Determinants of the Cannabinoid Receptor Life Cycle

描述（由申请人提供）：人大麻素受体1（CB 1）结合？9-四氢大麻酚，大麻的精神活性成分，和其他大麻模拟化合物。它是一种G蛋白偶联受体（GPCR），与中枢神经系统相关，主要通过与Gi/Go蛋白偶联发挥作用。CB 1激动剂的药理学作用包括镇痛、抑制恶心、刺激食欲、止吐活性和支气管扩张，而反向激动剂减轻过度进食障碍。任何靶向CB 1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明确的理解：ER整合和细胞表面表达，受体活化，脱敏和内化。在目标1中，我们将研究CB 1氨基末端（N-尾）的弱ER易位的基础。将确定N-末端的结构，并鉴定N-和C-末端的辅助蛋白及其识别基序。这些图案在细胞定位，包括sematodendritic和轴突膜表面定位在神经元中的作用将被检查。在目标2中，我们将建立在我们识别的受体的结构元件的关键区分激动剂和反向激动剂的相互作用和充分映射的关键接触点TM域和细胞外区域的CB 1参与。我们将定义对平衡CB 1的配体非依赖性平衡至关重要的残基，以及负责该中间受体状态相互转化为受体的静息和活化形式的残基。在目标3中，我们将利用新型受体突变体，模拟不同的受体结构状态，以研究受体激活，脱敏和细胞定位之间的联系。模拟CB 1的非活性和活性形式的突变体将提供用于分析用反向激动剂和激动剂长期治疗的后果的工具。我们将利用我们在开发结构分析和纯化成分结合试验方面的专业知识，以研究这些过程的分子基础，重点是受体的羧基末端（C-尾）。在这项工作的过程中，我们将确定的决定因素，提高细胞表面表达的CB 1和战略的大规模制备域的受体，和它们的结构分析，这可以应用于其他GPCR和膜蛋白的结构研究一般。大麻素受体1（CB 1）是一种与中枢神经系统相关的G蛋白偶联受体。集中于CB 1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CB 1生命周期关键阶段的结构特征，包括细胞表面表达，受体活化和内化，并最终影响其细胞表面暴露，以便其可用于治疗策略。

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.