Endosomal Platforms for Neuropeptide Receptor Signaling

项目摘要 该提案研究了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。虽然GPCR 介导伤害感受的所有方面，并且是主要的治疗靶点，GPCR信号传导的机制 对持续性疼痛的了解很少，GPCR拮抗剂在慢性疼痛中的临床试验经常失败， 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信号仅来自细胞表面。2.内体仅仅是GPCR再循环的管道， 降解3.细胞表面GPCR是最佳的治疗靶点。该提案假设：1。 内体GPCR产生介导脊髓神经元持续兴奋的持续信号， 伤害感受2.靶向核内体而不是细胞表面GPCR是理想的治疗策略， 常规拮抗剂的临床失败与它们不能抑制内体受体有关。实验 将集中在P物质和降钙素基因相关肽受体，介导中枢疼痛 在疼痛刺激后被内化。受体内吞在伤害感受中的作用 将使用药理学和遗传学方法进行评估，以破坏网格蛋白、发动蛋白和β-抑制蛋白， 以及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 包封将用于将拮抗剂递送至内体GPCR。目标1将决定 内吞作用对清醒小鼠躯体和结肠伤害性感受的影响。目标2将确定以下方面的重要性： 内吞作用用于脊髓神经元的兴奋，这将使用电生理学在完整组织中进行分析。 目的3将确定内吞作用对亚细胞区室信号产生的要求 这是神经元兴奋和伤害感受的基础，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。这些结果将提供有关疼痛信号的基本信息 和心理治疗由于GPCR是最大的一类信号蛋白，并且是蛋白质的一半的靶点， 治疗药物，结果将是广泛的显着。

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.