Post-translational regulation of the thyroid sodium(+)/iodine(-) symporter (NIS) by thyrotropin
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The Na+/I- symporter (NIS) is the intrinsic plasma membrane glycoprotein that mediates the active transport of iodide (I-) in the thyroid gland.;In this thesis we have thoroughly analyzed NIS thyroid protein expression, its biogenesis, half-life, subcellular localization, post-transcriptional modifications such as glycosylation and phosphorylation, and the effect of TSH in the regulation of NIS protein.;These studies were performed in FRTL-5 cells. By using a high affinity anti-NIS Ab, developed in our laboratory, we demonstrated conclusively that NIS is present in FRTL-5 cells as late as 10 days after TSH withdrawal and that de novo NIS biosynthesis requires TSH. These results clearly show that any NIS molecules detected in FRTL-5 cells kept in the absence of TSH had to be synthesized prior to TSH withdrawal, and suggest that NIS has a long half-life. Consistent with that, we determined NIS half-life in the presence (t1/2: 5 days) and absence (t1/2: 3 days) of TSH. Even though the NIS half-life in the absence of TSH is 40% shorter that in the presence of the hormone, it is still sufficiently long to account for the persistence of significant I- uptake activity in membrane vesicles from cells deprived of TSH.;To explain the observation that in the absence of TSH NIS is present but not functionally active in the intact cell, we tested the hypothesis of NIS redistribution, which proposes that in the absence of TSH NIS molecules would reside in intracellular organelles and not in the plasma membrane, thus NIS would not be able to mediate I- transport activity in the intact cell. We demonstrate a close correlation between NIS plasma membrane content and NIS activity, indicating that decreased I- transport after TSH withdrawal is due to a decrease in the amount of NIS at the cell surface. After 3 days of TSH deprivation, NIS molecules are recruited to intracellular compartments.;We have demonstrated for the first time that NIS is phosphorylated in vivo and that its phosphorylation pattern is modulated by TSH. We identified that the COOH terminus is the predominantly phosphorylated region of NIS, and that phosphorylation of NIS occurs mainly at serine residues independently of whether cells are kept in the presence or absence of TSH. We determined that S551 and S552, residues that localize at the COOH terminus, are phosphorylated in vivo. However, substitution of these serine residues for alanine did not impair either NIS functional expression or its phosphorylation level. Given that the COOH terminus was still the predominant region that is phosphorylated, and that serine residues were the only residues phosphorylated in these NIS mutants, we mutated S568 and S581 in the background of the S551 and S552 double mutant, named S568A-S581A*. (Abstract shortened by UMI.).
Source: Dissertation Abstracts International, Volume: 64-09, Section: B, page: 4157.;Advisors: Nancy Carrasco.