ABCMdb

PMID: 8995353

Loo TW, Clarke DM
Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.
J Biol Chem. 1997 Jan 10;272(2):709-12., 1997-01-10 [PubMed]

Sentences

No. Mutations Sentence Comment

51 ABCB1 p.Gly268Val RESULTS Effect of Drug Substrates on Processing of Misfolded Mutants-The effect of substrates and modulators of P-glycoprotein on the biosynthesis of two processing mutants were initially studied; G268V in the NH2-terminal transmembrane domain (16) and ⌬Y490 in the NH2-terminal nucleotide-binding domain (17). Login to comment 52 ABCB1 p.Gly268Val Mutant G268V is a temperature-insensitive processing mutant, whereas mutant ⌬Y490 contains a deletion at an equivalent position to the ⌬F508 mutation in CFTR. Login to comment 64 ABCC7 p.Ser434Cys ABCB1 p.Gly268Val ABCB1 p.Gly427Cys ABCB1 p.Ala841Leu ABCB1 p.Gly251Val ABCB1 p.Gly854Val ABCB1 p.Gly54Val ABCB1 p.Glu707Ala ABCB1 p.Ala718Leu ABCB1 p.Trp803Ala In addition to the mutants G268V and ⌬Y490, we were able to facilitate processing of P-glycoproteins with mutations in the predicted transmembrane segments (TM1, G54V; TM5, G300V; TM7, A718L; and TM9, A841L), in the extracellular loops between transmembrane segments (G854V), in the cytoplasmic loops (G251V and W803A), in the nucleotide-binding domains (G427C and S434C), and in the linker region connecting the two halves of the molecule (E707A) (data not shown). Login to comment 73 ABCB1 p.Gly268Val Fig. 2A shows that after 4 h in the presence of 15 ␮M cyclosporin A, about 50% of mutant G268V was present as the fully mature (170-kDa) form of the enzyme and that after 24 h, more than 80% of the mutant protein was present in the fully mature form. Login to comment 77 ABCB1 p.Gly268Val Fig. 2B shows that in the absence of cyclosporin A, the 150-kDa P-glycoprotein of mutant G268V was not processed to the mature enzyme. Login to comment 79 ABCB1 p.Gly268Val In the presence of cyclosporin A, however, the kinetics of maturation of the P-glycoprotein of mutant G268V was similar to that of wild-type enzyme. Login to comment 102 ABCB1 p.Gly268Val By contrast, most of the P-glycoprotein of mutants G268V and ⌬Y490 grown without drug substrate were recovered in the flow-through fractions during nickel-chelate chromatography. Login to comment 107 ABCB1 p.Gly268Val Similarly, drug-stimulated ATPase activity was detected in the mutant G268V after expression in the presence of cyclosporin A. Login to comment 108 ABCB1 p.Gly268Val The observation that mutant G268V exhibits reduced activity is consistent with previous observations that several glycine to valine mutations in the cytoplasmic loops of P-glycoprotein also alter the substrate specificity of the enzyme (16). Login to comment 110 ABCB1 p.Gly268Val Time-dependent appearance of the 170-kDa (mature) form of mutant P-glycoprotein-A52 (G268V). Login to comment 111 ABCB1 p.Gly268Val A, HEK 293 cells were transfected with A52-tagged mutant G268V cDNA and incubated for 24 h at 37 °C. Login to comment 114 ABCB1 p.Gly268Val B, HEK 293 cells were transfected with wild-type or mutant G268V P-glycoprotein-A52 cDNAs or vector alone (Control). Login to comment 137 ABCB1 p.Gly714Ala For example, some misfolded P-glycoprotein mutants such as G714A are temperatureand glycerol- sensitive only when stably expressed in NIH 3T3 cells but not when expressed in HEK 293 cells (data not shown). Login to comment 140 ABCB1 p.Gly268Val ABCB1 p.Gly268Val ABCB1 p.Gly251Val ABCB1 p.Gly251Val ABCB1 p.Glu707Ala ABCB1 p.Glu707Ala Another interesting observation is that misfolded mutants that are temperatureand glycerol-insensitive, such as G251V, G268V, and E707A could also be rescued by these drug substrates when expressed in either HEK 293 or NIH 3T3 cells (data not shown). Login to comment