Novel site of de novo triiodothyronine (T3) formation in thyroglobulin secreted from stimulated thyrocytes

TORRES, MAURICIO; CITTERIO, CINTIA E.; ARVAN, PETER; CAROLINA M. IBÁÑEZ PADILLA

Chicago

Congreso; 89th Annual Meeting of the American Thyroid Association (ATA); 2019

American Thyroid Association

Introduction:De novo T3 hormonogenesis involves coupling of mono- and di-iodotyrosine, famously occurring at the C-terminus of thyroglobulin (Tg), whereas T4 is formed primarily at Y-5 near the N-terminus. Upon in vitro iodination of Tg secreted from TSH-receptor (TSHR) hyperstimulated thyrocytes, T3 formation increases. Interestingly, hyperstimulation of thyroidal TSHRs promotes upregulation of Fam20C, a Golgi-localized serine (?casein?) kinase. Overexpression of Fam20C increases de novo T3 formation in secreted Tg, whereas Fam20C knockdown decreases T3 formation in Tg. Mutation of mTg-S2718A (equivalent to hTg-S2721A, an established phosphorylation site) inhibits Fam20C-stimulated T3 formation, while enhancement is observed for the phosphomimetic mTg-S2718E mutant. To our surprise, we found that Fam20C-stimulated T3-formation in mTg still occurred when the classic T3-formation sites were eliminated at the C-terminus of Tg: Y2519F, Y2552F and Y2744F (called Tg-3xF).Methods / Case Presentation:Our hypothesis is that TSH/Fam20C-stimulated T3-formation occurs at secondary T3 hormonogenic sites within Tg. To consider T3 formation at Y-5, or Y-973, or Y-1290, we utilized Tg-3xF and further mutagenized selective additional Tyr residues. In parallel, we also created a single mutation of Tg-Y5F. We co-expressed each Tg construct +/- Fam20C, performed enzymatic iodination of the secreted Tg, and monitored de novo T3-formation by immunoblotting with anti-T3 and anti-Tg antibodies. Results / Discussion:Our data reveal that Fam20C could still increase de novo T3-formation in Tg even in the Tg-Y973F-3xF or Tg-Y1290F-3xF substitution mutants. However, when Fam20C and Tg-Y5F-3xF were co-expressed, de novo T3 formation was inhibited nearly to control levels. Moreover, a prominent effect on de novo Fam20C-stimulated T3-formation was observed in the single Tg-Y5F substitution mutant, suggesting that Fam20C shifts Tg to favor T3-formation at the classic T4-formation site at Y-5. Conclusions: Our results suggest that a main candidate site of TSH/Fam20C-stimulated de novo T3 formation in Tg involves a coupling reaction that includes Y-5. These studies suggest that the donor iodotyrosine(s) to Y-5 may exert major control over the T4 to T3 ratio of Tg in different thyroid disease states.