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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK038772-08
Application #
3238272
Study Section
Endocrinology Study Section (END)
Project Start
1986-08-01
Project End
1996-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
8
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Leonard, J L; Leonard, D M; Safran, M et al. (1999) The mammalian homolog of the frog type II selenodeiodinase does not encode a functional enzyme in the rat. Endocrinology 140:2206-15
Shen, Q; Wu, R; Leonard, J L et al. (1998) Identification and molecular cloning of a human selenocysteine insertion sequence-binding protein. A bifunctional role for DNA-binding protein B. J Biol Chem 273:5443-6
Leonard, J L; Farwell, A P (1997) Thyroid hormone-regulated actin polymerization in brain. Thyroid 7:147-51
Safran, M; Farwell, A P; Leonard, J L (1996) Catalytic activity of type II iodothyronine 5'-deiodinase polypeptide is dependent upon a cyclic AMP activation factor. J Biol Chem 271:16363-8
Leonard, J L; Leonard, D M; Shen, Q et al. (1996) Selenium-regulated translation control of heterologous gene expression: normal function of selenocysteine-substituted gene products. J Cell Biochem 61:410-9
Farwell, A P; Safran, M; Dubord, S et al. (1996) Degradation and recycling of the substrate-binding subunit of type II iodothyronine 5'-deiodinase in astrocytes. J Biol Chem 271:16369-74
Farwell, A P; Tranter, M P; Leonard, J L (1995) Thyroxine-dependent regulation of integrin-laminin interactions in astrocytes. Endocrinology 136:3909-15
Shen, Q; Leonard, J L; Newburger, P E (1995) Structure and function of the selenium translation element in the 3'-untranslated region of human cellular glutathione peroxidase mRNA. RNA 1:519-25
Leonard, J L; Farwell, A P; Yen, P M et al. (1994) Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells. Endocrinology 135:548-55
Bick, T; Frick, G P; Leonard, D et al. (1994) Overexpression of the short form of the growth hormone receptor in 3T3-L1 mouse preadipocytes. Proc Soc Exp Biol Med 206:185-9

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