Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase
Graduate School of Biomedical Sciences; Department of Physiology; Department of Molecular Genetics and Microbiology; Department of Medicine, Division of Endocrinology & Metabolism
Life Sciences | Medicine and Health Sciences
Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.
DOI of Published Version
J Biol Chem. 2000 Aug 18;275(33):25194-201. Link to article on publisher's site
The Journal of biological chemistry
Leonard DM, Stachelek SJ, Safran M, Farwell AP, Kowalik TF, Leonard JL. (2000). Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase. GSBS Student Publications. https://doi.org/10.1074/jbc.M002036200. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/675