The purpose of this proposal is to characterize the molecular event(s) regulating T4 to T3 conversion in the brain and to clone and sequence type II iodothyronine 5'deiodinase (5'D-II), the enzyme catalyzing this reaction. This enzyme catalyzed reaction is the essential first step in the mechanism of action of thyroid hormone. In brain, 5'D-II activity is dynamically regulated by circulating T4 levels and the inverse relationship between the enzyme and thyroid hormone constitutes a homeostatic mechanism that insures that intracerebral T3 levels are kept within narrow limits. T4 accelerates the rate of 5'D-II inactivation by adjusting the physical state of cell microfilaments; a mechanism that is independent of the classical nuclear T3 receptor. Cerebrocortical astrocytes provide a cell culture model in which the enzyme can be differentially expressed and specific affinity labeling with BrAcT4 permits ready enzyme identification. Two dimensional PAGE gel analysis will examine the subunit composition of 5'D-II holoenzyme after cross-linking with cleavible reagents, and after the binding of the enzyme to F-actin. T4 initiated re-distribution of 5'D- II will be visualized using anti-T4 antibodies that recognize the affinity labeled enzyme, permitting the composition of the internalized enzyme and role of endocytosis in this process to be evaluated. Iodothyronine structural requirements of the cellular protein(s) initiating this T4 effect will be determined and 2-D gel analysis will identify accessory protein(s) bound to F-actin. Cloning of 5'D-II is possible because of the ability to differentially express the enzyme and the abundant quantities of 5'D-II in cAMP-induced glial cells. Enzyme clones will be selected from glial cell cDNA libraries by i) oligonucleotides constructed from the amino acid sequence of the purified enzyme, ii) by similarities of functional domains with the cloned isozyme, 5'D-I; or by iii) eukaryotic expression in transfected glial cells or iv) in Xenopus oocytes. The cloned 5'D-II will be sequenced, expressed in vitro and its catalytic identity confirmed. The primary structure of 5'D-II will then be compared to that of the renal 5'D- I isozyme. Conserved functional domains will be identified and chimeric enzymes constructed by inter-changing these functional domains. Definitive characterization of 5'D-II will allow the inter-relationships between these two enzymes to be established. Clarification of the events that modulate this extra-nuclear action of thyroid hormone will delineate how target tissues modulate their responsiveness to thyroid hormone and will provide a basis for understanding the essential role of thyroid hormone in the growth and development of the brain.
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