutant P-glycoproteins were investigated by comparing their relative resistance to Vbl, Col, VP16, and Adr (Fig. 3). Login to comment 75 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg The drastic alterations in the drug resistance profile were observed with Gly64 -> Arg or Leu65 -> Arg mutant as well as His61 -»Arg mutant. Login to comment 77 ABCB1 p.Ala63Arg In contrast, the resistance profile of He60 -»Arg or Ala63 -> Arg mutant P-glycoprotein did not altered much from that of the wild type. Login to comment 82 ABCB1 p.Leu65Arg From each of membrane fraction, a band migrating at about 170 kDa was detected, indicating that He60 -> Arg, His61 -»Arg, Ala63 -»Arg, Gly64 -»Arg, and Leu65 -> Arg mutant P-glycoproteins were normally processed and expressed in the plasma membrane. Login to comment 86 ABCB1 p.Ala57Arg ABCB1 p.Ala58Arg However, from cells transfected with Ala57 -> Arg, Ala58 -> Arg, He59 -> Arg, or Gly62 -> Arg mutant cDNA, no Vbl- or Col-resistant colonies were obtained (Fig. 1). Login to comment 87 ABCB1 p.Gly54Val ABCB1 p.Ala58Leu It was reported [21] that no Vbl- or Col-resistant colonies were obtained from cells transfected with either Gly54 -> Val, Ala58 -> Leu, or Gly62 -> Val mutant P-glycoprotein cDNA, and that the major products from these three mutant cDNAs were protein with an apparent mass of 150 kDa when their cDNAs were transiently expressed in HEK 293 cells, suggesting that these mutations in TMl affected the proper folding In this study, we examined whether the substitution of amino acids near His61 in TMl affect substrate specificity of P-glycoprotein. Login to comment 89 ABCB1 p.Leu65Arg The mutant P-glycoproteins, in which either He60 , Ala63 , Gly64 , or Leu65 was replaced by Arg were successfully expressed in the plasma membrane of KB3-1 cells and were functional as drug transporters as the His61 mutants. Login to comment 90 ABCB1 p.Leu65Arg ABCB1 p.Ala63Arg The substrate specificities of Gly64 -»Arg and Leu65 -> Arg mutant P-glycoproteins were quite different from that of the wild type, and similar to that of His61 -»Arg mutant, while He60 and Ala63 -> Arg mutant P-glycoproteins showed similar substrate specificities to that of the wild-type P-glycoprotein. Login to comment 92 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg The similarity among the substrate specificities of Gly64 -> Arg, Leu65 -> Arg, and His61 -> Arg mutant P-glycoproteins suggest that the amino acid residues at position 64 and 65 are also important in deciding the substrate specificity. Login to comment 106 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg The substrate specificities of the Gly64 -> Arg and Leu65 -> Arg mutants were similar to, but not exactly same with that of the His61 -> Arg mutant. Login to comment 107 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg The cells expressing Gly64 -> Arg or Leu65 -> Arg mutant were more resistant to VP16 than to Vbl, while cells expressing the His61 -* Arg mutant were more resistant to Vbl than to VP16 (Fig. 3). Login to comment