The pituitary receptor for thyrotropin-releasing hormone (TRH) serves as a model for the family of G-protein coupled Ca2+-mobilizing receptors. The proposed studies aim to establish how the [Ca2+]i response to TRH is controlled and how receptor localization affects the TRH response. The first goal is to determine the molecular mechanisms of the entire [Ca2+]i response to TRH. The observation that TRH directly activates Ca2+ efflux from cells forms the basis for the working hypothesis: that the TRH receptor is coupled to a Ca2+ pump, likely through a G-protein, i.e., that a plasma membrane Ca2+-transporting ATPase is a novel effector, and that activation of Ca2+ afflux controls the TRH response. The biochemical basis for TRH activation of Ca2+ afflux will be established by determining if TRH activates a Ca2+ pump directly, how the activation occurs, and which Ca2+ pump is activated. The importance of TRH activation of Ca2+ afflux to the [Ca2+]i and secretory responses will be measured in cells that are over expressing the hormone-responsive Ca2+ pump and in cells in which the Ca2+ pump has been inhibited. A limited analysis of TRH action in normal cells will be performed to determine which cells bind TRH, whether these internalize the TRH receptor; and whether key findings about TRH control of [Ca2+]i apply to normal lactotrophs and thyrotrophs. The second goal is to determine the mechanism of agonist-induced changes in TRH receptor localization and the importance of receptor trafficking to the TRH response. The TRH receptor undergoes extensive internalization when agonist binds and undergoes extensive recycling when agonist is removed. The hypothesis that the cycling of receptors controls the cellular responses to TRH will be tested. Agonist-activated receptor redistribution will be characterized with the objective of learning the mechanism and, most importantly, the consequences of receptor trafficking to the cell. The pathways of ligand-induced sequestration and recycling of TRH receptors will be determined by co-localizing receptor and ligand, localizing receptors at the EM level, and seeing if receptors concentrate in caveolae. The mechanism of receptor sequestration will be determined by altering residues potentially important in targeting, testing the importance of phosphorylation, and measuring the association of wild type and internalization defective receptors with adaptor proteins.
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