CELL AND MOLECULAR BIOLOGY OF THE TRH RECEPTOR

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The pituitary receptor for the hypothalamic peptide thyrotropin releasing hormone (TRH) is a G-protein coupled, calcium-mobilizing receptor. In response to agonist binding, the TRH-receptor complex undergoes rapid and extensive internalization. The receptor recycles to the plasma membrane following removal of the ligand over longer times, the receptor undergoes homologous downregulation. We have demonstrated receptor cycling directly using fluorescently labeled TRH agonists to follow the ligand, and using immunocytochemistry to localize epitope-tagged receptors stably expressed in pituitary lactotrophs. The function of this rapid and extensive cycling is completely unknown. We hypothesize that ligand-dependent endocytosis and recycling of the TRH receptor are important in the TRH response; specifically, we will test whether internalization is necessary for desensitization, resensitization, or downregulation of the TRH receptor. We have shown that a mutant TRH receptor with a truncated C- terminus fails to internalize TRH but can generate an increase in intracellular calcium in response to agonists. We will first determine which regions of the TRH receptor are required for ligand-dependent internalization and then develop pituitary cell lines expressing a variety of mutant receptors that are abnormal in internalization or recycling. We will then test the possibility that ligand-dependent internalization is responsible for either the desensitization or resensitization of the TRH response. We have shown that cells display an impaired calcium response to TRH if they are exposed to pulses of TRH spaced close together but a full response to pulses of TRH spaced at least ten minutes apart. We will compare the calcium responses of cells expressing normal or internalization-defective receptors. We will also measure TRH responses following selective inhibition of internalization or recycling. Finally, we hypothesize that while most receptor recycles following internalization, a fraction of the receptors are sorted to a degradative, lysosomal pathway, accounting for gradual homologous downregulation. We will test this hypothesis by measuring the ability of TRH to regulate receptor density in cells expressing internalization- defective receptors, and in cells in which endocytosis and recycling have been selectively inhibited.