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.
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