Two different regions of P-glycoprotein [corrected] are photoaffinity-labeled by azidopine.
Cells that express P-glycoprotein are resistant to many unrelated anticancer drugs. All evidence suggests that P-glycoprotein is a plasma membrane protein that confers multidrug resistance by actively transporting these cytotoxic drugs out of cells. The objective of our work is to locate drug binding sites on P-glycoprotein. Azidopine is a photoaffinity drug analog that specifically labels P-glycoprotein. To determine the region of P-glycoprotein that binds azidopine, we labeled P-glycoprotein with azidopine and digested the labeled protein into fragments. We then identified the labeled fragments with specific antibodies. We have determined that azidopine labels two different regions of P-glycoprotein: one region is in the amino half of P-glycoprotein, and the other is in the carboxyl half of the protein. Our results suggest that P-glycoprotein contains either two binding sites for azidopine or a single site formed by the two homologous halves of the protein.
Bruggemann
E P
EP
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Germann
U A
UA
Gottesman
M M
MM
Pastan
I
I
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D004095:Dihydropyridines / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007106:Immune Sera; D007624:KB Cells; D008562:Membrane Glycoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D012697:Serine Endopeptidases; D014357:Trypsin
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D001798:Blood Proteins; D004095:Dihydropyridines; D000939:Epitopes; D007106:Immune Sera; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C041130:azidopine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin