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 similar to 200-kDa, membrane-boun d enzyme is composed of at least one p29 subunit, an similar to 60-kDa, cAM P-induced activation protein, and one or more unidentified catalytic subuni t(s), Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II seleno deiodinase polypeptide is translated in the brain or brown adipose tissue o f rats. These data suggest that the native type II 5'-deiodinase in rat bra in is unrelated to this artificial selenoprotein. In this report, we descri be the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambda zapII cDNA library prepared from cAMP-induced astrocytes, The 3.3-ki lobase (kb) cDNA encodes an similar to 30-kDa, 277-amino acid long, hydroph obic protein lacking selenocysteine, Northern blot analysis showed that a 3 .5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activit y such as brain and cAMP-stimulated astrocytes, Domain-specific, anti-p29 a ntibodies specifically immunoprecipitated enzyme activity. Overexpression o f exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion pro tein led to a > 100-fold increase in deiodinating activity in cAMP-stimulat ed astrocytes, and the increased activity was specifically immunoprecipitat ed by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native e nzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) viru s particles into the cerebral cortex of neonatal rats leads to a similar to 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 io dothyronine 5'-deiodinase and 2) identify the first non-selenocysteine cont aining subunit of the deiodinase family of enzymes.