THYROTROPIN RELEASING HORMONE RECEPTOR MOLECULAR BIOLOGY

这项研究计划的广泛，长期目标是在一个 分子水平的信号转导机制和调控， 促甲状腺激素释放激素（TRH）及其GTP结合调节因子（G） 蛋白偶联受体（TRH-R）。这些研究将导致更好的 了解生理和病理条件下的细胞调节 TRH和所有激素，生长因子和 通过这个家族的受体传递信号的神经递质。这些发现 可能会导致合理设计新药来治疗这些疾病 信号系统。这一建议是及时的，因为新的见解 这些疾病现在正在获得， 分子生物学和计算机建模为更深入的研究提供了巨大的希望。 了解这些过程。TRH-R的三个相关方面 生物学将被探索。具体目标是：#1。为了模拟3D TRH-R的结构1A.为了进一步定义原子之间的相互作用， TRH和TRH-R。1B.为了进一步确定分子间的相互作用， TRH-R的跨膜螺旋。1C.开发一个模型， TRH-R的细胞外环，并确定这些环是否用于 引导TRH进入结合口袋。#2.为了阐明TRH的作用机制， R激活。2A.鉴定TRH-R的G蛋白偶联结构域 并确定受体/G蛋白所涉及的特定氨基酸 偶合器. 2B.为了鉴定TRH-R中的跨膜残基， 对于激活很重要。2C.为了开发一个计算机模拟 TRH-R的构象变化与从 从非活动状态到活动状态。#3.为了鉴定TRH的序列- R介导的翻译后内化过程， 下调，并描绘这些机制。针对特定目标#1 和#2，使用分子生物学工具的交互式方法， 结合TRH-R结构-功能实验分析， 基于实验观察的计算机模拟， 将指导进一步的实验。对于特定目的#3，诱变 TRH-R将与细胞生物学研究相结合。实验将 通过将天然和突变的蛋白质基因转移到细胞中进行 文化体系。

The broad, long term objective of this research program is to define at a molecular level the mechanism(s) and regulation of signal transduction by thyrotropin-releasing hormone (TRH) and its GTP-binding regulatory (G) protein-coupled receptor (TRH-R). These studies will lead to a better understanding of cell regulation under physiologic and pathologic circumstances by TRH and all hormones, growth factors and neurotransmitters that signal via receptors of this family. The findings may lead to rational design of new drugs to treat diseases of these signalling systems. This proposal is timely because new insights into these diseases are now being gained and a combined approach using molecular biology and computer modelling holds great promise for a deeper understanding of these processes. Three interrelated aspects of TRH-R biology will be explored. The Specific Aims are: #1. To model the 3-D structure of TRH-R. 1A. To further define the atomic interactions between TRH and TRH-R. 1B. To further define the intramolecular interactions among transmembrane helices of TRH-R. 1C. To develop a model of the extracellular loops of TRH-R and determine whether these loops serve to direct TRH into the binding pocket. #2. To delineate the mechanism of TRH- R activation. 2A. To identify the G protein coupling domain(s) of TRH-R and determine the specific amino acids involved in receptor/G protein coupling. 2B. To identify transmembrane residues in TRH-R that are important for activation. 2C. To develop a computer simulation of the conformational changes in TRH-R that are associated with transition from the inactive to the activated state. #3. To identify the sequences in TRH- R that mediate the post-translational processes of internalization and downregulation, and to delineate these mechanisms. For Specific Aims #1 and #2, an interactive approach using tools of molecular biology to experimentally analyze structure-function of TRH-R will be combined with computer simulations that are based on the experimental observations and will guide further experimentation. For Specific Aim #3, mutagenesis of TRH-R will be combined with cell biologic studies. Experiments will be performed by gene transfer of native and mutated proteins into cell culture systems.