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Antibodies against a novel adrenocorticotropic hormone-inducible cytochrome P450 (P450RAP), responsible for polycyclic aromatic hydrocarbon metabolism in rat adrenal microsomes (Otto, S., Bhattacharyya, K.K., and Jefcoate, C.R. (1992) Endocrinology 131, 3067-3076), identified a cDNA clone encoding a partial cytochrome P450 sequence from a rat adrenal cDNA library. Rescreening a second cDNA library yielded several clones up to 5.0 kilobases (kb) encoding a 1629-base pair open reading frame. The deduced amino acid sequence (543 residues) matched completely with five peptides cleaved from P450RAP. The amino acid sequence of P450RAP is 92% identical to a 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible CYP1B1, cloned from mouse C3H10T1/2 (10T1/2) embryo fibroblast cells (Savas, U., Bhattacharyya, K. K., Christou, M., Alexander, D.L., and Jefcoate, C. R. (1994) J. Biol. Chem. 269, 14905-14911), which shows nearly the same characteristics in polycyclic aromatic hydrocarbon metabolism. The available 5'- and 3'-noncoding regions show, respectively, 93 and 83% sequence identity. We conclude that P450RAP protein is encoded by a rat CYP1B1 gene orthologous to the mouse CYP1B1 gene. Alignment of rat CYP1B1 amino acid sequences with rat CYP1A1 (39% identical) indicated eight regions of high identity for each (60-78%), interspersed by extensive regions of less than 30% similarity. The CYP1B1 cDNAs hybridize a 5.2-kb mRNA in rat adrenals, consistent with the length of the longest clones and the mRNA recognized in 10T1/2 cells. CYP1B1 mRNA was elevated by a 2-day adrenocorticotropic hormone treatment but much less than CYP11A1 (cytochrome P450 side chain cleavage) mRNA (2-fold versus 4-fold). The lower levels of the 5.2-kb mRNA in other steroidogenic cells (ovary) was consistent with the amount of immunodetectable CYP1B1 protein and, unlike the adrenal, expression in the ovary was stimulated 5-fold by beta-naphthoflavone, an aryl hydrocarbon receptor agonist, in parallel with CYP1A1 induction. In several other tissues (liver > lung > uterus >> kidney), CYP1B1 mRNA and protein were constitutively undetectable but highly induced by beta-naphthoflavone, although at much lower levels than CYP1A1. Rat CYP1B1, therefore, exhibits regulation through hormonal signaling and the aryl hydrocarbon receptor in a cell-specific manner.