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 17alpha-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.
Lund
J
J
Department of Anatomy and Cell Biology, University of Bergen, Norway.
Jacob
A
A
Aesöy
R
R
Yri
O E
OE
Mellgren
G
G
D000306:Adrenal Cortex Neoplasms / Q000473:pathology; D000818:Animals; D050882:CREB-Binding Protein; D002417:Cattle; D000242:Cyclic AMP / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D051717:Fushi Tarazu Transcription Factors; D017353:Gene Deletion; D005799:Genes, Dominant / Q000502:physiology; D051548:Histone Acetyltransferases; D018398:Homeodomain Proteins; D051379:Mice; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000502:physiology; D056920:Nuclear Receptor Coactivator 1; D017354:Point Mutation / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D013254:Steroid 17-alpha-Hydroxylase / Q000502:physiology*; D054339:Steroidogenic Factor 1; D015534:Trans-Activators / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D015534:Trans-Activators; D014157:Transcription Factors; C515058:steroidogenic factor 1, mouse; D000242:Cyclic AMP; D013254:Steroid 17-alpha-Hydroxylase; D050882:CREB-Binding Protein; C494364:Crebbp protein, mouse; D051548:Histone Acetyltransferases; C538861:Ncoa1 protein, mouse; D056920:Nuclear Receptor Coactivator 1