Control of nuclear thyroid hormone signaling by cell surface proteins

描述（由申请人提供）：甲状腺疾病影响超过2500万美国人，是第二常见的代谢性疾病。甲状腺激素（TH）对生长、发育和代谢至关重要，通过与细胞核内TH受体结合并改变基因表达而起作用。重要的是，TH必须从血液中穿过质膜，与细胞核中的TH受体结合。这种易位过程中的一种细胞表面蛋白，l型氨基酸转运蛋白LAT1，在th依赖性发育过程中被强烈上调。有趣的是，LAT1的表达也与快速分裂的细胞有关，如活化的淋巴细胞和癌症。尽管已经开展了许多关于LAT1功能的体外研究，但LAT1在体内发育中的作用尚未得到很好的了解。当前项目的目的是研究LAT1在发育过程中如何调节组织对TH的反应。该项目补充了研究者的长期目标，即阐明细胞质蛋白控制TH信号的分子机制。为了实现这一目标，该计划是，在具体目标1，开发和表征一个改进的四环素诱导转基因系统。他将设计，生产和表征转基因诱导剂和报告系，当交叉时，将能够高水平的转基因诱导。这种改进的系统将使我们能够最大限度地减少泄漏的转基因表达，减少由插入位点引起的转基因表达水平的可变性，并通过不同剂量的诱导化学多西环素（Dox）“调节”转基因表达水平。此外，该系统可以扩展到组织特异性转基因诱导。初步数据显示，在没有Dox的情况下，泄漏表达最小，而在有Dox的情况下，转基因诱导率很高。特异性目的2将检查LAT1在th依赖性发育中的体内作用。研究者将使用来自Aim 1的转基因技术来验证LAT1在体内调节TH信号的假设。他将比较经Dox和未经Dox处理的转基因蝌蚪诱导LAT1过表达时th诱导的形态学和基因调控变化的速率和幅度。该项目有望揭示LAT1如何在体内调节TH信号的基本见解。特别是，TH转运体LAT1的过表达将增加TH对核TH受体的可用性，这可以通过增加TH依赖基因表达和形态变化来测量。本项目的一个显著的积极影响是，研究者将扩展研究th依赖性发育、青蛙变态的成熟系统的有用性，并为青蛙群落提供新的转基因资源。这些资源对于各种各样的实验方法都是有用的。此外，当考虑将LAT1作为治疗甲状腺疾病和癌症的药物开发靶点时，了解LAT1如何调节体内组织对TH的反应是重要的。

DESCRIPTION (provided by applicant): Thyroid diseases affect over 25 million Americans and constitute the second most common group of metabolic disorders. Thyroid hormone (TH) is critical for growth, development, and metabolism, and works by binding TH receptors in the cell nucleus and changing gene expression. Importantly, TH must cross the plasma membrane from the blood to bind TH receptors in the nucleus. One cell surface protein in this translocation process, the L-type amino acid transporter LAT1, is strongly up-regulated during TH-dependent development. Interestingly, LAT1 expression is also associated with rapidly dividing cells, such as activated lymphocytes and cancer. Even though many in vitro studies on LAT1 function have been carried out, the in-vivo developmental role of LAT1 is not well understood. The objective of the current project is to study how LAT1 regulates tissue responses to TH during development. This project complements the investigator's long-term goal of elucidating molecular mechanisms underlying control of TH signaling by cytoplasmic proteins. To achieve this objective, the plan is to, in Specific Aim 1, develop and characterize an improved tetracycline-inducible transgenic system. He will design, produce, and characterize transgenic inducer and reporter lines that, when crossed, will be capable of high levels of transgene induction. This improved system will enable us to minimize leaky transgene expression, reduce variability in transgene expression levels caused by insertion sites, and "dial in" transgene expression levels with the inducing chemical doxycycline (Dox) administered at different doses. In addition, this system can expand to enable tissue-specific transgene induction. Preliminary data showed minimal leaky expression in the absence of Dox and high transgene induction in the presence of Dox. Specific Aim 2 will then examine the in-vivo role of LAT1 in TH-dependent development. The investigator will use the transgenic technology from Aim 1 to test the hypothesis that LAT1 modulates TH signaling in vivo. He will compare rate and magnitude of TH-induced morphological and gene regulation changes in transgenic tadpoles treated with and without Dox to induce LAT1 overexpression. This project is expected to reveal fundamental insights about how LAT1 regulates TH signaling in vivo. In particular, it is expected that overexpression of the TH transporter LAT1 will increase availability of TH to nuclear TH receptors as measured by increased TH-dependent gene expression and morphological change. A significant positive impact of this project is that the investigator will extend the usefulness of a well-established system for studying TH-dependent development, frog metamorphosis, and provide new transgenic resources to the frog community. These resources will be useful for a wide variety of experimental approaches. In addition, knowledge of how LAT1 regulates tissue responses to TH in-vivo is significant when considering LAT1 as a target for drug development in the treatment of thyroid disorders and cancer. PROJECT NARRATIVE: The investigator is studying how the cell surface protein LAT1 affects thyroid hormone signaling during development. Importantly, LAT1 has been identified as a drug target in the treatment of thyroid diseases and related disorders and complications, such as obesity, cardiovascular disease, and cancer. Thus, knowledge of developmental roles of LAT1 is important when considering use of drugs targeting LAT1 to evaluate potential benefits and side-effects.