Functional consequences of glycine mutations in the predicted cytoplasmic loops of P-glycoprotein.
Site-directed mutagenesis was used to investigate whether glycine residues in the predicted cytoplasmic loops play essential roles in the structure and function of human P-glycoprotein. Mutant cDNAs in which codons for each of the 20 glycine residues were changed to valine, were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutation of Gly-251, -268, -269, or -781 yielded mutant proteins which were unable to confer drug resistance in transfected cells. Each of these mutant P-glycoproteins had an apparent mass of 150 kDa, compared with 170 kDa for wild-type P-glycoprotein and the apparent mass was altered by endoglycosidase H digestion. These observations suggest that these mutant proteins were improperly processed so that they were located in the endoplasmic reticulum and were not targeted correctly to the plasma membrane. The in vivo processing of mutants Gly-269 to Val and Gly-781 to Val was temperature-sensitive. When cells expressing these mutants were grown at a lower temperature (26 degrees C), the mature 170-kDa form of P-glycoprotein was the major product. Substitution of glycine with alanine at positions 251, 268, 269, or 781 yielded mutants with structural and functional characteristics similar to wild-type enzyme. Mutation of Gly-141, 187, 288, 812, or 830 to Val, altered the drug resistance profile conferred by P-glycoproteins expressed in transfected cells. All five mutations increased relative resistance to colchicine or adriamycin, without altering relative resistance to vinblastine. These results demonstrate that glycines located in the cytoplasmic loops play important roles in structure and function of P-glycoprotein.
Loo
T W
TW
Department of Medicine, University of Toronto, Ontario, Canada.
Clarke
D M
DM
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004095:Dihydropyridines / Q000737:chemistry; D004351:Drug Resistance; D005998:Glycine / Q000235:genetics / Q000502:physiology*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006358:Hot Temperature; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011499:Protein Processing, Post-Translational
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D018076:DNA, Complementary; D004095:Dihydropyridines; D008562:Membrane Glycoproteins; C041130:azidopine; D006026:Glycoside Hydrolases; D005998:Glycine