THYROTROPIN RELEASING HORMONE RECEPTOR MOLECULAR BIOLOGY

这项研究计划的广泛、长期目标是在一个 分子水平信号转导的机制与调控(S) 促甲状腺激素释放激素及其GTP结合调节(G) 蛋白偶联受体(TRH-R)。这些研究将导致更好的 对生理和病理条件下细胞调控的认识 由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。调查结果 可能导致合理设计治疗这些疾病的新药 信号系统。这一建议是及时的，因为对 这些疾病现在正在获得，并使用一种综合方法 分子生物学和计算机建模为更深层次的 对这些过程的理解。TRH-R的三个相互关联的方面 我们将探索生物学。具体目标是：#1.模拟3-D 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-R1B.为了进一步定义分子内相互作用 TRH-R的跨膜螺旋1C。要开发一种模型， TRH-R的胞外环，并确定这些环是否用于 将TRH直接放入装订袋中。#2.描述TRH的机制- R激活。2a.TRH-R的G蛋白偶联结构域(S)的鉴定 并测定参与受体/G蛋白的特定氨基酸 耦合。2B。鉴定TRH-R中的跨膜残基 对激活很重要。2C。开发一种计算机模拟的方法 TRH-R的构象变化与 从非激活状态变为激活状态。#3.要确定TRH中的序列- R调解翻译后的内化过程和 下调监管，并勾勒出这些机制。针对具体目标#1 和#2，一种使用分子生物学工具的交互方法 实验分析TRH-R的结构-功能将与 基于实验观测的计算机模拟和 将指导进一步的实验。针对特定目标#3，诱变 TRH-R将与细胞生物学研究相结合。实验将是 通过将天然和突变的蛋白质基因转移到细胞中来实现的 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.