Beta-Arrestin Signaling from the Cannabinoid 2 and mu Opioid Receptors

来自大麻素 2 和 mu 阿片受体的 Beta-Arrestin 信号传导

• 批准号: 9176213
• 负责人: DEBRA A KENDALL
• 金额: $ 20.72万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9176213
• 关键词: Adverse effects; Affect; Antibodies; Area; Bar Codes; Biochemical; Biological; Biological Assay; CNR1 gene; CNR2 gene; Cannabinoids; Cell membrane; Cells; Clathrin; Color; Disease; Drug Addiction; Drug abuse; Endocytosis; Event; Fluorescence; Fluorescence Microscopy; Future; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Health; Heterotrimeric GTP-Binding Proteins; Human; Imaging technology; Individual; Kinetics; Life; Ligand Binding; Ligands; Link; Mediating; Microscopy; Modeling; Molecular; Neurons; Opioid; Opioid Receptor; Outcome; Pain; Pathway interactions; Pattern; Phosphorylation; Phosphotransferases; Physiological; Plants; Post-Translational Protein Processing; Process; Receptor Activation; Receptor Signaling; Regulation; Role; Signal Transduction; Staging; Stimulus; System; Technology; Testing; Therapeutic; Therapeutic Agents; Time; Transducers; Work; base; beta-arrestin; cell type; clinically relevant; coated pit; design; drug discovery; genetic manipulation; live cell imaging; mu opioid receptors; novel therapeutics; preference; protein activation; psychostimulant; receptor; receptor binding; receptor function; receptor-mediated signaling; response; therapeutic target; trafficking

PROJECT SUMMARY The classic model of G protein coupled receptor (GPCR) activation centers on ligand binding, G protein activation, and signal transduction via G protein-mediated signaling events. This paradigm has been called into question however, with the finding that some ligands –including endogenous ligands and therapeutic agents– have a preference for beta-arrestin mediated pathways. However, the information flow from receptor activation to signaling cascades and the mechanism of beta-arrestin signaling are not well understood. This proposal will elucidate, at the molecular level, the fundamental mechanisms controlling ligand-specific induction of beta- arrestin signaling with two highly clinically relevant GPCRs, the cannabinoid 2 receptor (CB2R) and the mu opioid receptor (MOR). These human receptors bind the plant-derived cannabinoids and opioids leading to psychostimulant effects and reduction of pain. Precise control of receptor activation and signaling is critical to obtain only the desired therapeutic results, however, and not the undesired side effects such as tolerance, drug abuse and dependence. Substantial preliminary studies identified ligand-specific dwell times, i.e. the time receptors are clustered into clathrin coated pits with beta-arrestins before endocytosis, as a mechanism controlling beta-arrestin signaling. This trafficking event can be chemically and genetically modulated to selectively control beta-arrestin signaling, providing novel therapeutic strategies. This project will combine state-of-the-art live cell imaging technologies (total internal reflection fluorescence and spinning disk microscopies), and biochemical approaches to determine if ligand-specific dwell times are a general event controlling beta-arrestin signaling.  Multiple ligands for these receptors will be investigated in heterologous systems and in cells endogenously expressing the receptors. Preliminary results in primary cultures strongly support our hypothesis that long dwell times correlate with beta-arrestin signaling. The aims are to: (1) examine endocytosis of the CBR2 and MOR at the single endocytic pit level, and (2) define the impact on cellular mechanisms of CB2R and MOR mediated beta-arrestin signaling, including whether endocytic dwell times can modulate these pathways. Results will provide a physiological role for the previously described variability in endocytic dwell times. These findings may be extended to future drug discovery efforts, including for other GPCRs, to rationally design therapeutic agents with specific outcomes in areas intractable via current technology.

项目总结 G蛋白偶联受体(GPCR)激活的经典模型以配体结合为中心，即G蛋白 激活，并通过G蛋白介导的信号事件进行信号转导。这一范式已经被称为 然而，问题是发现一些配体-包括内源性配体和治疗剂- 偏爱β-arrestin介导的途径。然而，来自受体激活的信息流 对信号级联反应和β-arrestin信号转导机制还不是很清楚。这项提议将 在分子水平上阐明控制配体特异性诱导β-内酰胺酶的基本机制。 Arrestin与两个临床上高度相关的GPCRs，即大麻素2受体(CB2R)和MU 阿片受体(MOR)。这些人类受体与植物来源的大麻素和阿片类药物结合，导致 心理刺激作用和减轻疼痛。精确控制受体激活和信号转导对于 然而，只获得预期的治疗结果，而不是不良的副作用，如耐受性、药物 虐待和依赖。大量的初步研究确定了特定于配体的停留时间，即 作为一种机制，受体在内吞作用之前聚集在带有β-阻滞素的笼状蛋白包被的凹坑中。 控制β-arrestin信号。这一人口贩运事件可以通过化学和基因调节来 选择性地控制β-arrestin信号，提供新的治疗策略。这个项目将结合 最先进的活细胞成像技术(全内反射荧光和旋转磁盘 显微镜)，以及确定特定配基的停留时间是否是一般事件的生化方法 控制β-arrestin信号。这些受体的多个配体将在异源 系统和细胞内源性地表达受体。初步结果在原代培养中表现强劲 支持我们的假设，即长驻留时间与β-arrestin信号相关。目标是：(1) 检查CBR2和MOR在单个内吞小窝水平的内吞作用，以及(2)确定对 CB2R和MOR介导的β-arrestin信号的细胞机制，包括胞内是否滞留 时间可以调节这些途径。结果将为前面描述的 胞内滞留时间的可变性。这些发现可能会扩展到未来的药物发现努力，包括 对于其他GPCR，合理设计治疗药物，在目前难以治愈的领域具有特定的疗效 技术