G PROTEINS AND OPIOID RECEPTOR FUNCTIONS

G 蛋白和阿片受体功能

基本信息

批准号：
7228936
负责人：
PING-YEE LAW
金额：
$ 29.56万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-07-01 至 2009-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7228936
关键词：
5&apos Flanking Region Address Aden Adenovirus Vector Adenoviruses Adenylate Cyclase Affect Agonist Amino Acid Sequence Arrestin Arrestins Brain Cell model Cells Chimera organism Chronic Complex Cultured Cells Cyclic AMP Data Development Dimerization Dominant-Negative Mutation Elements Funding G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Gene Silencing Genes Goals Hippocampus (Brain)Homeostasis In Vitro Individual Laboratories LacZ Genes Laws Libraries Mass Spectrum Analysis Mediating Mus Neuroblastoma Neurons Opioid Opioid Receptor Peptide Sequence Determination Pertussis Toxin Phospholipase Plant Resins Potassium Channel Production Progress Reports Property Protein Subunits Proteins Proteomics Reagent Receptor Signaling Recruitment Activity Regulation Reporter Role Ryanodine Scaffolding Protein Screening procedure Signal Transduction Small Interfering RNA Specificity Structure System Tacrolimus Binding Protein 1A Tacrolimus Binding Proteins Tail Technology Testing Transgenic Mice Virus Wheat Germ Agglutinins Yeasts arrestin B base beta-arrestin design dimer in vivo interest mu opioid receptors mutant nano-electrospray promoter protein activation protein aminoacid sequence protein expression receptor receptor expression receptor function research study restoration success tandem mass spectrometry three dimensional structure trafficking vector voltage yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Opioid receptor regulates multiple effector systems in same cells with similar but distinct properties. Our previous findings indicate that the opioid agonist activates the Gi/Go proteins mediating the receptor functions with similar potencies. Hence, we hypothesize that integration of the opioid receptor signals must occur and this is accomplished by the formation of protein scaffolds or receptor signaling units. By recruiting different proteins to the receptor vicinity, e.g., beta-arrestin, Src, RGS, AGS etc., the magnitude and duration of the signals between effector systems could be modulated. During the past funding period, we have initiated studies to demonstrate our hypotheses. We examined the receptor domains involved in the G protein activation by mutational analyses and receptor chimera studies. Using adenoviruses to deliver the PTX-insensitive G-alpha subunits, we demonstrated the Get specificity in mediating opioid receptor functions. We have established the existing of signaling units by demonstrating the existence of heterodimeric receptor-G protein complexes and in identifying cellular proteins, e.g., the FK506 binding protein FKBP12 that interacted specifically with the carboxyl tail domains of MOR (mu-opioid receptor). The alteration in FKBP12 level could affect MOR regulation of intracellular Ca2+ homeostasis. Hence, in the current proposal, we will continue these on-going projects in order to determine and characterize the components of opioid receptor signaling units. We will use both the yeast two-hybrid and proteomic approaches to identify proteins that require multiple receptor domains for interaction. The receptor mutants that are defective in either signaling or trafficking will be used to characterize the roles of these proteins in opioid receptor functions. Over-expression of these proteins with adenoviruses, or the knockdown of these proteins levels in neuroblastoma N2A cells by siRNA approach will be carried out to determine their effects on two of the effectors regulated by opioid receptor, i.e., adenylyl cyclase and intracellular Ca2+ homeostasis. The alteration of these proteins levels in primary hippocampal cell cultures enriched in neurons expressing endogenous MOR will be carried out also. An inducible vector based siRNA silencing of these genes will be developed and used in our studies. These experiments are designed to address the mechanism in the neuronal integration of opioid receptor signals. Understanding of the mechanism will allow the eventual elucidation of the in vivo regulation of opioid receptor signaling during chronic agonist administration.

描述（由申请人提供）：阿片类受体调节具有相似但不同特性的相同细胞中的多个效应系统。我们以前的发现表明，阿片类动力学家激活了具有相似效力的受体功能的GI/GO蛋白。因此，我们假设必须发生阿片受体信号的整合，这是通过形成蛋白质支架或受体信号单元来完成的。通过募集不同的蛋白质与受体附近，例如Beta-arrestin，SRC，RGS，AGS等，可以调节效应器系统之间信号的幅度和持续时间。在过去的资金期间，我们开始了研究以证明我们的假设。我们通过突变分析和受体嵌合体研究检查了参与G蛋白激活的受体结构域。使用腺病毒传递PTX不敏感的G-α亚基，我们证明了介导阿片类受体功能的特异性。我们通过证明存在异二聚体受体-G蛋白复合物的存在以及鉴定细胞蛋白（例如，例如FK506结合蛋白FKBP12）来确定现有的信号传导单元的现有。 FKBP12水平的改变可能会影响细胞内Ca2+稳态的MOR调节。因此，在当前的提案中，我们将继续进行这些持续的项目，以确定和表征阿片受体信号单元的组成部分。我们将使用酵母两杂交和蛋白质组学方法来识别需要多个受体结构域进行相互作用的蛋白质。在信号传导或运输中有缺陷的受体突变体将用于表征这些蛋白在阿片类受体功能中的作用。将通过siRNA方法对这些蛋白质的过表达，或通过siRNA方法敲低神经母细胞瘤N2A细胞中的这些蛋白质水平，以确定它们对阿片受体调节的两个效应子的影响，即腺苷酸环酶和细胞内CA2+稳态。这些蛋白质水平在富含表达内源性MOR的神经元的原发性海马细胞培养物中的改变也将进行。这些基因的基于诱导载体的siRNA沉默将在我们的研究中开发和使用。这些实验旨在解决阿片受体信号神经元整合的机制。对机制的理解将允许在慢性激动剂给药期间最终阐明阿片受体信号传导的体内调节。