Expression cloning of the aldosterone target cell-specific 11 beta-hydroxysteroid dehydrogenase from rabbit...

11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor from occupancy by endogenous glucocorticoids. We have recently described a novel isoform of 11-OHSD in the renal aldosterone target cells (11 beta-OHSD/CD) that differs from the previously characterized isoform (11 beta-OHSD-1). Unlike 11-OHSD-1, the collecting duct enzyme catalyzes irreversible dehydrogenation, has a very high affinity for its substrate, and is tissue-specific. We report here the isolation, sequence, and characterization of a complementary DNA (cDNA) encoding the rabbit collecting duct 11 beta-OHSD/CD or 11 beta-OHSD type 2. The cDNA, isolated using expression screening in Xenopus oocytes, is 1.9 kilobases in length and encodes a protein of 406 amino acids with a predicted molecular mass of 44,130 daltons. The cloned enzyme has a Michaelis constant (Km) for corticosterone of 6.6 +/- 3 nM, catalyzes exclusively dehydrogenation, and uses only NAD as cofactor. The cloned enzyme shows 85% and 75% amino acid identity to the recently cloned human type 2 11 beta-OHSD and sheep kidney 11 beta-OHSD, respectively, whereas the overall homology to rat liver 11 beta-OHSD-1 is less than 20% The messenger RNA for this 11 beta-OHSD is expressed at very high levels in the renal collecting duct and at much lower levels in the colon. The intrarenal distribution was determined by reverse-transcription polymerase chain reaction in isolated nephron segments or cell types. The messenger RNA is present only in aldosterone target cells within the kidney, at highest levels in principal cells, at lower levels in intercalated cells, and in inner medullary cells. These data suggest that the 11 beta-OHSD cDNA from rabbit collecting duct cells encodes the enzyme that confers aldosterone selectivity to mineralocorticoid target cells.