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，以及其他类似大麻的化合物。它是一种与中枢神经系统相关的g蛋白偶联受体（GPCR），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、止吐活性和支气管扩张，而逆激动剂可减轻过度饮食失调。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结构特征有一个坚定的理解：内质网整合和细胞表面表达、受体激活、脱敏和内化。在目的1中，我们将研究CB1氨基端（n -尾）弱ER易位的基础。将确定N端结构，并确定N端和c端中的辅助蛋白及其识别基序。这些基序在细胞定位中的作用，包括神经元的半树突和轴突膜表面定位。在目标2中，我们将建立在我们对受体结构元件的鉴定基础上，这些结构元件对区分激动剂和反激动剂相互作用至关重要，并全面绘制CB1的TM结构域和涉及的细胞外区域的关键接触点。我们将定义对CB1不依赖配体的非典型激活平衡至关重要的残基，以及那些负责将这种中间受体状态相互转化为受体的静止和激活形式的残基。在Aim 3中，我们将利用新型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为分析长期使用逆激动剂和激动剂治疗的后果提供工具。我们将利用我们在开发结构分析和纯化组分结合分析方面的专业知识来研究这些过程的分子基础，重点研究受体的羧基端（c尾）。在这项工作的过程中，我们将确定增强CB1细胞表面表达的决定因素和受体结构域的大规模制备策略，以及它们的结构分析，这可以应用于其他gpcr和膜蛋白的结构研究。相关性：大麻素受体1 （CB1）是一种与中枢神经系统相关的g蛋白偶联受体。关注CB1在信号转导中的作用的研究活动强调了其作为治疗剂靶点的巨大潜力。该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.