Insulin treatment increases the transcription of the endogenous prolactin gene and the expression of chimeric plasmids consisting of 5'-flanking DNA from the prolactin gene ligated to the bacterial chloramphenicol acetyl transferase (CAT) gene in GH cells. Thus, this cell culture system accurately reflects the physiological regulation of prolactin by insulin. Defects in prolactin expression due to diabetes have been suggested to account for several disorders including infant respiratory distress syndrome and impotence. The long-term goal of this research is to discover all of the components of this regulation and how they interact to cause increased prolactin gene expression. Results to date have identified a consensus insulin response element (IRE). This IRE is the Ets-motif-related sequence CGGAA and it mediates 100% of the >10-fold increase in Prolactin-CAT expression caused by insulin. GABP was identified as the transcription factor that probably mediates the increase in prolactin-CAT expression due to insulin. The studies proposed in this application focus on insulin signaling to prolactin gene expression. Studies done by others and by us have not identified the signaling pathway that leads to hormone-regulated gene expression. These studies were performed with inhibitors of the various signaling pathways or used overexpression of dominant negative or wild type signaling molecules. These protocols can never completely prove that a signaling pathway is involved in a particular process and they can not reveal unknown pathways. Therefore, this proposal uses a genetic approach that a) does not require any previous knowledge of signaling pathways and b) that will unequivocally establish the role of any molecule that is isolated as a participant in signaling to gene expression. First, cell lines were established that express a selectable marker under control of the prolactin promoter. The hormonal response of this promoter is identical with the wild type prolactin promoter. These cell lines will be mutagenized to produce mutants that are hormonally unresponsive. These mutants will then be analyzed to identify the mutant molecules that produce the hormonally non-responsive phenotype. Finally, complementation will be performed to confirm their signaling role. This application may he perceived to be high risk, but it is the only protocol with a reasonable likelihood of success and the potential benefits are also high. The alternative is to laboriously examine and eliminate each known signaling pathway with no guarantee of knowing more at the end then at the beginning of the studies.