THYROID Na+/I SYMPORTER CHARACTERIZATION

描述（由申请人提供）：该项目的广泛目标是 Na+/I-同向转运体（NIS）的详细分子表征是 一种内在质膜转运蛋白，介导活性 甲状腺中碘离子的移位。NIS在甲状腺中起着至关重要的作用， 激素生成和甲状腺疾病的评估、诊断和治疗 疾病，包括癌症。在这个项目的早期，NIS cDNA被分离出来， 从而在NIS的分子表征方面取得了显著进展。的 阐明了NIS的生物起源和翻译后修饰，2：1 Na+/I-转运化学计量和有序的离子结合机制， 通过电生理分析证实，NIS的调节 转录已确定。功能重要氨基酸的作用 NIS中的残基已经通过表征引起 先天性I-运输缺陷。NIS也介导碘转运在极少数 其他组织，包括唾液腺、胃粘膜和泌乳期乳腺 和乳腺癌。NIS在乳腺癌中的表达具有潜在的 诊断和治疗意义。为了达到这个广泛的目标， 项目将实现以下具体目标：1.阐明 NIS的调节机制，通过检查的作用：a）NIS磷酸化 B）TSH在调节NIS的比率中的作用 靶向质膜和/或内化; c）PDZ基序， NIS对基底外侧膜的极化靶向;以及d）NIS对基底外侧膜的极化靶向。 蛋白酶体调节NIS靶向质膜， 贩运和退化。2.建立结构-功能关系 NIS，通过a）扩展NIS拓扑的分析; B）执行 突变NIS蛋白的电生理学分析，以评估 Na+和I-转运中功能重要的氨基酸残基 c）鉴定氨基酸侧链; 通过NIS抑制剂高氯酸盐改性; d）优化条件 用于NIS溶解、重构和纯化，从而使未来 可以进行结构研究。3.为了评估NIS在胃中的表达， 生理和病理生理条件，通过解决差异 NIS在胃的不同区域、不同类型的胃粘膜中的表达， 胃细胞和癌前病变。连续分子 对国家创新系统的分析显然有可能对更广泛的 从转运蛋白的结构-功能到 癌症的诊断和治疗，无论是在甲状腺和超越。

DESCRIPTION (provided by applicant): The broad objective of this project is the detailed molecular characterization of the Na+/I- symporter (NIS) a key intrinsic plasma membrane transport protein that mediates the active translocation of I- in the thyroid. NIS plays a crucial role in thyroid hormogenesis and in the evaluation, diagnosis, and treatment of thyroid diseases, including cancer. Earlier in this project, the NIS cDNA was isolated, resulting in remarkable progress in the molecular characterization of NIS. The biogenesis and post-translational modifications of NIS were elucidated, a 2:1 Na+/I- transport stoichiometry and an ordered ionic binding mechanism were demonstrated by electrophysiological analysis, and the regulation of NIS transcription was determined. The roles of functionally important amino acid residues in NIS have been identified by characterizing NIS mutations that cause congenital I- transport defect. NIS also mediates iodide transport in very few other tissues, including salivary glands, gastric mucosa and lactating breast and in breast cancer. NIS expression in breast cancer is of potential diagnostic and therapeutic significance. To reach the broad objective of this project, the following specific aims will be pursued: 1. To elucidate regulatory mechanisms of NIS, by examining the roles of: a) NIS phosphorylation in NIS trafficking and activity; b) TSH in modulating the rates of NIS targeting to the plasma membrane and/or internalization; c) the PDZ motif in the polarized targeting of NIS to the basolateral membrane; and d) the proteasome in the regulation of NIS targeting to the plasma membrane, trafficking, and degradation. 2. To establish structure-function relations in NIS, by a) extending the analysis of NIS topology; b) performing an electrophysiological analysis of mutant NIS proteins to assess the role of functionally important amino acid residues in the Na+ and I- translocation pathways, as well as in Na+/I- coupling; c) identifying amino acid side chains that are modified by the NIS inhibitor perchlorate; d) optimizing conditions for NIS solubilization, reconstitution and purification, thus making future structural studies possible. 3. To assess NIS expression in the stomach under physiological and pathophysiological conditions by addressing the differences in NIS expression among different regions of the stomach, in different types of gastric cells, and in premalignant gastric lesions. The continued molecular analysis of NIS clearly holds the potential of an even greater impact on a wide spectrum of fields, ranging from structure-function of transport proteins to the diagnosis and treatment of cancer, both in the thyroid and beyond.