Effects of mutating different steroidogenic factor-1 protein regions on gene regulation

The involvement of cyclic adenosine monophosphate cAMP-dependent protein ki nase A (PKA) in the regulation of the steroidogenic acute regulatory protei n (StAR) and the high-density lipoprotein receptor (HDL-R) genes by steroid ogenic factor-1 (SF-1) and cAMP were examined. Cotransfection studies carri ed out in Kin 8 cells, a Y1 cell line (mouse adrenal) with a mutation in th e type I PKA regulatory subunit, demonstrated that an intact PKA is require d for maximal activation and that SF-1 participates in cAMP regulation of t hese genes. Site-directed mutational analysis was performed to examine whic h SF-1 regions could be involved in SF-1 transcriptional activation of the StAR and H DL-R genes. SF-1 regions protein analyzed were amino acids Thr 6 0, Ser 203, Ser 431, Thr 462, and the activation function-2 domain (amino a cids 449-462). Plasmids encoding each of the mutated SF-1 proteins were cot ransfected with the StAR and HDL-R promoter constructs into human bladder c arcinoma (HTB-9) cells in the presence or absence of dibutyryl cAMP. The re sults of these studies suggest that although SF-1 is required for optimal p romoter response to cAMP, transcriptional activation of genes by SF-1 and c AMP are promoter dependent, perhaps resulting from gene-specific interactio ns of this transcription factor with other regulatory proteins.