Novel Molecular Mechanism of De Novo Triiodothyronine (T3) Formation Induced by Fam20C in Stimulated Thyrocytes

CAROLINA IGLESIAS; HÉCTOR M. TARGOVNIK; ALICIA T. GAUNA; CINTIA E. CITTERIO; PETER ARVAN

Virtual

Congreso; Reunión Anual de Sociedades de Biociencias SAIC-SAI-SAFIS 2020; 2020

Sociedad Argentina de Investigación Clínica

T4 derives from the N-terminus of thyroglobulin (Tg), whereas de novo formation of T3 involves coupling of mono- and di-iodotyrosine famously occurring at the opposite end of the Tg protein. Stimulation of thyroidal TSH receptors enhances T3 formation in secreted Tg, as occurs in autoimmune hyperthyroidism of Graves´ disease, and promotes the expression of Fam20C, a Golgi-localized secretory (casein) kinase. Knockdown of Fam20C or 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. We found that Fam20C-stimulated T3-formation in mTg still occurred when the classic T3-forming sites were eliminated: Y2519F, Y2552F and Y2744F (Tg-3xF). We hypothesize that Fam20C-stimulated T3-formation occurs at secondary T3 hormonogenic sites in Tg. To consider T3 formation involving Y5, Y130, Y239, Y973, or Y1290, we utilized Tg-3xF and further mutagenized selective additional Y residue to F. In parallel, we also created a single mutation of Tg-Y5F, Tg-Y130F, or Tg-Y239F. 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 Abs. Our data reveal that Fam20C could still increase de novo T3-formation in Tg even in the Tg-Y973F-3xF or Tg-Y1290F-3xF mutants. However, Fam20C-stimulated T3-formation was partially reduced when either Y130 or Y239 (iodotyrosyl donors to Y5) were disrupted. Moreover, when Fam20C was co-expressed with Tg-Y5F-3xF or Tg-Y5F, de novo T3 formation was inhibited nearly to control levels, suggesting that Fam20C shifts Tg to favor T3-formation at the classic T4-forming site at Y5. Our results show that TSH/Fam20C-stimulated de novo T3 formation in Tg entails a coupling reaction that includes Y5. Donor iodotyrosines, such as Y130 and Y239, may control the T4:T3 ratio of Tg in different thyroid diseases.