mdr2 encodes P-glycoprotein expressed in the bile canalicular membrane as determined by isoform-specific antibodies.
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.
Buschman
E
E
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Arceci
R J
RJ
Croop
J M
JM
Che
M
M
Arias
I M
IM
Housman
D E
DE
Gros
P
P
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D003001:Cloning, Molecular; D006224:Cricetinae; D004351:Drug Resistance / Q000235:genetics*; D000939:Epitopes / Q000032:analysis; D005704:Gallbladder / Q000502:physiology*; D007150:Immunohistochemistry; D008099:Liver / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000276:immunology; D015183:Restriction Mapping; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000939:Epitopes; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins