Thyrotropin (TSH) is the pituitary glycoprotein hormone which controls thyroid gland function. Abnormalities of TSH production occur in many diseases of the thyroid-pituitary-hypothalamic axis. The purposes of the investigations outlined in the current proposal are to make basic advances in our understanding of the regulation of TSH biosynthesis and secretion. TSH is a glycoprotein hormone composed of two nonidentical subunits, Alpha and TSH-Beta, which are noncovalently associated. These two subunit arise independently from separate messenger RNA species and ultimately from separate genes. The steps of subunit biosynthesis and secretion are classified sequentially as transcriptional, post-transcriptional, translational, post-translational, and secretory. We will determine the mechanisms by which these steps are regulated. Our proposal is divided into three sections. In Section I, we described the methodology which will be utilized to explore the regulation of TSH and subunit biosynthesis. Experiments will be performed to delineate whether primary gene transcription can be assessed accurately and whether this process is regulated. Determination of steady-state mRNA levels for the subunits of TSH will be analyzed as a function of their respective transcriptional rate and stability within the cell. The translational activity of these mRNAs in cell-free systems will be correlated with polysome run-off analysis. Finally, post-translational processing of the subunits will be investigated to determine whether differential processing is a regulated biosynthetic event. In Section II, the mouse thyrotropic tumor and hypothyroid mouse pituitaries will be treated in vivo with known regulators of TSH and subunit secretion to determine the characteristics of their effects upon biosynthetic events. These studies will investigate the molecular basis for TSH and subunit regulation by thyroid hormone, glucocorticoids, and dopamine. In Section III, cell cultures of normal bovine pituitaries will be used in vitro to determine whether direct application of hypothalamic hormones (TRH, SRIF, and catecholamines) can regulate TSH and subunit biosynthesis in addition to release. These projects will utilize the most modern techniques of cell biology and molecular endocrinology. Cell cultures, tumor transplantation and propagation, and radio-immunoassays will be combined with novel biosynthetic studies including cell-free translation, pulse-chase analysis, and cDNA/mRNA hybridization assays. These studies should provide fundamental information on glycoprotein hormone biosynthesis and regulation which is directly applicable to human thyroid and pituitary disease.
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