Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase

J Biol Chem. 2000 Aug 18;275(33):25194-201. doi: 10.1074/jbc.M002036200.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Astrocytes / metabolism
  • Base Sequence
  • Blotting, Northern
  • Brain / metabolism
  • Cell-Free System
  • Cells, Cultured
  • Cerebral Cortex / metabolism
  • Cloning, Molecular
  • Cyclic AMP / metabolism
  • DNA, Complementary / metabolism
  • Gene Library
  • Green Fluorescent Proteins
  • Immunohistochemistry
  • Iodide Peroxidase / biosynthesis
  • Iodide Peroxidase / chemistry*
  • Iodide Peroxidase / genetics
  • Kinetics
  • Luminescent Proteins / metabolism
  • Models, Genetic
  • Molecular Sequence Data
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Biosynthesis
  • Protein Structure, Tertiary
  • Rats
  • Recombinant Fusion Proteins / metabolism
  • Sequence Analysis, DNA
  • Tissue Distribution

Substances

  • DNA, Complementary
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Cyclic AMP
  • iodothyronine deiodinase type II
  • Iodide Peroxidase

Associated data

  • GENBANK/AF245040