Insulin-like growth factor I (IGF-I) is important in the regulation of normal ovarian function in women. IGF-I regulates the rate-limiting enzyme in the steroidogenic pathway, P-450 cholesterol side-chain cleavage (P450scc). Dr. Urban isolated and sequenced the upstream region of the porcine P450scc gene and found that IGF-I stimulates porcine P450scc gene expression through a 30 base pair GC rich responsive region (IGFRE). The ubiquitous transcription factor Sp1 and other transcription factor, designated P2, bind to the IGFRE. Mutant oligonucleotides to the GC box of the IGFRE showed that Sp1 and P2 binding are necessary for activity of the IGFRE. However, tumor necrosis factor alpha inhibits activity of the IGFRE by specifically preventing IGF-I-induced binding of P2. P2 was identified as PTB-associated splicing protein (PSF). PSF binding to the IGFRE was confirmed by affinity chromatography and electrophoretic mobility supershift assay with an antibody to PSF. This proposal will determine how PSF contributes to the activity of the porcine P450scc IGFRE. The PI will express recombinant PSF and determine its footprinting on the IGFRE and on selective mutants of the IGFRE. Moreover, he will determine the competitive effects of recombinant Sp1 and PSF on binding to the IGFRE through affinity chromatography. PSF will be expressed in a mouse fibroblast cell line that over expresses the IGF-I receptor to assess PSF control of IGFRE transcriptional activity. Nested and end terminal deletions will be done to the PSF cDNA to map the DNA binding domain and IGF-I activation motif. Additional studies will investigate the interactions between the DNA and RNA binding domains of this unique, multifunctional protein. This work is significant in that it is the first to identify a transcription factor that mediates gene expression by IFG-I (PSF) and to show that a protein can find both DNA and RNA and mediate gene expression. Further study of the P450scc IGFRE and how PSF regulates its activity will result in an understanding of the mechanism of action of IGF-I in the ovary and the changes that occur in ovarian disease.