TH-Dependent, Actin-Based Vesicle Trafficking

TH 依赖性、基于肌动蛋白的囊泡运输

• 批准号: 6517957
• 负责人: JACK L. LEONARD
• 金额: $ 33.99万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-15 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517957
• 关键词: actins; brain; developmental neurobiology; endocytosis; expression cloning; gene targeting; glia; hormone receptor; hormone regulation /control mechanism; laboratory mouse; myosins; neurons; thyroid hormone binding protein; thyroid hormones; vesicle /vacuole

DESCRIPTION (provided by applicant): Thyroid hormone (TH) is essential for the normal growth and development of the brain, but the molecular events that mediate these actions are poorly understood. While TH-regulated gene expression is widely assumed to play a key role in this process, the recent revelation that both TH receptor genes (TRa and TRb) can be deleted without affecting brain development raises the possibility that other nongenomic TH-dependent processes contribute to the actions of TH in brain. One of the best-characterized, nongenomic actions of TH is the dynamic regulation of brain type 2 deiodinase (D2). Both thyroxine (T4) and the metabolically inert TH, reverse T3 (rT3), dynamically regulate brain D2 levels in vivo by modulating the rate of D2 removal from the plasma membrane through actin-based endocytosis. A key component of this regulatory cascade is a high affinity TH Initiator Protein (TIP) that facilitates the docking of the D2 containing endosome to the actin based molecular motor, Myosin 5a (Myo5a). We hypothesize the high affinity T4-binding protein(s) (TIP) that tethers the endocytotic vesicle to Myo5a is derived from the C-terminus of the TRa gene, known as TRAa. We will establish the role of TRAa(s) in TH-dependent vesicle trafficking using TH-displacement analysis, in vitro actin-binding assays, real time analysis of TH-initiated vesicle trafficking and pull-down assays with the vesicle binding domain of Myo 5a (SA #1). We will also survey the cell lysate for additional TIP proteins using bait:prey with the C-terminus of Myo5a, expression cloning and TH-binding assays (SA#II). We will then develop targeted gene constructs designed to delete the TRAdelta-alphas using homologous recombination and the LoxP/Cre system. We will examine the consequences of this targeted TRa intron 7 deletion on TH-dependent :actin-based endocytosis in neurons and glial cells derived in vitro from TRAa-nul ES cells and create TRda-nul :mice (SA#III). The biology of T4-dependent actin-based endocytosis of p29 is a unique experimental model for understanding the nongenomic actions(s) of TH in brain and uses cellular machinery essential to normal synaptic function which may account for the neurological abnormalities of hypothyroidism.

描述（由申请人提供）： 甲状腺激素（Th）对于正常生长和发展至关重要 大脑，但是介导这些动作的分子事件很差 理解。而TH调控的基因表达被广泛假定播放键 在这一过程中的作用，最近两个受体基因的启示（tra 可以删除TRB）而不会影响大脑发育会增加 其他非核心TH依赖性过程有可能有助于 在大脑中的动作。 TH的特征最佳，非原始组动作之一 是大脑2脱碘酶（D2）的动态调节。甲状腺素（T4） 以及代谢惰性Th，反向T3（RT3），动态调节脑D2 通过调节从质膜去除D2的速率，体内水平 通过基于肌动蛋白的内吞作用。此监管级联的关键组成部分是 高亲和力TH启动蛋白（TIP），可促进对接 D2包含基于肌动蛋白的分子运动体的内体，肌球蛋白5a（MyO5a）。 我们假设高亲和力T4结合蛋白（S）（尖端） 到肌5a的内吞囊泡源自Tra基因的C末端， 被称为Traa。我们将确定Traa在Th依赖性囊泡中的作用 使用Th-Displacement分析，体外肌动蛋白结合测定法进行贩运 用刺激囊泡运输和下拉测定的时间分析 Myo 5a的囊泡结合域（SA＃1）。我们还将调查细胞裂解物 对于使用诱饵的其他尖端蛋白质：猎物与myo5a的C末端 表达克隆和TH结合测定（SA＃II）。然后，我们将发展目标 旨在使用同源的基因结构 重组和LOXP/CRE系统。我们将研究这一点的后果 靶向TRA内含子7对TH依赖性的缺失：基于肌动蛋白的内吞作用 神经元和神经胶质细胞在体外从Traa-Nul ES细胞中得出，并创建 trda-nul：小鼠（SA＃III）。基于T4依赖性肌动蛋白的生物学的生物学 P29是一个独特的实验模型，用于理解非原始组作用（S） 在大脑中的TH，并使用正常突触的必不可少的蜂窝机械 可能解释神经系统异常的功能 甲状腺功能减退症。