The studies proposed are designed to investigate two closely related issues: (A) the form, cellular content, and function of the various thyroid hormone receptors and receptor variants and (B) the molecular mechanisms by which thyroid hormone assures normal development of the central nervous system. (A) T3 receptors: Studies within the past four years have established that there are at least four distinctive species of receptors which are coded by two genes, designated alpha1, alpha2, beta1, beta1. Whereas the beta1 receptor appears to be specific for the pituitary, alpha1 and alpha2 have a wider distribution. In contrast to the others, alpha2 forms do not bind T3 since the ligand-binding domain is spliced out of the primary transcript. The development of specific polyclonal antibodies to the alpha2 and beta1 proteins and the application of Western blot techniques have shown even further diversity in size of these receptors. Studies are planned to determine the basis of this heterogeneity whether due to posttranslational modification, multiple translational start sites or other factors. Methods are also proposed to extend these studies to an analysis of the alpha1 receptor and to quantitate the concentration of the various isoforms in tissues and cell lines. The availability of specific antibodies should allow precise measurements of the specific thyroid hormone receptor components in homo- and heterodimers which have been shown to play an important role in thyroid hormone action. The proposed studies should be helpful in clarifying the specific role of the various receptor isoforms in initiating the physiological action of the hormones. (B) Thyroid hormone-dependent development of the central nervous system. Thyroid hormones play a decisive role in normal brain development. Recent studies have shown that a 40-fold surge in the level of the beta1 mRNA occurs in the first 10 neonatal days of rats. This period is coincident with the rise in plasma and tissue T3 and the timing of T3-dependent changes in CNS development. Immunoprecipitation studies have shown that the rise in beta1 mRNA is accompanied by a comparable increase in the beta1 receptor level. Further, immunocytochemical studies have shown a high concentration of beta1 receptor in cerebellar Purkinje cells, consonant with the essential role of thyroid hormones in the normal development of this cell type. Three genes expressed selectively in Purkinje cells have been shown to be regulated either directly or indirectly by T3 in the developmental process. Transient transfection experiments are designed to define the nature of the target gene TREs, the potential role of auxiliary trans- and cis-acting factors, and to determine whether the beta1 receptor plays a unique role in regulating expression. These studies should provide fresh insights not only into the specific mechanism by which thyroid hormones govern brain development but into the general patterns by which other nuclear-acting hormones exert their ontogenic effects.
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