Determinants of the Cannabinoid Receptor Life Cycle

描述(由申请人提供)：人大麻素受体1(CB1)结合9-四氢大麻酚、大麻的精神活性成分和其他拟大麻化合物。它是一种G蛋白偶联受体，与中枢神经系统相关，主要通过偶联Gi/Go蛋白发挥作用。CB1激动剂的药理作用包括镇痛、抑制恶心、刺激食欲、止吐和扩张支气管，而反向激动剂则减轻过度进食障碍。任何针对CB1的治疗策略都需要我们对影响其生命周期关键点的受体的结构特征有一个确切的了解：内质网整合和细胞表面表达，受体激活，以及脱敏和内化。在目标1中，我们将研究CB1氨基末端(N-Tail)弱ER易位的基础。将确定N-末端的结构，并鉴定辅助蛋白及其在N-末端和C-末端的识别基序。这些基序在细胞定位中的作用，包括神经元中半树突状细胞和轴突膜表面的定位，将被研究。在目标2中，我们将在识别对区分激动剂和反向激动剂相互作用至关重要的受体结构元件的基础上，全面定位参与其中的TM结构域和CB1胞外区的关键接触点。我们将定义对于使CB1非典型的配体非依赖平衡走向激活至关重要的残基，以及负责将这种中间受体状态相互转化为静止和激活形式的残基。在目标3中，我们将利用新的受体突变体来模拟受体的不同结构状态，以检查受体激活、脱敏和细胞定位之间的联系。模拟CB1非活性和活性形式的突变体将提供工具，用于分析长期使用反向激动剂和激动剂治疗的后果。我们将利用我们在开发结构分析和与纯化成分的结合分析方面的专业知识来研究这些过程的分子基础，重点是受体的羧基末端(C-Tail)。在这项工作中，我们将确定提高CB1细胞表面表达的决定因素，大规模制备受体结构域的策略，以及它们的结构分析，这可应用于其他GPCRs和膜蛋白的结构研究。相关：大麻素受体1(CB1)是一种G蛋白偶联受体，与中枢神经系统有关。集中在CB1在信号转导中的作用的研究活动强调了它作为治疗药物靶点的巨大潜力。本项目的目标是了解影响CB1生命周期关键阶段的结构特征，包括细胞表面表达、受体激活和内化，并最终影响其细胞表面暴露，以便为治疗策略提供依据。

DESCRIPTION (provided by applicant): The human cannabinoid receptor one (CB1) binds ?9-tetrahydrocannabinol, the psychoactive component of Cannabis sativa L., and other cannabimimetic compounds. It is a G-protein coupled receptor (GPCR) that is associated with the central nervous system and exerts its effects primarily via coupling to Gi/Go proteins. The pharmacological effects of CB1 agonists include analgesia, inhibition of nausea, appetite stimulation, antiemetic activity and bronchial dilation while inverse agonists attenuate excessive eating disorders. Any therapeutic strategy targeting CB1 will require that we have a firm understanding of the structural features of the receptor that impact key points in its life cycle: ER integration and cell surface expression, receptor activation, and desensitization and internalization. In Aim 1, we will examine the basis for the weak ER translocation of the CB1 amino terminus (N-tail). The structure of the N-terminus will be determined and accessory proteins and their recognition motifs in the N- and C-terminus will be identified. The role of these motifs in cellular localization including sematodendritic and axonal membrane surface localization in neurons will be examined. In Aim 2, we will build on our identification of structural elements of the receptor critical for distinguishing agonist and inverse agonist interactions and fully map the key contact points in the TM domain and the extracellular region of CB1 that are involved. We will define residues critical for poising the ligand- independent equilibrium of CB1 atypically toward activation and those responsible for the interconversion of this intermediate receptor state to the resting and activated forms of the receptor. In Aim 3, we will take advantage of novel receptor mutants that model different structural states of the receptor to examine linkages between receptor activation, desensitization, and cellular localization. Mutants that model the inactive and active forms of CB1 will provide tools for analyzing the consequences of prolonged treatment with inverse agonists and agonists. We will utilize our expertise in developing structural analyses and binding assays with purified components to examine the molecular basis of these processes with emphasis on the carboxyl terminus (C-tail) of the receptor. In the course of this work we will identify determinants that enhance the cell surface expression of CB1 and strategies for the large-scale preparation of domains of the receptor, and their structural analysis, which could be applied to structural studies of other GPCRs and membrane proteins in general. RELEVANCE: The cannabinoid receptor one (CB1) is a G-protein coupled receptor that is associated with the central nervous system. Research activities that have focused on the role of CB1 in signal transduction have underscored its enormous potential as a target for therapeutic agents. The goal of this project is to understand the structural features that influence key stages in the life cycle of CB1 including cell surface expression, receptor activation, and internalization and ultimately impact its cell surface exposure so that it will be accessible for therapeutic strategies.