This application is to continue the study of thyroxine (T4) activation under a grant which is now in its 27th year. The first step in thyroid hormone action is the 5' deiodination of T4 to form the active hormone, 3,5,3'-triiodothyronine (T3). Recent progress has led to the cloning of the type 2 iodothyronine deiodinase cDNA (D2), a low Km (1 x 10-9 M), propylthiouracil-insensitive selenodeiodinase which catalyzes this reaction. New information indicates that there are important differences in the expression of this enzyme between humans and rats. These include the expression of D2 mRNA in skeletal, cardiac muscle, and thyroid tissue in humans but little or no expression in these tissues in the rat. This indicates that D2 has a much more important role in the production of plasma T3 in humans than we had previously thought due to our use of the rat as a model for human thyroid hormone metabolism. The presence of D2 mRNA in human myocardium suggests that the heart, like the brain and pituitary, may respond to plasma T4 independent of the plasma T3 due to local T4 to T3 conversion within the tissue. Thus, both hormones may contribute to the thyroid status of the heart, as well as the hypothalamic-pituitary axis. We propose to use molecular biological techniques to evaluate the transcriptional regulation of the human dio2 gene, the control of Ds mRNA translation, and the post-translational regulation of D2 activity by its substrates. Modifications will be made in critically important amino acids in the D2 molecule to analyze the mechanism of T4 deiodination by D2. We will explore the mechanism for important amino acids in the D2 molecule to analyze the mechanism of T4 deodination by D2. We will explore the mechanism for and significance of D2 expression in the human thyroid and compare this with that of the type 1 iodothyronine deiodinase also present in this tissue. Lastly, we will evaluate the role of D2 in the central nervous system as the critical transducer in the feedback regulation of thyroid function. The information from these studies is vital to understanding the physiology of T4 activation in humans. This is relevant for human disease since levothyroxine is used in the treatment of the over two million hypothyroid persons, mostly women, in the U.S.A. alone. Furthermore, it seems likely that compensatory changes in D2 are the major mechanism by which the more than 250 million individuals living under iodine- deficient conditions adapt to this circumstance.