Endosomal Platforms for Neuropeptide Receptor Signaling

神经肽受体信号转导的内体平台

• 批准号: 10093292
• 负责人: NIGEL W BUNNETT
• 金额: $ 28.3万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10093292
• 关键词: Endosomal; Platforms; Neuropeptide; Receptor; Signaling

PROJECT SUMMARY This proposal examines the mechanisms by which G protein-coupled receptors (GPCRs) signal pain. Chronic pain is a hallmark of disease, a side effect of therapy, and a major cause of suffering. Although GPCRs mediate all aspects of nociception and are major therapeutic targets, the mechanisms by which GPCRs signal sustained pain are poorly understood, and clinical trials of GPCR antagonists in chronic pain often fail for unexplained reasons. The proposal challenges three dogmas that contribute to this lack of understanding: 1. GPCRs signal only from the cell-surface. 2. Endosomes are merely a conduit for GPCR recycling or degradation. 3. Cell-surface GPCRs are the optimal therapeutic target. The proposal hypothesizes that: 1. Endosomal GPCRs generate sustained signals that mediate persistent excitation of spinal neurons and nociception. 2. Targeting endosomal rather than cell-surface GPCRs is the ideal therapeutic strategy, and the clinical failure of conventional antagonists relates to their inability to inhibit endosomal receptors. Experiments will focus on substance P and calcitonin gene-related peptide receptors, which mediate central pain transmission and are internalized after painful stimuli. The contribution of receptor endocytosis to nociception will be evaluated using pharmacological and genetic approaches to disrupt clathrin, dynamin and β-arrestin, and by studying transgenic mice expressing non-internalizing receptors. Lipid-conjugation and nanoparticle- encapsulation will be used to deliver antagonists to endosomal GPCRs. Aim 1 will determine the contribution of endocytosis to somatic and colonic nociception in conscious mice. Aim 2 will define the importance of endocytosis for excitation of spinal neurons, which will be analyzed in intact tissues using electrophysiology. Aim 3 will determine the requirement of endocytosis for the generation of signals in subcellular compartments that underlie neuronal excitation and nociception, which will be studied in isolated neurons using biophysical, imaging and proteomic approaches. The results will provide fundamental information about pain signaling and therapy. Since GPCRs are the largest class of signaling proteins and the target of one half of therapeutic drugs, the outcomes will be broadly significant.

项目概要 该提案研究了 G 蛋白偶联受体 (GPCR) 发出疼痛信号的机制。慢性的 疼痛是疾病的标志，是治疗的副作用，也是造成痛苦的主要原因。虽然 GPCR 介导伤害感受的各个方面，是主要的治疗靶点，即 GPCR 发出信号的机制 人们对持续性疼痛知之甚少，GPCR 拮抗剂治疗慢性疼痛的临床试验常常因以下原因而失败： 无法解释的原因。该提案挑战了导致这种缺乏理解的三个教条：1. GPCR 仅从细胞表面发出信号。 2. 内体只是 GPCR 回收的管道或 降解。 3. 细胞表面GPCR是最佳治疗靶点。该提案假设：1. 内体 GPCR 产生持续的信号，介导脊髓神经元的持续兴奋和 伤害感受。 2. 靶向内体而非细胞表面 GPCR 是理想的治疗策略，并且 常规拮抗剂的临床失败与其不能抑制内体受体有关。实验 将重点关注介导中枢疼痛的 P 物质和降钙素基因相关肽受体 传递并在痛苦刺激后内化。受体内吞作用对伤害感受的贡献 将使用药理学和遗传学方法来破坏网格蛋白、动力和β-抑制蛋白进行评估， 并通过研究表达非内化受体的转基因小鼠。脂质缀合和纳米颗粒 封装将用于将拮抗剂递送至内体 GPCR。目标 1 将确定的贡献 清醒小鼠的躯体和结肠伤害感受的内吞作用。目标 2 将定义以下内容的重要性： 用于兴奋脊髓神经元的内吞作用，将使用电生理学在完整组织中进行分析。 目标 3 将确定内吞作用对亚细胞区室中信号生成的要求 这是神经元兴奋和伤害感受的基础，将使用生物物理学在孤立的神经元中进行研究， 成像和蛋白质组学方法。结果将提供有关疼痛信号传导的基本信息 和治疗。由于 GPCR 是最大一类信号蛋白，并且是一半的信号蛋白的靶标 治疗药物，其结果将具有广泛意义。