THYROTROPIN RELEASING HORMONE RECEPTOR MOLECULAR BIOLOGY

该研究计划的广泛、长期目标是确定 分子水平的信号转导机制和调节 促甲状腺素释放激素 (TRH) 及其 GTP 结合调节因子 (G) 蛋白偶联受体（TRH-R）。这些研究将带来更好的结果 了解生理和病理下的细胞调节 TRH 和所有激素、生长因子和 通过该家族的受体发出信号的神经递质。研究结果 可能会导致合理设计新药来治疗这些疾病 信号系统。这个提议是及时的，因为新的见解 现在正在研究这些疾病并采用综合方法 分子生物学和计算机建模为更深入的研究提供了广阔的前景 了解这些过程。 TRH-R 的三个相互关联的方面 将探索生物学。具体目标是：#1。 3D 建模 TRH-R的结构。 1A。进一步定义之间的原子相互作用 TRH 和 TRH-R。 1B.进一步明确分子内相互作用 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 将 TRH 直接放入绑定口袋中。 #2.描述TRH-的机制 R激活。 2A。鉴定 TRH-R 的 G 蛋白偶联结构域 并确定参与受体/G蛋白的特定氨基酸 耦合。 2B。鉴定 TRH-R 中的跨膜残基 对于激活很重要。 2C。开发计算机模拟 TRH-R 的构象变化与从 从不活动状态到激活状态。 ＃3。鉴定TRH-中的序列 R 介导内化和翻译后过程 下调，并描述这些机制。为了具体目标#1 #2，一种使用分子生物学工具的交互式方法 实验分析TRH-R的结构-功能将与 基于实验观察的计算机模拟 将指导进一步的实验。对于具体目标#3，诱变 TRH-R将与细胞生物学研究相结合。实验将是 通过将天然和突变蛋白质基因转移到细胞中来进行 文化系统。

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.