REGULATION OF CELLULAR RESPONSES TO NEUROPEPTIDES

这一建议检验了一种假设，即激动剂诱导的G蛋白偶联受体(GPCRs)的运输是信号转导的启动和终止所必需的，并且缺陷会导致 不受控制的刺激和疾病。P物质(SP)诱导的神经激肽1受体(NKIR)转运的分子机制和生理作用将在细胞和整个动物水平上进行研究。鉴于NK1R受体在神经源性炎症、疼痛和肠道运动中的病理生理作用，了解NK1R信号转导机制是非常重要的。NK1R的调控将在短期培养的转基因细胞系、内皮细胞和肠道神经元中进行研究，并在基因敲除和转基因小鼠中进行研究。目的1明确SP刺激NK1R的内吞作用和细胞内转运的分子机制。将通过显性负性突变体的表达以及通过研究p-arrestin基因敲除小鼠的神经元来检验笼状蛋白适配器p-arrestin以及Dynamin和Rab GTP酶在NK1R转运中的作用。将使用特定药物研究细胞骨架、内体酸化和磷酸酶的作用，目的2将确定NK1R运输对于启动信号转导的重要性。P-arrestins、RABS和细胞骨架在NKlR介导的MAP激酶激活中的作用将使用Aim 1中开发的试剂来确定。这些研究将确定p-arrestins作为分子支架的作用，招募和组织MAPK级联的组件。目标3将定义以下内容的重要性 NK1R交易在信号的脱敏和再增敏中的作用。G蛋白受体激酶(GRKs)和p-arrestins在脱敏中的作用将通过显性负性突变体的表达和对基因敲除小鼠神经元的研究来确定。RABS、细胞骨架和内体分选对再增敏的重要性将使用AIM 1的试剂进行检查。AIM 4将确定NK1R脱敏机制的缺陷是否导致SP信号转导延长和疾病。SP信号将在缺乏GRK或p-arrestins或表达脱敏和内化缺陷的小鼠中进行检测 突变型NK1R(NK1R8325，自然产生的NK1R变异体)。将研究SP对肠道运动和神经源性炎症的影响，期望脱敏缺陷将导致夸大的运动和炎症反应。总之，这些实验的结果将提供有关GPCR信号的新信息，并将定义信号转导中的缺陷如何导致疾病。

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.