Molecular Characterization of the Sodium/lodide Symporter (NIS)

描述(申请人提供)：这个项目的主要目标是详细的分子表征钠/L转运蛋白(NIS)，一个关键的内在质膜转运蛋白，介导I-到甲状腺的主动转位。NIS在甲状腺兴奋发生和甲状腺疾病的诊断和治疗中起着至关重要的作用，例如在甲状腺癌的放射性碘治疗中。从本项目早期分离NIS cDNA开始，在所有水平的蛋白质鉴定方面都取得了很大进展，包括通过鉴定导致I-运输缺陷的NIS突变来鉴定功能重要的残基。NIS还调节其他组织中的I-转运，包括哺乳期乳腺癌和乳腺癌转移，因此NIS在乳腺癌中具有潜在的治疗意义。通过基因转移的方式，NIS也可能在治疗其他组织中没有内源性表达NIS的癌症方面有价值。这里提出的研究强调了NIS进入和离开质膜的关键过程，对其阐明具有重要的基础和医学意义。具体目标如下：1.阐明I-调控NIS自身转运的分子机制：a)高浓度的I-是否影响NIS的降解？B)NIS的降解途径是什么？C)I-诱导NIS内化的途径是什么？D)I-诱导的NIS二聚反应是通过分子间二硫键形成的吗？E)甲状腺DUOX氧化酶是否参与了I-抑制NIS介导的I-转运？2.为了确定NIS极化转运到基侧质膜的决定因素：a)NIS Ct内有哪些信号将蛋白质靶向基侧膜？B)NIS基侧靶向的决定因素能否将钠/单羧酸转运体(SMCT)的靶向从心尖部逆转到基边部？3.为了在NIS中建立新的结构/功能关系：我们将a)确定跨膜片段(TMS)IX上的氨基酸残基在钠依赖中所起的作用；b)对嵌合体NIS/SMCT蛋白进行功能分析，以评估功能重要区域和/或氨基酸残基在Na和I易位途径以及Na/L偶联中的作用；c)承担在大肠杆菌中表达的NIS的溶解、重组和纯化。

DESCRIPTION (provided by applicant): The broad objective of this project is the detailed molecular characterization of the Na+/l- symporter (NIS), a key intrinsic plasma membrane transport protein that mediates active translocation of I- into the thyroid. NIS plays a crucial role in thyroid hormogenesis and in the diagnosis and treatment of thyroid diseases, such as the use of radioiodide therapy in thyroid cancer. Starting with the isolation of the NIS cDNA earlier in this project, much progress has been made in the characterization of the protein at all levels, including the identification of functionally important residues through the characterization of NIS mutations that cause I- transport defect. NIS also mediates I- transport in other tissues, including lactating breast and breast cancer metastases, so NIS is of potential therapeutic significance in breast cancer. By means of gene transfer, NIS may also be valuable in the treatment of cancer in other tissues that do not express NIS endogenously. The studies proposed here emphasize the key process of trafficking of NIS to and from the plasma membrane, the elucidation of which is of major basic and medical relevance. The following specific aims are proposed: 1. To elucidate the molecular mechanism by which I- regulates its own NIS-mediated transport: a) does I- at high concentrations affect NIS degradation? b) what is the NIS degradation pathway? c) what is the pathway of I- -induced NIS internalization? d) does I- -induced NIS dimerization occur by intermolecular disulfide bond formation? e) is the thyroid Duox oxidase involved in the inhibition of NIS-mediated I- transport by I-? 2. To identify the determinants for polarized trafficking of NIS to the basolateral plasma membrane: a) what are the signals within the NIS Ct that target the protein to the basolateral membrane? b) can the determinants of NIS basolateral targeting reverse the targeting of the sodium/monocarboxylate transporter (SMCT) from apical to basolateral? c) is the PDZ-domain-containing protein hScrib involved in NIS targeting to and/or retention at the plasma membrane? 3. To establish new structure/function relations in NIS: we will a) ascertain the role played by amino acid residues present in transmembrane segment (TMS) IX in Na+ dependence; b) perform functional analyses of chimera NIS/SMCT proteins to assess the role of functionally important regions and/or amino acid residues in the Na+ and I- translocation pathways, as well as in Na+/l- coupling; c) undertake the solubilization, reconstitution, and purification of NIS expressed in E. coli.