Biochemical and functional analysis of nuclear receptors as targets in cAMP-dependent control of bovine CYP17

Several lines of evidence have suggested that the nuclear receptor Steroidogenic Factor-1 (SF-1 or Ad4BP) may be directly involved in the cAMP-dependent regulation of steroid hydroxylase genes in adrenocortical cells. In the bovine CYP17 gene, which encodes the cytochrome P450 17 alpha-hydroxylase, an SF-1 site is present within cAMP-responsive sequence 2 (CRS2) and mutations which interfere with SF-1 binding correlate with decreases in cAMP-stimulated transcription of a linked reporter gene. In order to determine whether the cAMP response relies on structures within SF-1 itself, mutations and deletions were introduced. We demonstrate that even a single point mutation (E454A) in the transactivating AF-2 domain drastically reduces the ability of SF-1 to mediate cAMP-dependent transcription. Furthermore, the mutation results in a protein which acts in a dominant negative fashion with respect to cAMP-dependent regulation of the bovine CYP17 gene. Finally, we demonstrate that the coactivators CBP and SRC-1 are limiting with respect to cAMP-induced CRS2-dependent transcription in Y1 adrenocortical tumor cells, suggesting that part of the action of cAMP may be to influence the interaction of SF-1 with other cofactors via the AF-2 domain.