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

本提案延续了两年前启动的一个项目。它的 总体目标是甲状腺的分子特征。 钠/碘转运体与促甲状腺激素(TSH)作用机制的研究 规范符号商的活动。我们对分子的了解 几种甲状腺特异蛋白的性质，包括甲状腺 过氧化物酶、甲状腺球蛋白和促甲状腺激素受体增加 基本上是在最近几年。然而，一个主要的差距存在于 对生理上重要的血浆Na+/I-转运蛋白的认识 在甲状腺中介导碘蓄积的膜蛋白。I-摄取 是甲状腺激素T3和T4生物发生的第一步。至 到目前为止，共转运体分子尚未被表征；它的大小和 氨基酸序列未知，也没有抗同向转运蛋白抗体。 Na~+/I~-转运蛋白已在乳突圆线虫卵母细胞中表达 高功能大鼠甲状腺FRTL-5细胞的Poly A+RNA 细胞。经蔗糖梯度离心法分级后， 注入产生的组分，编码 在含有2.8-4.0kb信息的片段中发现了符号转运子 长度。已获得约10(6)个独立克隆的cDNA库 由这一部分构成的。以沙门氏菌DNA为模板体外制备RNA 一个由3,600个克隆组成的池被证明可以诱导I-摄取活性 卵母细胞。Harmaline和Trp-P-2是两个萜类化合物，已被鉴定为 使用FRTL-5的新型Na+位竞争性抑制剂的研究 细胞和小牛甲状腺膜囊泡作为模型系统。Trp-P-2有 被用于亲和层析研究，目的是识别 辛波特。选择性地保留了大约120 kDa的多肽。 亲和力矩阵。这种多肽可能是转运蛋白，但更多的 决定性的实验是必要的。已经有观察到 提示Na+/I-结合过程不是电生的，而Na+/I-结合过程 化学计量比为1：1。出乎意料的是，从 在无TSH的培养液中维持的FRTL-5细胞[TSH](-)]积累I-， 即使相应的TSH(-)完整细胞没有显示 运输。该提案的具体目标是：1.确定 Na+/I-转运蛋白及其衍生一级序列的测定 通过对3,600个克隆的cDNA库进行持续的功能筛选。 2.共转运体的分子表征，通过以下性能：a) 结构/功能研究，采用抗体和修饰 B)生物发生研究，通过脉冲追逐分析。3. 通过检验两个提出的TSH假说，TSH对转运蛋白的调节 通过亚细胞分级来发挥作用。

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.