The long-term objective of this project is to understand the molecular mechanisms of thyroid hormone action. Thyroid hormone is an important regulator of development and homeostasis, and abnormalities in thyroid status correlate with many human diseases ranging from cancer to birth defects to metabolic disorders. Thyroxine (T4) is the major form of thyroid hormone that is secreted from the thyroid gland, and T4 is metabolized to the active hormone T3 by enzymatic deiodination. T3 regulates the transcription of thyroid hormone responsive genes by binding to and activating nuclear thyroid hormone receptors. However, there are many effects of thyroid hormone that occur much faster than the timescale of transcriptional regulation, and the molecular mechanisms of these rapid effects are unknown. This research plan is constructed around the hypothesis that rapid thyroid hormone signaling involves a novel metabolite of T4 that is chemically distinct from T3. This hypothesis is supported by experiments showing the existence of this novel metabolite in tissue, as well as the induction of rapid physiological responses when the metabolite is administered in vivo. In addition, this T4 metabolite is a potent agonist of a newly discovered orphan G protein-coupled receptor (GPCR) related to the family of a biogenic amine GPCRs. This research plan seeks to further explore this novel pathway of thyroid hormone signaling. Specific aim 1 involves chemical modification of the metabolite to delineate the structural features important to GPCR activation. Specific aim 2 is a study to understand the enzymology responsible for generating the rapid-acting T4 metabolite. Specific aim 3 involves the isolation, expression, and pharmacological characterization of the different subtypes of the GPCR that responds to the metabolite. Specific aim 4 is an approach to examine different tissues for the presence of the metabolite and perform quantitative analysis. Specific aim 5 is a plan to develop a chemical antagonist of the metabolite that will be useful for studying the in vivo biology of this new thyroid hormone signaling pathway.