REGULATION OF CELLULAR RESPONSES TO NEUROPEPTIDES

细胞对神经肽反应的调节

基本信息

批准号：
8004317
负责人：
NIGEL W BUNNETT
金额：
$ 10万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-01 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8004317
关键词：
Abbreviations Acids Adrenergic Receptor Agonist Animals Arrestins Binding California Cell Line Cell membrane Cells Cities Clathrin Coupled Cyclic GMP-Dependent Protein Kinases Cytoskeleton Cytosol Defect Disease Doctor of Medicine Doctor of Philosophy Dominant-Negative Mutation Drug usage Dynamin Endocytosis Endosomes Endothelial Cells Ensure Enteral Extracellular Signal Regulated Kinases Face G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Green Fluorescent Proteins Heterotrimeric GTP-Binding Proteins Human Resources Inflammation Inflammatory Response Instruction Intestinal Motility Knock-out Knockout Mice Los Angeles Mediating Mental Depression Mitogen-Activated Protein Kinases Molecular Motor Mus Names Neprilysin Neurogenic Inflammation Neurons Neuropeptides PAR-2 Receptor Pain Pathway interactions Performance Peroxidases Pharmaceutical Preparations Phosphoric Monoester Hydrolases Phosphotransferases Physiological Postdoctoral Fellow Principal Investigator Printing Process Protein Kinase C Reagent Recruitment Activity Recycling Regulation Research Personnel Research Project Grants Resistance Role San Francisco Scaffolding Protein Second Messenger Systems Signal Transduction Signaling Molecule Sorting - Cell Movement Specificity Substance P Substance P Receptor Testing Transgenic Mice Transport Vesicles Universities Variant Vesicle cell growth regulation cell motility citrate carrier desensitization expectation human disease mutant programs protein kinase C kinase rab GTP-Binding Proteins receptor research study response scaffold second messenger trafficking

项目摘要

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.

该提案研究了一个假设，即激动剂诱导的G蛋白偶联受体（GPCR）的运输是信号转导和终止所必需的，并且缺陷导致不受控制的刺激和疾病。 P（SP）的分子机制和生理作用 - 将在细胞和整个动物的水平上检查神经蛋白1受体（NKIR）的运输。鉴于该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信号传导的机制很重要。 NK1R调节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进行研究。 AIM 1将定义NK1R的SP刺激内吞作用和细胞内运输的分子机制。网格蛋白衔接子P-甲蛋白以及Dynamin和rab GTPases在NK1R运输中的作用将通过表达显性阴性突变体的表达以及研究P-抑制蛋白基因敲除小鼠的神经元进行检查。细胞骨架和内体酸化和磷酸酶的作用将使用特定药物AIM 2检查将定义NK1R运输对启动信号转导的重要性。 p- arrestin，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中开发的试剂确定。这些研究将定义P- arrectins作为募集和组织MAPK级联组件的分子支架的作用。 AIM 3将定义的重要性 NK1R运输在信号传导的脱敏和敏化方面。 G蛋白受体激酶（GRK）和P- arre蛋白在脱敏中的作用将取决于显性阴性突变体的表达和研究敲除小鼠的神经元的作用。将使用AIM 1中的试剂检查RAB，细胞骨架和内体分类对敏化的重要性。AIM4将确定NK1R脱敏机制缺陷是否导致SP信号传导和疾病的延长。将在缺乏GRK或P-arrestin的小鼠中检查SP信号，或表达脱敏和内在化缺陷突变NK1R（NK1R8325，一种天然存在的NK1R变体）。将检查SP对肠运动性和神经源性炎症的影响，期望脱敏的缺陷会导致运动和炎症反应夸大。这些实验的结果一起将提供有关GPCR信号传导的新信息，并将定义信号转导的缺陷如何导致疾病。