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Molecular characterization of a multidrug resistance-associated protein, Mrp2, from the little skate
Shi-Ying Cai1, Carol J Soroka, Nazzareno Ballatori
1Liver Center, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Abstract:
Multidrug resistance protein Mrp2 (symbol Abcc2) in liver plays a significant role in the biliary excretion of organic anionic conjugates. Mutations in human MRP2 result in defects in excretion of conjugated bilirubin and other cholephiles known as the Dubin-Johnson syndrome. Previous studies indicate that transporters with Mrp2-like functions are present in ancient vertebrates. We have now characterized an Mrp2 ortholog at the molecular level from the liver of the small skate, Raja erinacea, a marine vertebrate that evolved approximately 200 million years ago. The full-length skate Mrp2 cDNA is 6 kb and encodes for a 1,564-amino acid peptide with 56% identity to human Mrp2. Northern blot analysis demonstrated that skate Mrp2 is abundantly expressed in skate liver, intestine, and kidney. Immunoblots reveal a 180-kDa protein in skate liver. Immunofluorescence studies locate skate Mrp2 to the apical membrane of hepatocytes, renal tubules, and intestine. A PDZ-interacting motif is also found at its COOH terminus. Further sequence analysis indicates that transmembrane domains 1, 9, 11, 16, and 17 are the most highly conserved transmembrane domains between skate Mrp2 and mammalian MRP2/Mrp2s. This analysis indicates that Mrp2 orthologs evolved early in vertebrate evolution and that conserved domains may be important determinants of Mrp2 substrate specificity.
Insights
Researchers identified a multidrug resistance protein Mrp2 (ABCC2) ortholog in the small skate, a primitive vertebrate. This discovery reveals early evolution of Mrp2 transporters and conserved domains crucial for their function in excretion.
Area of Science:
- * Comparative genomics and molecular biology.
- * Evolutionary biology and transporter function.
Background:
- * Multidrug resistance-associated protein 2 (MRP2/ABCC2) is vital for excreting organic anions from the liver.
- * Human MRP2 defects cause Dubin-Johnson syndrome, highlighting its role in bile excretion.
- * MRP2-like transporters are known in ancient vertebrates, suggesting early evolutionary origins.
Purpose of the Study:
- * To characterize the Mrp2 ortholog from the liver of the small skate (Raja erinacea), an ancient marine vertebrate.
- * To investigate the molecular and evolutionary aspects of Mrp2 in early vertebrate species.
Main Methods:
- * Molecular cloning of the full-length skate Mrp2 cDNA.
- * Northern blot and immunoblot analyses for gene and protein expression.
- * Immunofluorescence microscopy to determine protein localization.
- * Sequence analysis to identify conserved domains and motifs.
Main Results:
- * A 6-kb skate Mrp2 cDNA encoding a 1,564-amino acid protein (56% identity to human Mrp2) was identified.
- * Skate Mrp2 is highly expressed in the liver, intestine, and kidney, with a 180-kDa protein detected in the liver.
- * The protein localizes to the apical membrane of hepatocytes, renal tubules, and intestinal cells, and possesses a PDZ-interacting motif.
- * Highly conserved transmembrane domains (1, 9, 11, 16, 17) were identified between skate and mammalian Mrp2.
Conclusions:
- * MRP2 orthologs evolved early in vertebrate evolution, predating mammalian divergence.
- * Conserved transmembrane domains in skate Mrp2 likely play a critical role in substrate specificity and transporter function.
- * This study provides insights into the ancient origins and conserved functions of Mrp2 transporters in vertebrates.