Major photoaffinity drug labeling sites for iodoaryl azidoprazosin in P-glycoprotein are within, or immediately C-terminal to, transmembrane domains 6 and 12.
P-glycoprotein can mediate multidrug resistance in tumor cells and is hypothesized to be an energy-dependent drug efflux pump. The protein is composed of two cassettes; each cassette contains six transmembrane domains followed by a nucleotide binding fold. The [125I]iodoaryl azidoprazosin photoaffinity drug binding sites in P-glycoprotein have been mapped by immunological analysis. After complete digestion of P-glycoprotein with trypsin, two major photolabeled fragments have been mapped. The 5- and 4-kDa fragments are located within, or immediately C-terminal to, the last transmembrane domain of each cassette: transmembranes 6 and 12 of P-glycoprotein, respectively. The 4-kDa fragment maximally extends up to, but not including, the Walker A motif of the second nucleotide binding fold, whereas the 5-kDa fragment maximally extends a few residues beyond the Walker A motif of the first nucleotide binding fold. A minor photolabeling domain is also found between transmembrane domain 4 and up to, but not including, transmembrane domain 6. These data suggest that a portion of the drug binding site in P-glycoprotein is in close proximity to ATP binding regions. Furthermore, symmetrical regions in each cassette of P-glycoprotein are likely to participate in drug efflux.
Greenberger
L M
LM
Oncology and Immunology Research Section, Lederle Laboratories, American Cyanamid Company, Pearl River, New York 10965.
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002918:Chymotrypsin; D004351:Drug Resistance; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D008264:Macrophages; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008961:Models, Structural; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D011224:Prazosin / Q000031:analogs & derivatives* / Q000378:metabolism; D017433:Protein Structure, Secondary; D014357:Trypsin
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C041552:azidoprazosin; D002918:Chymotrypsin; D014357:Trypsin; D011224:Prazosin