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.

项目摘要 G蛋白偶联受体（GPCR）活化的经典模型集中于配体结合，G蛋白 活化和通过G蛋白介导的信号传导事件的信号转导。这种模式被称为 然而，随着一些配体-包括内源性配体和治疗剂- 对β-抑制蛋白介导的通路有偏好。然而，从受体激活的信息流 信号级联和β-抑制蛋白信号传导的机制还没有很好的理解。这项建议会 阐明，在分子水平上，控制配体特异性诱导β- 抑制蛋白信号传导与两个高度临床相关的GPCR，大麻素2受体（CB 2 R）和mu 阿片受体（莫尔）。这些人类受体结合植物来源的大麻素和阿片类物质， 精神刺激作用和减轻疼痛。受体激活和信号传导的精确控制对于 然而，仅获得期望的治疗结果，而不是不期望的副作用，例如耐受性、药物依赖性、药物耐受性和药物耐受性。 滥用和依赖。大量的初步研究确定了配体特异性停留时间，即 作为一种机制，在胞吞作用之前，受体被β-抑制蛋白聚集到网格蛋白包被的小凹中， 控制β-抑制蛋白信号传导。这种贩运事件可以通过化学和遗传调节， 选择性控制β-抑制蛋白信号传导，提供新的治疗策略。这个项目将联合收割机 最先进的活细胞成像技术（全内反射荧光和旋转盘 显微镜）和生物化学方法来确定配体特异性停留时间是否是一般事件 控制β-抑制蛋白信号传导。这些受体的多个配体将在异源 系统和内源性表达受体的细胞中。在原代培养物中的初步结果强烈 支持我们的假设，即长停留时间与β-抑制蛋白信号相关。其目的是：（1） 在单个内吞小窝水平检查CBR 2和莫尔的内吞作用，以及（2）确定对 CB 2 R和莫尔介导的β-抑制蛋白信号传导的细胞机制，包括内吞停留是否 时间可以调节这些途径。结果将为先前描述的生物学行为提供生理作用。 内吞停留时间的可变性。这些发现可能会扩展到未来的药物发现工作，包括 对于其他GPCR，合理设计治疗药物，在目前难以治疗的领域具有特定的结果， 技术.