Polypeptide hormones exert rapid, specific effects on protein biosynthesis and gene expression in their target tissues, critical for normal development and homeostasis. Defining the precise biochemical mechanisms responsible for this regulation would provide a conceptual framework with which to investigate many diseases of disorders regulation. Thyrotropin releasing hormone (TRH), epidermal growth factor (EGF), bombesin, phorbol esters (TPA), and cAMP act within minutes of their addition to clonal rat pituitary cell cultures (GH4) to produce specific transcriptional stimulation of the prolactin gene; growth hormone releasing factor exerts comparable actions on the GH gene. The site of regulation of rat prolactin and growth hormone (GH) biosynthesis by polypeptide hormones is therefore at the level of gene transcription. We propose to define the precise genomic sequences in the less than 200 bp of 5' flanking information which quantitatively transfer polypeptide and steroid hormonal regulation characteristic of prolatin and GH gene regulation using a combinatorial analysis including DNA-mediated gene transfer of fusion genes containing genetically modified prolactin and growth hormone genomic fragments into homologous cell lines and into primary pituitary cell cultures, and into normal tissues using appropriate retroviral vector systems. Several approaches will be utilized to define the sequences imparting tissue-specific neuroendocrine gene expression and to identify the critical trans-acting factors. Affinity chromatography and a novel expression strategy will be adapted to isolate the transactive regulatory factors critical for hormonally- and developmentally- mediated patterns of gene expression, and to clone their encoding mRNAs. Transgenic animal approaches will be used to further define the complex patterns of sequence recognition and chromosomal position effects which adjudicate the tissue-specific developmental patterns of gene expression characteristic of the neuroendocrine system. The structure and regulatory function of two novel basic chromatin associated proteins which are rapidly phosphorylated in response to polypeptide hormones aned cAMPL will be elucidated via recombinant DNA technology. Based upon the cloning of the EGF receptor cDNA in this laboratory and the identification of polymorphic RNA products, we propose genetic evaluation of the structural components of the EGF receptor required for growth, protein synthesis, and transformation.
