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mdr2 encodes P-glycoprotein expressed in the bile canalicular membrane as determined by isoform-specific antibodies
E Buschman1, R J Arceci, J M Croop
1Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Abstract:
We have produced antibodies specific for the three P-glycoprotein (P-gp) isoforms encoded by the mouse mdr1, mdr2, and mdr3 genes. The anti-Mdr2 and anti-Mdr3 antibodies were generated against synthetic peptides derived from the "linker" region, whereas the anti-Mdr1 antibody was raised against a fusion protein containing the amino terminus of Mdr1. Western blot analysis showed that the three antibodies could discriminate between the three isoforms in membrane fractions from Hamster cells transfected with the corresponding full-length or chimeric mdr cDNAs. Immunocytochemistry studies of mdr-transfected cells showed that the three antibodies specifically recognized each P-gp isoform expressed in whole cells. Immunoblotting of normal mouse tissues revealed that the Mdr2 isoform was expressed at very high levels in liver canalicular membrane vesicles (CMV) but not in membrane vesicles prepared from the basolateral (sinusoidal) domain (SMV). Mdr3 was detected in intestinal brush border membrane vesicles and also in CMV, although at levels much lower than Mdr2. Mdr1 was not detected in CMV or SMV but was detected in endometrial tissue from the gravid uterus. Photolabeling experiments with [125I]iodoarylazidoprazosin followed by immunoprecipitation with isoform-specific antibodies indicated that, in CMV, Mdr3 but not Mdr2 could bind the drug analogue.
Insights
Researchers developed specific antibodies for mouse P-glycoprotein (P-gp) isoforms Mdr1, Mdr2, and Mdr3. These tools enable precise detection and study of P-gp distribution and drug-binding capabilities in various mouse tissues.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- P-glycoprotein (P-gp) is a crucial efflux pump involved in drug transport.
- Mouse genome contains three P-gp isoforms: Mdr1, Mdr2, and Mdr3, with distinct functions.
- Understanding the specific roles and localizations of each isoform is essential for drug development.
Purpose of the Study:
- To generate isoform-specific antibodies for mouse P-glycoprotein (P-gp).
- To characterize the tissue distribution and drug-binding properties of Mdr1, Mdr2, and Mdr3.
Main Methods:
- Antibody generation using synthetic peptides and fusion proteins.
- Western blot and immunocytochemistry for isoform discrimination in transfected cells.
- Immunoblotting of mouse tissues and photolabeling with a drug analogue.
Main Results:
- Developed antibodies successfully distinguished between Mdr1, Mdr2, and Mdr3 isoforms.
- Mdr2 highly expressed in liver canalicular membrane vesicles; Mdr3 in intestinal and liver vesicles.
- Mdr1 found in gravid uterus endometrium; Mdr3, but not Mdr2, binds a drug analogue in liver vesicles.
Conclusions:
- Isoform-specific antibodies are valuable tools for P-gp research.
- Distinct tissue localization patterns suggest specialized functions for each P-gp isoform.
- Mdr3's drug-binding ability in the liver warrants further investigation for therapeutic implications.