THYROID Na+/I SYMPORTER CHARACTERIZATION

描述(由申请人提供)：本项目的总体目标是 Na+/I-转运蛋白(NIS)A的详细分子特征 介导活性的内源性质膜转运蛋白 I-在甲状腺中的移位。NIS在甲状腺中起着至关重要的作用 甲状腺的兴奋性及其在评估、诊断和治疗中的作用 疾病，包括癌症。在这个项目的早期，NIS的cDNA被分离出来， 从而在NIS的分子表征方面取得了显著的进展。这个 阐明了NIS的生物发生和翻译后修饰，比例为2：1 Na+/I-转运的化学计量和有序离子结合机制 通过电生理分析和NIS的调节来证明 转录测定。功能重要氨基酸的作用 NIS中的残留物已经通过表征NIS突变来鉴定，这些突变导致 先天性i-转运缺陷。NIS还在极少数情况下调节碘的运输 其他组织，包括唾液腺、胃粘膜和哺乳期乳房 在乳腺癌方面。NIS在乳腺癌中的表达具有潜在意义 具有诊断和治疗意义。为了实现这一宏伟目标 该项目将追求以下具体目标：1.澄清 NIS的调节机制，通过研究：A)NIS磷酸化的作用 在新谢克尔的贩运和活动中；b)TSH在调节新谢克尔的比率中 靶向质膜和/或内化；c)PDZ基序 NIS对基底膜的极化靶向；以及d) 蛋白酶体在调节NIS靶向质膜中的作用 贩卖和堕落。2.建立结构-功能关系 NIS，通过a)扩展NIS拓扑的分析；b)执行 电生理学分析突变型NIS蛋白的作用 Na+和I-转位中的重要氨基酸残基 途径，以及在Na+/I偶联中；c)识别氨基酸侧链 由NIS抑制剂高氯酸盐修饰的；d)优化条件 用于NIS的增溶、重组和提纯，从而使未来 结构研究是可能的。3.评估NIS在胃中的表达 通过解决不同的生理和病理生理学条件 NIS在胃的不同区域、不同类型的 胃细胞，以及癌前病变中。连续的分子 对NIS的分析显然有可能对广泛的 领域的光谱，从运输蛋白的结构-功能到 甲状腺癌的诊断和治疗，包括甲状腺癌和甲状腺以外的癌症。

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.