The hypothalamic peptide corticotropin-releasing factor (CRF) mediates the central control of ACTH release. To examine the cascade of molecular events initiated by CRF to stimulate ACTH release, we have used a tumor cell line (AtT-20) derived from the mouse anterior pituitary. These cells provide a model system for investigating stimulus-secretion coupling in corticotrophs as they are a homogeneous population of ACTH-secreting cells. CRF stimulates ACTH release through a cAMP-dependent mechanism since blockade of cAMP-dependent protein kinase activity, by the insertion of an inhibitor of protein kinase into intact AtT-20 cells, prevents the release of ACTH in response to CRF. The link between cAMP-dependent protein kinase and biological responses usually involves protein phosphorylation. Therefore, to identify the biochemical events following cAMP-dependent protein kinase activation that lead to the ACTH release response to CRF, the phosphoproteins regulated by CRF in AtT-20 cells will be characterized by two-dimensional gel electrophoresis and phosphopeptide mapping. Those phosphoproteins regulated by CRF through a cAMP-dependent mechanism will be identified by inserting the protein kinase inhibitor into AtT-20 cells prior to CRF stimulation. In addition to stimulating ACTH release, CRF induces the synthesis of pro-opiomelanocortin (POMC), the precursor of ACTH. CRF raises POMC mRNA levels in AtT-20 cells. This effect could be due to activation of the POMC gene or to a stabilization of POMC mRNA. To test the former possibility, we will use recombinant DNA methodologies to examine the effect of CRF on the rate of POMC gene transcription and the affinity of the POMC gene promoter for RNA polymerase. For the POMC gene promoter studies, AtT-20 cells will be transfected with a recombinant plasmid in which bacterial chloramphenicol acetyltransferase coding sequences are under the control of the POMC gene promoter. If CRF is found to regulate the POMC gene-promoter affinity for RNA polymerase, then mutagenesis of portions of the promoter will be initiated to determine the region of the promoter under CRF control. Since CRF also stimulates POMC gene expression through a cAMP-dependent mechanism, we will attempt to determine whether the regulation of ACTH release and synthesis by this peptide is through similar or distinct phosphorylation events.
