ABCMdb

PMID: 8104183

Loo TW, Clarke DM
Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein.
J Biol Chem. 1993 Sep 25;268(27):19965-72., 1993-09-25 [PubMed]

Sentences

No. Mutations Sentence Comment

37 ABCB1 p.Phe777Ala ABCB1 p.Phe777Ala We found that changes to three phenylalanineresidues altered the activityof P-glycoprotein. Mutationof either Phe335 or Phe-978to Ala resultedinmutantsthatconferred altereddrugresistance profilessuggesting thatthese two residues play critical roles in drug transport activity.A Phe-777 to Ala changeresultedin a mutantproteinthat was defective in processing, suggesting that this residue is important for the biosynthesisof the protein. Login to comment 119 ABCB1 p.Phe978Ala ABCB1 p.Phe335Ala Functional Consequencesof Other Changes to Phe-335 and Phe-978"Mutation of either Phe-335 or Phe-978 to alanine had aprofound effecton the ability of P-glycoprotein to confer resistance to various cytotoxic compounds. Login to comment 120 ABCB1 p.Phe335Ser ABCB1 p.Phe978Ser To test theeffects of other changes to these two residues, site-directed mutagenesis was used to change either Phe-335 or Phe-978 to serine (polar residue), leucine (small non-polar residue),or to tyrosine (aromatic residue withpolar side chain). Login to comment 126 ABCB1 p.Phe978Ser ABCB1 p.Phe978Tyr ABCB1 p.Phe978Leu Mutants with changes of Phe-978 to Ser, Leu, or Tyr all retained the ability to confer resistance to vinblastine, since drug-resistantcolonies were obtained after transfection with the corresponding mutant cDNAs. Login to comment 127 ABCB1 p.Phe978Ser No colchicine-resistant colonies, however, were observed after transfection of cells with cDNAs coding for mutants Phe-978 Ser or Leu. Login to comment 134 ABCB1 p.Phe335Ala Mutation of Phe-335 to Ala orSer actually reversed the preferential resistance of P-glycoprotein to vinblastine relative to colchicine. Login to comment 135 ABCB1 p.Phe335Ala ABCB1 p.Phe335Ser A vinblastine/colchicine ratio of 4.1 was obtained for the wild-type enzyme, whereas mutants Phe335 to Ala or Ser hadvalues of 0.59and 0.44respectively. Login to comment 160 ABCB1 p.Phe335Ala Vinblastine Phe335-->Ala Wild-type -->Leu -->Ser Phe978-->Ah -->Tyr -->Ser ">Leu -->Tyr Colchicine 1 Adriamycin Actinomycin D 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 Relative Resistance FIG. 5. Comparision of relative resistances of stable cell lines expressing mutants Phe-335or Phe-978 +Ala, Ser, Leu, or Tyr. Login to comment 162 ABCB1 p.Phe335Leu ABCB1 p.Phe335Tyr Phe335 to Leu or Tyr gave vinblastine/colchicine ratios of 1.1and 3.5, respectively. Login to comment 166 ABCB1 p.Phe978Leu Substitution of Phe-978 with Leu had an intermediate effect; drug transport was impaired for all four drugs with the greatest reduction occurring in the presence of colchicine and adriamycin. Login to comment 179 ABCB1 p.Phe335Ala 100 F335A -80 60 40 20 0 ' 1I I I I 10 100 1000 Vinblastine(ug/ml) FIG.7. Login to comment 183 ABCB1 p.Phe978Ala 0,wild-type;0,mutant Phe-335+Ala;W, mutant Phe-978 to Ala. Login to comment 221 ABCB1 p.Pro223Ala We found that mutation of Pro-223 to Ala altered the substrate specificity of the transporter. Mutationof Pro-866, which resides in an equivalent position in the COOH-terminal half of P-glycoprotein when the two halves are aligned, also altered the substrate specificity of the enzyme. Login to comment 243 ABCB1 p.Phe335Ala ABCB1 p.Phe335Ser It has been found that mutations of Pro-223 toAla, Pro-866 toAla (Loo and Clarke, 1993), Phe-978 toAla or Ser (this study), and Ser-941 to Phe (Groset al., 1991) yield proteins that have reduced ability to conferresistanceto colchicine butretainthecapacity to conferresistancetovinblastine.The oppositeeffect is observed for mutation of Phe-335 to Ala or Ser (this study) or Gly-185 to Val (Choi et al., 1988). Login to comment