Molecular Characterization of the Sodium/lodide Symporter (NIS)

描述（由申请人提供）：本项目的主要目标是详细描述Na+/l-同调体（NIS）的分子特征，NIS是一种关键的内在质膜转运蛋白，可介导I-在甲状腺中的主动转运。NIS在甲状腺激素形成和甲状腺疾病的诊断和治疗方面发挥着至关重要的作用，例如在甲状腺癌中使用放射性碘化物治疗。从本项目早期NIS cDNA的分离开始，在各个水平上对该蛋白的表征取得了很大进展，包括通过表征导致I-转运缺陷的NIS突变来鉴定功能重要的残基。NIS还介导I-在其他组织中的转运，包括哺乳期乳腺和乳腺癌转移灶，因此NIS在乳腺癌中具有潜在的治疗意义。通过基因转移，NIS也可能在治疗其他不内源性表达NIS的组织中的癌症中有价值。本文提出的研究强调了NIS进出质膜的关键过程，阐明这一过程具有重要的基础和医学意义。提出了以下具体目标：为了阐明I-调节其自身NIS介导的转运的分子机制：a)高浓度I-是否影响NIS降解？b) NIS的降解途径是什么？c) I-诱导的NIS内化途径是什么？d) I-诱导的NIS二聚化是否通过分子间二硫键形成发生？e)甲状腺二氧化酶是否参与I-抑制nis介导的I-转运？2. 为了确定NIS向基底外侧质膜极化运输的决定因素：a) NIS Ct内将蛋白质靶向到基底外侧膜的信号是什么？b) NIS基底外侧靶向的决定因素能否逆转钠/单羧酸转运蛋白（SMCT）从根尖到基底外侧的靶向？c)含有pdz结构域的蛋白hScrib是否参与靶向和/或滞留在质膜上的NIS ？3. 为了在NIS中建立新的结构/功能关系：我们将a)确定跨膜段（TMS） IX中存在的氨基酸残基在Na+依赖性中的作用；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)对大肠杆菌中表达的NIS进行增溶、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.