Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes.

Abstract

Abstract.We have previously shown that the multidrug resistance protein (MRP) mediates the ATPdependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes.The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATPdependent conjugate transport in canalicular membranes from TR-mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transportdeficient TR-mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte.XCRETION into bile is a major pathway for the elimination of endogenous and xenobiotic lipophilic compounds from the mammalian organism. Drugmetabolizing transferases in the hepatocyte convert many of these compounds into amphiphilic anionic conjugates with glutathione, glucuronate, or sulfate. Excretion of these conjugates across the hepatocyte canalicular membrane into bile is mediated by a primary-active ATP-dependent export pump which has been characterized functionally and termed multispecific organic anion transporter (MOAT; 1 Oude Elferink and Jansen, 1994), non-bile acid organic anion transporter (Arias et al., 1993), glutathione S-conju-

The apical conjugate efflux pump ABCC2 (MRP2)

“…Abcc2 cDNA was initially identified and cloned as a fragment from rat liver, using degenerate oligonucleotides complementary to human ABCC1 [21] and Leishmania ltpgpA [114]. This Abcc2 cDNA fragment was amplified from normal rat liver, but not from the liver of mutant rats that were deficient in the hepatobiliary excretion of anionic conjugates [114].…”

“…This Abcc2 cDNA fragment was amplified from normal rat liver, but not from the liver of mutant rats that were deficient in the hepatobiliary excretion of anionic conjugates [114]. The subsequent cloning and analysis of the full-length rat Abcc2 cDNA identified Abcc2 [Mrp2; formerly described as cMrp and canalicular multispecific organic anion transporter (cMoat)] as an ABCC1-related protein localized in the canalicular membrane of hepatocytes [13,133] (Fig.…”

“…In consequence of the molecular identification and cloning of rat Abcc2 as the apical conjugate efflux pump [13,60,133], which is defective in these rat strains [13,60,133], both can now be considered as animal models of human Dubin-Johnson syndrome. Two distinct naturally occurring sequence variants in the rat Abcc2 gene, either at codon 401 (GY/ TR − ) [133] or at codon 855 (EHBR) [60], lead to premature stop codons and to a lack of the Abcc2 protein from the hepatocyte canalicular membrane [13,114,133]. Recently, Abcc2-knock-out mice have been generated and characterized by several groups [18,121,177].…”

“…The multidrug resistance protein 2 (ABCC2) was the second member of the subfamily of MRP efflux pumps to be cloned from rat and human tissues [13,60,114,133,165]. The function of rat Abcc2 was recognized long before its cloning by studies on the hepatobiliary elimination of organic anions in normal and transport-deficient mutant rats [54,55,64,131,163].…”

Second International Ringberg Conference: “Cell Biology and Molecular Basis of Liver Transport”

“…W. Wolkoff (New York, NY) analyzed the expression and tissue distribution of the oatp carrier by the use of polyclonal pressed, whereas expression is altered in GY/TR 0 mutant rats. 19 In basolateral and canalicular membrane preparations from hu-antibodies. 35,36 In Western blots, the antibodies stain an 80-kd protein.…”