Thyroid hormone (T3) regulates growth and differentiation during development and modulates the activity of a wide variety of metabolic pathways in adults. Thyroid hormone acts via nuclear receptors that function as transcription factors in increase or decrease levels of gene expression. The circulating levels of thyroid hormone are determined by thyroid stimulating hormone (TSH). In a classical negative feedback loop, thyroid hormone inhibits TSH alpha and beta subunit gene transcription. Thus, regulation of TSH by T3 represents a critical "set- point" that established the metabolic status of an individual. The mechanisms by which thyroid hormone receptors turn off TSH gene transcription is not well-understood, but represents an important paradigm for understanding this pathway of thyroid hormone action. This syndrome of thyroid hormone resistance (THR) is characterized by elevated levels of thyroid hormone with incomplete suppression of TSH and blunted hormone responses in most tissues. Thyroid hormone resistance is caused by mutations in the hormone binding domain of the thyroid hormone receptor beta gene. Consistent with the autosomal dominant pattern of inheritance, the mutant receptors are proposed to function in a dominant negative manner to block the action of normal thyroid hormone receptors. We have shown that the ability of the mutant receptors to function as antagonists requires preservation of receptor dimerization and DNA binding with selective impairments of T3 binding or transactivation. The locations of the THR mutants therefore provide valuable insights into thyroid hormone receptor structure-function. The purpose of this proposal is to continue an integrated series of studies that address the promoter and receptor determinants of thyroid hormone action. The three specific aims are: 1) To delineate further the TSH alpha gene regulatory sequences that are involved in transcriptional repression by T3. Site- directed mutagenesis will be performed, focusing on promoter regions previously identified to be important for T3 repression; 2) To develop experimental models for the molecular pathophysiology of thyroid hormone resistance. Transient expression assays will be used to understand the mechanisms by which THR mutants antagonize normal receptors; and 3) To demonstrate, by analogy with mutants that cause thyroid hormone resistance, that the naturally occurring thyroid hormone receptor variant alpha2, acts as a dominant negative inhibitor of a restricted subset of thyroid hormone response elements. These studies will further our understanding of how genes are repressed by thyroid hormone and will clarify the mechanisms by which naturally occurring mutants and receptor variant antagonize thyroid hormone action.