Domain mapping of the photoaffinity drug-binding sites in P-glycoprotein encoded by mouse mdr1b.
P-glycoprotein is an energy-dependent drug efflux pump with broad specificity for hydrophobic antitumor agents such as vinblastine, doxorubicin, and taxol. We have previously shown that [3H]azidopine and [125I] iodoaryl azidoprazosin, which are photoaffinity probes for the alpha 1-subunit of the L-type calcium channel and alpha 1-adrenergic receptor, respectively, specifically interact with P-glycoprotein, partially reverse multidrug resistance, and bind to a 6-kDa common domain in the 140-kDa P-glycoprotein molecule (Greenberger, L., Yang, C.-P. H., Gindin, E., and Horwitz, S. B. (1990) J. Biol. Chem. 265, 4394-4401). An immunological approach was used to identify the position of photoaffinity drug-binding domains in P-glycoprotein. Analysis was done with a series of site-specific rabbit polyclonal antibodies to peptides that mimic domains in the mouse mdr1b gene product. The antibodies were made against amino acid residues 269-284, 356-373, 665-682, 740-750, 907-924, and 1203-1222. Upon trypsin digestion, cleavage products of 95 and 55 kDa were obtained, which after further digestion migrated at 60 and 40 kDa, respectively. The 40-kDa fragment was recognized by the antibodies to residues 1203-1222 and 919-1276, while the 55-kDa fragment was recognized by these antibodies plus antibodies to residues 740-750 and 907-924. In contrast, the 95- and 60-kDa trypsin fragments were recognized only by the antibody to residues 269-284. The 55- and 40-kDa fragments, as well as the 95- and 60-kDa fragments, were major photolabeled species after digestion of P-glycoprotein. The previously identified 6-kDa photo-labeled P-glycoprotein fragment was within the 40-kDa trypsin fragment. These data suggest that there are two photoaffinity drug-binding domains in P-glycoprotein encoded by mouse mdr1b. The C-terminal site most likely resides within or in close proximity to putative transmembrane domains 11-12.
Lederle Laboratories, Medical Research Division, American Cyanamid Company, Pearl River, New York 10965.
L M
LM
Greenberger
C J
CJ
Lisanti
J T
JT
Silva
S B
SB
Horwitz
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D003429:Cross Reactions; D004351:Drug Resistance; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D010777:Photochemistry; D011224:Prazosin / Q000031:analogs & derivatives / Q000378:metabolism; D013045:Species Specificity
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D008562:Membrane Glycoproteins; D010455:Peptides; C041552:azidoprazosin; D011224:Prazosin