Abstract
ATP-dependent transport of glutathione and glucuronate conjugates from hepatocytes into bile is mediated by a distinct member of the ATP-binding cassette superfamily. We have cloned and sequenced the canalicular isoform of the multidrug resistance protein from rat liver, and termed it cMrp. This membrane glycoprotein is composed of 1541 amino acids with an identity of 47.8% with the human multidrug resistance protein (MRP) and of 41.9% with the yeast cadmium factor (YCF1). The carboxyl-terminal 130 amino acids of the human hepatocyte canalicular isoform of MRP (cMRP) were 80.2% identical with rat cMrp. cMrp was not expressed in the liver of two mutant rat strains, the Eisai hyperbilirubinemic rat and the GY/TR- mutant, which are deficient in the ATP-dependent transport of conjugates across the canalicular membrane. Immunoblotting using an antibody raised against the carboxyl terminus of cMrp detected the glycoprotein of about 190 kDa only in the canalicular membrane from normal liver. Double immunofluorescence and confocal laser scanning microscopy localized cMrp exclusively to the canalicular membrane domain of hepatocytes and demonstrated its loss in the hyperbilirubinemic mutant rat. The results identify cMrp as a canalicular transport protein with a novel sequence and with a function similar to the one of the MRP.
Highlights
CMrp was not expressed in the liver of two mutant rat strains, the Eisai hyperbilirubinemic rat and the GY/ TR؊ mutant, which are deficient in the ATP-dependent transport of conjugates across the canalicular membrane
1 The abbreviations used are: MRP, human multidrug resistance protein; MRP, gene encoding the human multidrug resistance protein; mrp, gene encoding the rat multidrug resistance protein; Mrp, rat multidrug resistance protein; ABC, ATP-binding cassette; canalicular isoform of MRP (cMRP), human canalicular isoform of the multidrug resistance protein; canalicular isoform of Mrp (cMrp), rat canalicular isoform of the multidrug resistance protein; cmrp, gene encoding the rat canalicular multidrug resistance protein; DPPIV, dipeptidyl peptidase IV (CD26; EC 3.4.14.5); EHBR, Eisai hyperbilirubinemic rat (9, 10); GY, Groningen Yellow; GY/TRϪ, transport-deficient Wistar rat (8); LT, leukotriene; MK 571, 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethyl-amino-3-oxopropyl)-thio}-methyl]thio) cloned and sequenced from multidrug-resistant human lung cancer cells and identified as an integral membrane glycoprotein of about 190 kDa belonging to the superfamily of ATPbinding cassette transporters (1)
We deduced the carboxyl-terminal sequence of the human hepatocyte canalicular MRP isoform, cMRP, from a cDNA clone isolated from human liver
Summary
Materials—[14,15,19,20-3H4]LTC4 (4.7 Tbq/mmol), [␣-32P]dCTP (111 Tbq/mmol), and ␣-35S-dATP (37 Tbq/mmol) were obtained from DuPont NEN.
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