Cloning, expression, and tissue distribution of the rat nicotinamide adenine dinucleotide-dependent 11...

A pcDNAI adult rat kidney complementary DNA (cDNA) library was screened using a sheep 11-hydroxysteroid dehydrogenase 2 (11 beta HSD-2) probe, and the isolated clones were sequenced. The 5'-end of the cDNA was determined by 5'-rapid amplification of cDNA ends. The rat 11 beta HSD-2 cDNA is 1864 base pair (bp) long. It consists of a 5'-untranslated region of 126 bp, an open reading frame of 1203 bp, and a 3'-untranslated region of 535 bp. The predicted protein contains 400 amino acid residues, with a calculated mol wt of 43,700. The rat 11 beta HSD-2 protein sequence is 85% homologous to human 11 beta HSD-2 and 76% to sheep 11 beta HSD-2. Expression of 11 beta HSD-2 messenger RNA by Northern blot and reverse transcription-polymerase chain reaction was high in kidney, distal colon, and adrenal and lower in the lung, hypothalamus, hippocampus, and midbrain. The rat 11 beta HSD-2 was transiently transfected into modified Chinese hamster ovary cells. Cells transfected with the 11 beta HSD-2 cDNA converted corticosterone into 11-dehydrocorticosterone. Conversion of corticosterone to 11-dehydrocorticosterone was NAD+ dependent and had a Km of 10.1 +/- 2.1 nM. In conclusion, we have cloned a rat NAD(+)-dependent 11 beta HSD with tissue distribution and kinetic characteristics suggesting that it could play a significant role in mineralocorticoid receptor selectivity.