Endosomal Platforms for Neuropeptide Receptor Signaling

项目总结 这项建议研究了G蛋白偶联受体(GPCRs)发出疼痛信号的机制。慢性 疼痛是疾病的标志，是治疗的副作用，也是痛苦的主要原因。尽管GPCRs 介导伤害性感觉的所有方面，是主要的治疗靶点，GPCRs信号转导的机制 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经常失败 无法解释的原因。该提案对导致这种缺乏理解的三种教条提出了挑战：1. GPCRs仅从细胞表面发出信号。2.内体只是gpcr循环利用的管道。 退化。3.细胞表面GPCRs是最佳的治疗靶点。该提案假设：1. 内体GPCRs产生持续的信号，介导脊髓神经元的持续兴奋和 伤害性感受。2.靶向内体而不是细胞表面的GPCRs是理想的治疗策略，并且 传统拮抗剂的临床失败与它们不能抑制内体受体有关。实验 将重点放在P物质和降钙素基因相关的多肽受体，它们介导中枢性疼痛 传递，并在痛苦的刺激后内化。受体内吞作用在伤害性感受中的作用 将使用药理学和遗传学方法来破坏笼蛋白、动力素和β-arrestin， 并通过研究表达非内化受体的转基因小鼠。脂质偶联和纳米颗粒- 胶囊化将被用于将拮抗剂输送到内体GPCRs。目标1将确定以下方面的贡献 清醒小鼠对躯体和结肠伤害性感受的内吞作用。目标2将定义以下内容的重要性 用于脊髓神经元兴奋的内吞作用，将在完整的组织中使用电生理学进行分析。 目标3将确定亚细胞隔室产生信号所需的内吞作用 这是神经元兴奋和伤害性感受的基础，这将在分离的神经元中使用生物物理学进行研究， 成像和蛋白质组学方法。研究结果将提供有关疼痛信号的基本信息 和心理治疗。由于GPCRs是最大的一类信号蛋白，并且有一半是 在治疗药物方面，结果将是广泛意义上的。

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.