Thyroid hormone (T3) regulates gene expression by binding to a nuclear receptor protein (T3R), which in turn binds to specific DNA sequences (T3) Response Elements, or TREs) in target genes. A region of the T3R that is required for transcriptional activation (gamma domain) has been identified, and it has been postulated that this domain is a site of interaction with another protein. In support of this a nuclear protein (T3 Receptor Auxiliary Protein, or TRAP) has been identified that interacts with T3Rs. One signal of this interaction is enhanced binding of T3Rs to TREs in a cell free assay. Mutations in the gamma domain that impair transcriptional activation also impair the ability of TRAP to enhance T3R-TRE binding. The data suggest that the T3R gamma domain is a site of direct contact with TRAP, and that interaction with TRAP is essential for hormone induced gene expression. The experiments in this proposal will further our understanding of the interaction between T3Rs, TRAP, and TREs. TRAP will be characterized in terms of molecular weight. Experiments will test whether TRAP binds the TRE, or other DNA sequences. The stoichiometry of the TRAP-T3R interaction will be studied. The T3R amino acids that are required for interaction with TRAP will be determined, and it also will be determined whether TrAP directly contacts these amino acids. The regulation of TRAP activity by thyroid hormone will be studied. Other experiments will test whether TRAP or TRAP-like proteins interact with other receptors in the erbA superfamily (the tau domain is conserved in all members). Finally, cDNA clones encoding TRAP will be isolated. Techniques employed to address these questions will include gel shift and avidin biotin complex to DNA assays, DNA footprinting, site directed mutagenesis, and screening of cDNA expression libraries. These studies are directly related to the regulation of thyroid hormone responsiveness in various tissues, and therefore to understanding the basis for the signs and symptoms of hypothyroidism and hyperthyroidism. Techniques employed to address these questions will include gel shift and avidin biotin complex to DNA assays, DNA footprinting, site directed mutagenesis, and screening of cDNA expression libraries. These studies are directly related to the regulation of thyroid hormone responsiveness in various tissues, and therefore to understanding the basis for the signs and symptoms of hypothyroidism and hyperthyroidism.
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