THYROTROPIN-RELEASING HORMONE RECEPTOR MOLECULAR BIOLOGY

本研究计划的长远目标是定义

The broad, long term objective of this research program is to define the mechanism of action of thyrotropin-releasing hormone (TRH) so as to better understand the mechanism of cell regulation by all extracellular signal molecules, such as hormones, neurotransmitters and growth factors and of some pathologic processes that disorder cell regulation. Specifically, the molecular biology of the TRH receptor from anterior pituitary cells will be studied. A complementary DNA (cDNA) for the TRH receptor has very nearly been cloned. The cloning strategy involved expression of a functional TRH receptor from a mouse pituitary thyroid-stimulating hormone-producing (TtT) tumor in Xenopus laevis oocytes. We first propose to sequence the cDNA and then use it and the deduced amino acid sequence of the receptor protein in studies to define the tissue distribution of the TRH receptor, the structure-function relations of the TRH receptor, in particular the molecular details of its relations of the TRH receptor, in particular the molecular details of its interaction with TRH and its coupling to a guanine nucleotide binding regulatory (G) protein, and the mechanism of modulation of TRH receptor number, which is a proven mechanism for regulation of TRH action. Studies will be performed using GH3 rat pituitary glands and rat tissues. A sequencing stategy using the dideoxynucleotide method has been devised. In situ hybridization will be performed with radiolabeled cDNA probes. Selective mutations of the cDNA will be made and then used to transform GH-Y cells, a subclone of GH3 without TRH receptors or detectable receptor mRNA activity, to define the functional domains of the TRH receptor. And, the mechanism of regulation of TRH receptor number will be studied by measuring the levels and synthesis and degradation rates of the receptor protein and mRNA, with complementary measurements of mRNA activity made in Xenopus oocytes to determine whether there are changes in translational efficiency. Phosphorylation of the TRH receptor will be studied as a possible mechanism of its regulation.