Molecular Characterization of the Sodium/lodide Symporter (NIS)

钠/碘同向转运体 (NIS) 的分子表征

• 批准号: 7892884
• 负责人: Nancy Carrasco
• 金额: $ 5.15万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-05-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7892884
• 关键词: Address; Affect; Amino Acids; Apical; Biogenesis; Breast; Carrier Proteins; Cell membrane; Cells; Chimera organism; Complementary DNA; Coupling; Cretinism; Defect; Degradation Pathway; Dependence; Diagnosis; Dimerization; Escherichia coli; Exhibits; Funding; Gastric mucosa; Gene Transfer; Genetic Transcription; Genomics; Human; Lateral; Malignant neoplasm of thyroid; Mediating; Medical; Membrane; Membrane Transport Proteins; Molecular; Molecular Analysis; Mutation; Neoplasm Metastasis; Oxidases; Pathway interactions; Physiological; Plasma; Play; Post-Translational Protein Processing; Process; Proteins; Regulation; Role; SLC5A5 gene; Salivary Glands; Signal Transduction; Sodium; Sterile coverings; Structure; Testing; Therapeutic; Thyroid Diseases; Thyroid Gland; Tissues; basolateral membrane; cancer therapy; clinically relevant; diagnosis evaluation; disulfide bond; insight; malignant breast neoplasm; oxidation; reconstitution; research study; symporter; trafficking

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.

描述（由申请人提供）：本项目的广泛目标是Na+/l-同向转运体（NIS）的详细分子表征，NIS是介导I-主动转运至甲状腺的关键内在质膜转运蛋白。NIS在甲状腺激素生成以及甲状腺疾病的诊断和治疗中起着至关重要的作用，例如在甲状腺癌中使用放射性碘治疗。从本项目早期分离NIS cDNA开始，在所有水平上对蛋白质进行表征方面取得了很大进展，包括通过表征引起I-转运缺陷的NIS突变来鉴定功能重要的残基。NIS还介导其他组织中的I-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。通过基因转移的方式，NIS也可能在治疗不内源性表达NIS的其他组织中的癌症中有价值。这里提出的研究强调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：1。为了阐明I-调节其自身NIS介导的转运的分子机制：a）高浓度的I-是否影响NIS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二聚化是否通过分子间二硫键的形成而发生？e）甲状腺Duox氧化酶是否参与I-对NIS介导的I-转运的抑制？2.为了确定NIS极化运输至基底外侧质膜的决定因素：a）NIS Ct内将蛋白质靶向基底外侧膜的信号是什么？B）NIS基底外侧靶向的决定因素是否可以逆转钠/单羧酸转运蛋白（SMCT）从顶端到基底外侧的靶向？c）含PDZ结构域的蛋白hScrib是否参与NIS靶向和/或保留在质膜上？3.为了建立NIS新的结构/功能关系，我们将a）确定跨膜片段（TMS）IX中存在的氨基酸残基在Na+依赖性中所起的作用; 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-转运途径以及Na+/I-偶联中的作用; c）对E.杆菌