Cloning of two alternatively spliced 21-hydroxylase CDNAs from rat adrenal

Interest in extra-adrenal corticosteroid synthesis has been revived by technological advances and the quest for answers to clinical problems. The cytochrome P450 21-hydroxylase converts progesterone to deoxycorticosterone, the obligatory substrate for the production of the main adrenal steroids aldosterone, cortisol and corticosterone. The rat P450 21-hydroxylase was cloned and two constructs, 21OH-5 and 21OH-6, sequenced. The constructs are similar, except that 21OH-6 has three additional major insertions of 64, 70 and 84 bp, a 3 bp deletion, and four extra base pairs immediately before the poly-A sequence. The entire coding region of 21OH-5 has 87 and 71% homology with the mouse and human 21-hydroxylase cDNA, respectively, whereas the encoded protein has 84 and 65% homology. Reverse transcriptase-polymerase chain reaction (RT-PCR) combined with Southern blot demonstrated expression of both transcripts in the kidney, aorta, liver, cerebellum, hypothalamus and brain stem, heart and cerebrum, but not the hippocampus, in addition to the adrenal. The entire coding region of 21OH-5 and the corresponding region of 21OH-6 including the three introns were cloned into pCR3 and the plasmids transiently transfected into COS-7 cells. Only 21OH-5 was translated into active protein, converting approximately 64% of 3H-progesterone to deoxycorticosterone in 2 h.