Thyrotropin-releasing hormone (TRH) acts at a G protein-coupled receptor to stimulate phospholipase CB (PLCB) leading to the release of calcium from the endoplasmic reticulum. The TRH receptor is a prototype of the calcium-mobilizing receptor family, and insights obtained in these studies should lead to a better understanding of the signal pathway used by many hormones. The results will add directly to the understanding of how hypothalamic peptides regulate pituitary function. Although many receptors use the same overall signal pathway, cells display distinct responses to TRH and to other hormones that activate PLCB Specificity in TRH signal transduction may be achieved in part because TRH activates calcium signaling in a spatially restricted manner. TRH stimulates calcium movement in both directions across the plasma membrane and into and Out of the endoplasmic reticulum. Studies will be carried out in pituitary cell lines.
The first aim i s to use confocal imaging to identify the sites of release of calcium from the endoplasmic reticulum and the sites of uptake of extracellular calcium for store refilling. Novel methods that permit measurement of calcium in the endoplasmic reticulum with green fluorescent protein-based calcium indicators will be employed. The mechanism by which TRH depletes calcium stores will be identified, as will the mechanism underlying the specificity in PLCI3 activation by TRH. TRH receptors are subject to a variety of post-translational modifications that are important for desensitization and receptor targeting, but the sites of receptor phosphoxylation are unknown.
The second aim i s to analyze basal and TRH-stimulated modifications of the TRH receptor directly and determine their significance in TRH signaling using phosphospecific antibodies and site-directed mutagenesis. The TRH receptor undergoes ligand-dependent dimerization and interacts with calmodulin.
The third aim i s to determine the mechanism and function of these interactions of the TRH receptor. The last aim is to identify proteins that interact with the TRH receptor, using yeast two-hybrid screens, glutathione S-transferasefusion proteins and immunoprecipitation assays to search for new interacting proteins and determine the function of the interactions in vitro and in cells.
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