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 α1-subunit of the L-type calcium channel and α1-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 photolabeled 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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1991|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology