Domain mapping of the photoaffinity drug-binding sites in P-glycoprotein encoded by mouse mdr1b

Lee M. Greenberger, Christopher J. Lisanti, Julian T. Silva, Susan Band Horwitz

Research output: Contribution to journalArticle

100 Citations (Scopus)

Abstract

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 languageEnglish (US)
Pages (from-to)20744-20751
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number31
StatePublished - Nov 5 1991
Externally publishedYes

Fingerprint

P-Glycoprotein
Binding Sites
Antibodies
Pharmaceutical Preparations
Trypsin
Digestion
L-Type Calcium Channels
Vinblastine
Multiple Drug Resistance
Paclitaxel
Antineoplastic Agents
Doxorubicin
Adrenergic Receptors
Genes
Pumps
Rabbits
Amino Acids
Peptides
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Domain mapping of the photoaffinity drug-binding sites in P-glycoprotein encoded by mouse mdr1b. / Greenberger, Lee M.; Lisanti, Christopher J.; Silva, Julian T.; Band Horwitz, Susan.

In: Journal of Biological Chemistry, Vol. 266, No. 31, 05.11.1991, p. 20744-20751.

Research output: Contribution to journalArticle

Greenberger, Lee M. ; Lisanti, Christopher J. ; Silva, Julian T. ; Band Horwitz, Susan. / Domain mapping of the photoaffinity drug-binding sites in P-glycoprotein encoded by mouse mdr1b. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 31. pp. 20744-20751.
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