REGULATION OF CELLULAR RESPONSES TO NEUROPEPTIDES

该提议检验了激动剂诱导的G蛋白偶联受体（GPCR）的运输是信号转导的起始和终止所需的假设，并且缺陷导致GPCR的缺失。 不受控制的刺激和疾病。本研究将在细胞和动物整体水平上探讨P物质（SP）诱导神经激肽1受体（NKIR）转运的分子机制和生理作用。鉴于NK 1 R受体在神经源性炎症、疼痛和肠动力中的病理生理作用，了解NK 1 R信号传导机制是重要的。将在短期培养的转染细胞系、内皮细胞和肠神经元以及敲除和转基因小鼠中研究NK 1 R调控。目的1阐明SP刺激NK 1 R内吞和胞内转运的分子机制。网格蛋白衔接子p-抑制蛋白以及发动蛋白和Rab GTP酶在NK 1 R运输中的作用将通过显性失活突变体的表达和通过研究来自p-抑制蛋白敲除小鼠的神经元来检查。将使用特定药物检查细胞骨架和内体酸化和磷酸酶的作用。目的2将定义NK 1 R运输对于信号转导起始的重要性。β-抑制蛋白、Rabs和细胞骨架在NKlR介导的MAP激酶活化中的作用将使用在目标1中开发的试剂来确定。这些研究将确定β-arrestins作为招募和组织MAPK级联反应组分的分子支架的作用。目标3将确定以下方面的重要性： NK 1 R在信号转导的脱敏和再敏化中的运输。G蛋白受体激酶（GRKs）和β-抑制蛋白在脱敏中的作用将通过显性失活突变体的表达和研究基因敲除小鼠的神经元来确定。将使用目标1的试剂检查Rabs、细胞骨架和内体分选对于再致敏的重要性。目的4将确定NK 1 R脱敏机制的缺陷是否导致SP信号传导延长和疾病。将在缺乏GRKs或β-抑制蛋白或表达脱敏和内化缺陷的GRKs或β-抑制蛋白的小鼠中检查SP信号传导。 突变型NK 1 R（NK 1 R8325，天然存在的NK 1 R变体）。将检查SP对肠运动和神经源性炎症的影响，预期脱敏缺陷将导致过度的运动和炎症反应。总之，这些实验的结果将提供有关GPCR信号转导的新信息，并将定义信号转导缺陷如何导致疾病。

This proposal examines the hypothesis that agonist-induced trafficking of G-protein coupled receptors (GPCRs) is required for the initiation and termination of signal transduction, and that defects result in uncontrolled stimulation and disease. The molecular mechanisms and physiological roles of substance P (SP)- induced trafficking of the neurokinin 1 receptor (NKIR) will be examined at the level of the cell and the whole animal. It is important to understand the mechanisms of NK1R signaling in view of the pathophysiological roles of this receptor in neurogenic inflammation, pain and intestinal motility. NK1R regulation will be studied in transfected cell lines, endothelial cells and enteric neurons in short-term culture, and in knockout and transgenic mice. Aim 1 will define the molecular mechanism of SP-stimulated endocytosis and intracellular trafficking of the NK1R. The role of the clathrin adapter p-arrestin, and of dynamin and Rab GTPases in NK1R trafficking will be examined by expression of dominant negative mutants, and by studying neurons from p- arrestin knockout mice. The role of the cytoskeleton and of endosomal acidification and phosphatases will be examined using specific drugs Aim 2 will define the importance of NK1R trafficking for the initiation of signal transduction. The role of p-arrestins, Rabs and the cytoskeleton in NKlR-mediated MAP kinase activation will be determined using reagents developed in Aim 1. These studies will define the role of p-arrestins as molecular scaffolds that recruit and organize components of the MAPK cascade. Aim 3 will define the importance of NK1R trafficking in desensitization and resensitization of signaling. The role of G-protein receptor kinases (GRKs) and p-arrestins in desensitization will be determined by expression of dominant negative mutants and by studying neurons from knockout mice. The importance of Rabs, the cytoskeleton and endosomal sorting for resensitization will be examined using reagents from Aim 1. Aim 4 will determine whether defects in mechanisms of NK1R desensilization result in prolonged SP signaling and disease. SP signaling will be examined in mice deficient in GRKs or p-arrestins, or expressing a desensitization and internalization-defective mutant NK1R (NK1R8325, a naturally occurring NK1R variant). The effects of SP on intestinal motility and neurogenic inflammation will be examined, with the expectation that defects in desensitization will result in exaggerated motor and inflammatory responses. Together, the results of these experiments will provide new information about GPCR signaling, and will define how defects in signal transduction can cause disease.