Molecular Characterization of a Multidrug Resistance Associated Protein from the Little Skate, Raja Erinacea

Multidrug resistance protein 2 (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 DubinJohnson syndrome. Previous studies indicate that transporters with Mrp2 like functions are present in ancient vertebrates. We have now characterized an Mrp2 orthologue at the molecular level from the liver of the small skate, Raja erinacea, a marine vertebrate that evolved about 200 million years ago. The full-length sMrp2 cDNA is 6 kb, and encodes for a 1564 amino acid peptide with 56% identity to human MRP2. Northern-blot analysis demonstrated that sMrp2 is abundantly expressed in skate liver, intestine, and kidney. Immunoblots reveal a 180 kD protein in skate liver. Immunofluorescence studies locate sMrp2 to the apical membrane of hepatocytes, renal tubules and intestine. A PDZinteracting motif is also found at its C-terminus. Further sequence analysis indicates that transmembrane domains 1, 9, 11, 16, 17 are the most highly conserved transmembrane domains between sMrp2 and mammalian MRP2/Mrp2s. This analysis indicates that Mrp2 orthologues evolved early in vertebrate evolution and that conserved domains may be important determinants of Mrp2 substrate specificity.