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P. Reed Larsen
Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA It is over 40 years since the first demonstration that thyroxine (T4), the principal secretory product of the thyroid, is deiodinated to 3,5,3'-triiodothyronine (T3) in humans(1,2). Subsequently it was shown in rats that the antithyroid drug propylthiouracil (PTU) blocked the metabolic effects of administered T4 in proportion to its inhibition of T4 to T3 conversion indicating that deiodination was critical for T4 activation(3). Type 1 iodothyronine deiodinase (D1) activity was later identified in mammalian liver, kidney, and thyroid and was shown to be sensitive to inhibition by PTU thus explaining the earlier results(4). This enzyme catalyzes both outer- and inner-ring deiodination of T4, although its preferred substrates are 3,3',5'-triiodothyronine (reverse T3) for outer-ring (5') deiodination and the sulfate ester of T3 for inner-ring (5) deiodination(5). Subsequently, a second, PTU-insensitive enzyme catalyzing only T4 to T3 5' deiodination of T4 (to T3) and rT3 (to 3,3' T2) conversion was identified (type 2 deiodinase or D2) which had a 1000-fold lower Km for T4 than D1(6, 7). Experiments in rats showed that the T3 produced by this enzyme was critically important as a source of nuclear receptor (TR) bound T3 in the pituitary, brain, and brown adipose tissue(8). A third deiodinase (D3) was also identified which inactivated T3 or T4 by removal of an iodine from the inner-ring producing either reverse T3 or 3,3'- diiodothyronine(9). This enzyme was found in placenta and the central nervous system. Both D1 and D3 activities are increased by thyroid hormone whereas D2 activity is negatively regulated. These results pointed to a family of enzymes the activities of which are carefully regulated to maintain T3 homeostasis both extra- and intracellularly. Despite the 60-fold higher levels of total T4 than T3 in human plasma, the T4 to T3 ratio of the free (metabolically available) hormones is only about 3 to 1. Nonetheless, since the binding affinity of the thyroid hormone receptor for T3 is 10 to 15-fold higher than that for T4, T3 is the active thyroid hormone. Physiological studies have shown that essentially all of the metabolic effects of T4 are derived from that fraction of this prohormone, about 35-40% in humans, which is monodeiodinated to T3. Thus, conversion of T4 to T3 is the first step in thyroid hormone action. In turn, the level of T3 in the circulation and its half-life is determined by inner-ring deiodination catalyzed by D3, and to a lesser extent, D1. The fact that all three of these enzymes are integral membrane proteins frustrated attempts to purify them to obtain protein sequence. The approach which eventually proved to be successful in obtaining a cDNA was a technique termed expression cloning which did not require obtaining protein sequence. This allowed the identification of a 2.1 kbp cDNA from rat liver which encoded D1. The deduced sequence provided several surprises (10). A novel feature of D1 enzyme was the presence of a rare amino acid, selenocysteine, in its active center (Figure 1). Later work has shown that the other deiodinases, D2 and D3, also contain selenocysteine in this position(11). Selenocysteine, the selenium analogue of cysteine, is encoded by UGA which presents a difficult problem for the cell since for virtually all eukaryotic mRNAs, UGA is a translation termination or "stop" codon. Further studies showed that recognition of the UGA codon as a selenocysteine codon, as opposed to a "stop" signal, is conferred by a specific feature of the 3' untranslated region of the mRNAs encoding this selenoprotein(12). This signal, a hairpin-shaped structure in the mRNA, is termed a "selenocysteine insertion sequence", abbreviated "SECIS". This element has been indentified in 3'ut of all eukaryotic selenoproteins for which complete cDNA sequences have been isolated. These include D1 and D3, the glutathione peroxidases and the D2 from Rana catesbeiana (13). The SECIS element has not yet been identified in the mammalian D2 mRNAs for which only partial cDNA sequences are currently available. |
