Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells.
An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1. Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1. Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein. Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K. The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H. These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid. P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.
Richert
N D
ND
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Aldwin
L
L
Nitecki
D
D
Gottesman
M M
MM
Pastan
I
I
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D000906:Antibodies; D004351:Drug Resistance; D006031:Glycosylation; D006801:Humans; D007624:KB Cells / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000906:Antibodies; D008562:Membrane Glycoproteins