THE NA+/I- SYMPORTER: CHARCTERIZATION & TSH REGULATION

本建议是两年前开始的一个项目的继续。 其 总体目标是甲状腺的分子特征 Na+/I-同向转运体与促甲状腺激素（TSH） 调节同向转运体的活性。 我们对分子的了解 几种甲状腺特异性蛋白质的性质，包括甲状腺 过氧化物酶，甲状腺球蛋白和TSH受体，增加了 近几年大幅。 然而，在这方面存在着重大差距。 了解Na+/i-同向转运体，一种重要的生理血浆 介导甲状腺中碘积累的膜蛋白。 I-摄取 是甲状腺激素T3和T4生物合成的第一步。到 迄今为止，同向转运体分子尚未被表征;其大小和 氨基酸序列是未知的，并且抗同向转运体Ab不可用。 Na ~+/I ~-同向转运体在X.卵母细胞注射 来自FRTL-5细胞（一种高功能大鼠甲状腺细胞系）的poly A+ RNA 细胞 在通过蔗糖梯度离心和 注射所得的级分，编码聚腺苷酸的poly A+ RNA 在含有2.8- 4.0 kb信息的片段中发现了同向转运体， 长度已经构建了大约10（6）个独立克隆的cDNA文库。 由这一小部分组成。 RNA在体外从cDNA模板 3，600个克隆的库已显示诱导I-摄取活性， 卵母细胞 骆驼蓬碱和TRP-P-2，两种萜烯，已被鉴定为 使用FRTL-5的新型Na+位点竞争性共转运体抑制剂 细胞和小牛甲状腺膜囊泡作为模型系统。 TRP-P-2具有 用于亲和层析研究，旨在鉴定 同向转运体。约120 kDA的多肽被选择性保留， 亲和矩阵 这种多肽可能是同向转运体，但更多的是 确定性的实验是必要的。 有人指出， 表明Na+/I-共输过程不是产电的，Na+/I-共输过程是一个非产电的过程。 化学计量比为1：1。 出乎意料的是，由 保持在无TSH培养基中的FRTL-5细胞[TSH]（-）]积累I-， 即使相应的TSH（-）完整细胞不显示这种 运输 该提案的具体目标是：1。鉴定 Na ~+/I ~-同向转运体及其一级序列测定 通过对3,600个克隆的cDNA库进行持续的功能筛选。 2.通过以下性能对同向转运体进行分子表征：a） 结构/功能研究，采用抗体和修饰的 共转运体分子，和B）生物发生研究，通过脉冲追踪分析。3. 促甲状腺激素调节的同向转运体，通过测试两个提出的假设，促甲状腺激素 通过亚细胞分级作用。

The present proposal continues a project initiated two years ago. Its overall objectives are the molecular characterization of the thyroid Na+/I- symporter and the study of the mechanism by which thyrotropin (TSH) regulates the symporter's activity. Our knowledge of the molecular properties of several thyroid-specific proteins, including thyroid peroxidase, thyroglobulin, and the TSH receptor, has increased substantially in recent years. However, a major gap exists in the understanding of the Na+/i- symporter, a physiologically important plasma membrane protein that mediates I- accumulation in the thyroid. I- uptake is the first step in the biogenesis of the thyroid hormones T3 and T4. To date, the symporter molecule has not been characterized; its size and amino acid sequence are unknown, and anti-symporter Abs are not available. The Na+/I- symporter has been expressed in X. laevis oocytes injected with poly A+ RNA from FRTL-5 cells, a line of highly functional rat thyroid cells. After fractionation by sucrose gradient centrifugation and injection of the resulting fractions, the poly A+ RNA encoding the symporter was found in a fraction containing messages of 2.8-4.O kb in length. A approximate 10 (6) independent-clone cDNA library has been constructed from this fraction. RNA made in vitro from cDNA template of a pool of 3,600 clones has been shown to induce I- uptake activity in oocytes. Harmaline and TRP-P-2, two terpenes, have been identified as novel Na+- site competitive inhibitors of the symporter, using FRTL-5 cells and calf thyroid membrane vesicles as model systems. TRP-P-2 has been employed in affinity chromatography studies aimed at identifying the symporter. A approximate 120 kDA polypeptide was selectively retained by the affinity matrix. This polypeptide might be the symporter, but more definitive experiments are necessary. Observations have been made that suggest the Na+/I- symport process is not-electrogenic and the Na+/I- stoichiometry is 1:1. Unexpectedly, mixed membrane vesicles prepared from FRTL-5 cells maintained in a TSH free medium [TSH] (-)] accumulate I-, even though the corresponding TSH (-) intact cells display no such transport. The Specific Aims of the proposal are: 1. Identification of the Na+/I- symporter and determination of its derived primary sequence through continued functional screening of the cDNA pool of 3,600 clones. 2. Molecular characterization of the symporter, by performance of : a) structure/function studies, employing Abs and modifications of the symporter molecule, and b) biogenesis studies, by pulse-chase analysis. 3. TSH regulation of the symporter, by testing two proposed hypotheses of TSH action via subcellular fractionation.