MOLECULAR CHARACTERIZATION OF THE IODIDE CARRIER

这个项目的总体目标是描述 甲状腺碘载体蛋白的结构与功能 腺体。这种转运蛋白负责活性的钠离子依赖 相对于其浓度梯度的碘的积累，是一种 位于上皮基底外侧端的膜蛋白 甲状腺滤泡的细胞。碘向这些细胞的转运 是合成甲状腺激素(T_3和T_3)的第一步 T4)。促甲状腺激素(TSH)调节其表达 通过激活腺苷环化酶-cAMP- 依赖蛋白激酶系统。尽管它的基本原理 在哺乳动物生理学中的重要性，目前对此知之甚少 碘化物的结构、生物化学和分子性质 载体蛋白。本提案所载的初步研究 表明非洲爪哇表达了碘的转运活性 显微注射从FRTL-5细胞(A)中分离的mRNA的卵母细胞 连续培养的全功能大鼠甲状腺细胞系 细胞)，编码转运蛋白的mRNA大小为2.8-4.0kb 在篇幅上。为此，提出了以下几点建议：1)确定 碘载体蛋白一级序列的推导 从2.8kb-4.0kb的mRNA片段构建cDNA文库。 B)非洲爪哇翻译中的功能分析 从阳性克隆中插入的系统和序列；2)至 研究碘载体的结构/功能关系 蛋白质，通过进行定点多克隆研究 抗体，部分蛋白分解，组特异性试剂和位点- 特异性诱变：3)研究生物合成和后诱变。 脉冲修饰I-载体蛋白的翻译修饰 Chase分析；4)确定碘化物的生电性 直接熔剂输运和测定Na~+/I-化学计量比 通过测量和应用热力学方法。

The overall objective of this project is to characterize the structure and function of the iodide carrier protein of the thyroid gland. This transporter' responsible for the active Na+-dependent accumulation of iodide against its concentration gradient, is a membrane protein located in the baso-lateral end of the epithelial cells of the thyroid follicles. Iodide transport into these cells is the first step in the synthesis of the thyroid hormones (T3 and T4). Thyroid stimulating hormone (TSH) regulates the expression of the iodide carrier by activating the adenylate cyclase-cAMP- dependent protein kinase system. Despite its fundamental importance in mammalian physiology, little is currently known about the structure, biochemistry and molecular properties of the iodide carrier protein. Preliminary studies contained in this proposal show that iodide transport activity is expressed in Xenopus laevis oocytes microinjected with mRNA isolated from FRTL-5 cells (a continuous line of cultured and fully functional rat thyroid cells), and that the mRNA encoding the transporter is 2.8-4.0 kb in length. Hence, the following is proposed: 1) to determine the derived primary sequence of the iodide carrier protein by constructing a cDNA library from the 2.8-4.0 kb fraction of mRNA. screen it b) functional analysis in the Xenopus laevis translation system and sequence cDNA inserts from positive clones; 2) to investigate structure/function relationships of the iodide carrier protein, by performing studies with site-directed polyclonal antibodies, partial proteolysis, group-specific reagents and site- specific mutagenesis: 3) to study the biosynthesis and post- translational modifications of the I- carrier protein by pulse- chase analysis; 4) to establish the electrogenicity of iodide transport and to determine the Na+/I-stoichiometry by direct flux measurements and by applying a thermodynamic approach.