This proposal addresses the molecular mechanism of thyrotropin-releasing hormone TRH, acting through its cell membrane receptor, modulates the phosphorylation state of a pituitary-specific transcription factor, Pit-I. Pit-I, in turn, regulates anterior pituitary expression of prolactin (Pr1), growth hormone (GH), and thyrotropin-beta subunit (TSH-beta) genes by binding to specific DNA response elements. [Four] Specific aims are proposed to explore further the role of Pit-1 in TRH action: (1) TRH- responsive regions of the thyrotropin beta-subunit gene (TSH-beta) will be mapped in vivo before and after TRH stimulation. (2) The importance of individual phosphorylation sites on Pit-1 for TRH action will be determined. [Purified protein kinases and TRH-treated rat pituitary somatotroph (GH3 cells) cellular extract will be used to phosphorylate recombinant wild type or mutant Pit-1 protein preparations in vitro and phosphorylation sites confirmed in vivo in GH3 cells.] The phosphorylation pattern and DNA-binding properties of these Pit-I proteins will then be determined. Transient transfection of GH3 cells with wild type of mutant Pit-1 and its isoforms will also be utilized to study TRH-stimulated expression of the TSH-beta subunit and prolactin (Pr1) genes. [(3) The role of CREB-binding protein (CBP) in mediating TRH-stimulated gene expression of Pr1 and TSH-beta subunit genes via Pit-1 will next be explored. CBP both binds to Pit-1 and enhances TRH stimulated gene expression. The location and phosphorylation dependence of this interaction will be characterized.] (4) Finally, the ability of human Pit- 1 mutations to disrupt TRH action in vitro will be evaluated. [The effect of human Pit-1 mutations on DNA-binding, CBP interaction, and TRH- stimulated expression of Pr1 and TSH-beta subunit genes will be determined. This proposal will provide strong evidence that TRH stimulates gene expression through a phosphorylation-dependent interaction between Pit-1 and CBP.] Moreover, the study of human mutations of the pit-I gene and their effects on TRH signaling will yield new insights into the molecular mechanisms of normal anterior pituitary development, and regulation in man.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Endocrinology Study Section (END)
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Sato, Sheryl M
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Beth Israel Deaconess Medical Center
United States
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