Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase.
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.
Leonard
D M
DM
Molecular Endocrinology Laboratories, Department of Cellular and Molecular Physiology, University of Massachusetts Medical School, Worcester 01655, USA.
Stachelek
S J
SJ
Safran
M
M
Farwell
A P
AP
Kowalik
T F
TF
Leonard
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D002474:Cell-Free System; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D007453:Iodide Peroxidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution; D000097564:Iodothyronine Deiodinase Type II
D000242:Cyclic AMP; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D007453:Iodide Peroxidase; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097564:Iodothyronine Deiodinase Type II