MECHANISM OF TRH SIGNALING PATHWAYS MEDIATED BY PIT-1

Project Details

Description

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.
StatusFinished
Effective start/end date9/15/975/31/01

Funding

  • National Institute of Diabetes and Digestive and Kidney Diseases: $223,912.00
  • National Institute of Diabetes and Digestive and Kidney Diseases
  • National Institute of Diabetes and Digestive and Kidney Diseases
  • National Institute of Diabetes and Digestive and Kidney Diseases

ASJC

  • Genetics
  • Molecular Biology

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