Identification and Characterization of mu Opioid Receptor Interacting Proteins

mu 阿片受体相互作用蛋白的鉴定和表征

• 批准号: 7595559
• 负责人: ROBERT LEVENSON
• 金额: $ 30.62万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7595559
• 关键词: Address; Adenylate Cyclase; Affect; Analgesics; Antibodies; Biochemical; Brain; Buprenorphine; Cell secretion; Cells; Complex; Coupling; Dependence; Disruption; Dominant-Negative Mutation; Drosophila genus; Drug abuse; Etiology; Etorphine; Future; G-Protein-Coupled Receptors; Generations; Goals; Heroin; Human; Instruction; Linkage Disequilibrium; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Morphine; Mus; Numbers; Opiate Addiction; Opiates; Opioid; Opioid Receptor; Orthologous Gene; Peptides; Pharmaceutical Preparations; Play; Property; Protein Binding Domain; Protein Kinase C; Proteins; Proteomics; Public Health; Receptor Cell; Receptor Mediated Signal Transduction; Receptor Signaling; Regulation; Rodent; Role; Series; Signal Transduction; Signal Transduction Pathway; Site; Small Interfering RNA; Synaptophysin; System; Techniques; Testing; Transgenic Mice; Ubiquitin; Validation; Withdrawal; Yeasts; cDNA Library; clinically relevant; cohort; desensitization; design; drug of abuse; filamin; human RIPK1 protein; inclusion criteria; insight; knock-down; mu opioid receptors; novel; novel therapeutics; protein function; protein protein interaction; receptor; receptor expression; research study; response; therapeutic target; trafficking; yeast two hybrid system

The mu-opioid receptor (MOR) mediates most of the actions of morphine and other clinically relevant analgesics as well as drugs of abuse such as heroin. The proteomic and functional studies proposed in this application are designed to elucidate the protein components of MOR signaling complexes. Identification of MOR interacting proteins will allow us to test our hypothesis that protein-protein interactions play an important role in regulating the MOR signal transduction pathway. In Aim I, we will identify and characterize MOR interacting proteins. A split-ubiquitin screen we have performed identified several novel MORIPs including GPR177, the mammalian ortholog of Drosophila Wntless. To identify additional MORIPs, each of the intracellular domains of the human MOR will be used as bait to separately screen a human brain cDNA library. We also propose to utilize highly selective anti-MOR antibodies combined with proteomics and mass spectrometry to identify a spectrum of MORIPs from immunoprecipitated mouse brain lysates. The major goal in Aim 2 will be to validate the MOR-protein interactions identified in Aim I. Cellular colocalization studies will confirm whether or not the MOR and candidate interactors are expressed within the same cells and intracellular compartments. Pull-down and coimmunoprecipitation will substantiate the MOR-protein interaction, while deletion mapping will permit identification of sites within the proteins that are necessary for the interactions to occur. Preliminary studies we have so far performed indicate that GPR177 meets all inclusion criteria and appears to represent a bona-fide MORIP. The goal in Aim 3 is to understand the functional significance of MOR/GPR177 interaction. We will examine the requirement for MOR/GPR177 interaction in MOR trafficking, desensitization, and signal transduction. We will also examine the role of the MOR/GPR177 interaction in regulating Wnt2 secretion from cells. To accomplish this goal, we will determine whether disrupting the MOR/GPR177 interaction, by expressing dominant negative forms of GPR177 or by knocking down GPR177 expression, affects the functional properties of the MOR. Identification of MOR interacting proteins will provide new insights into the etiology of drug abuse and dependence. RELEVANCE (See instructions): This project seeks to identify and evaluate the function of proteins that interact with and regulate the muopioid receptor (MOR), the cell associated receptor that mediates most of the analgesic actions of morphine as well as drugs of abuse. Understanding the function of GPR177, a novel interacting protein we identified, is likely to provide new insight into the etiology of drug abuse and dependence. MOR-interacting proteins may also represent novel therapeutic targets for the treatment of these important public health problems.

mu-阿片受体 (MOR) 介导吗啡和其他临床相关药物的大部分作用 镇痛药以及海洛因等滥用药物。本文提出的蛋白质组学和功能研究 该应用旨在阐明 MOR 信号复合物的蛋白质成分。鉴定 MOR 相互作用蛋白将使我们能够检验我们的假设，即蛋白质-蛋白质相互作用在 在调节MOR信号转导通路中发挥重要作用。在目标 I 中，我们将识别并描述 MOR 相互作用蛋白。我们进行的分裂泛素筛选鉴定出了几种新型 MORIP 包括 GPR177，果蝇 Wntless 的哺乳动物直系同源物。为了确定其他 MORIP，每个 人MOR的细胞内结构域将被用作诱饵，单独筛选人脑cDNA 图书馆。我们还建议利用高选择性抗 MOR 抗体结合蛋白质组学和质量 光谱测定法从免疫沉淀的小鼠脑裂解物中鉴定 MORIP 谱。主要 目标 2 的目标是验证目标 I 中确定的 MOR-蛋白质相互作用。细胞共定位 研究将确认 MOR 和候选相互作用因子是否在同一细胞内表达 和细胞内区室。 Pull-down 和免疫共沉淀将证实 MOR 蛋白 相互作用，而删除图谱将允许识别蛋白质内必要的位点 发生的相互作用。我们迄今为止进行的初步研究表明，GPR177 满足所有要求 纳入标准，似乎代表了真正的 MORIP。目标 3 的目标是了解 MOR/GPR177 相互作用的功能意义。我们将审查 MOR/GPR177 的要求 MOR 运输、脱敏和信号转导中的相互作用。我们还将研究该机构的作用 MOR/GPR177 相互作用调节细胞 Wnt2 分泌。为了实现这一目标，我们将确定 是否通过表达 GPR177 的显性失活形式或通过 敲低 GPR177 的表达，会影响 MOR 的功能特性。铁道部的鉴定 相互作用的蛋白质将为药物滥用和依赖性的病因学提供新的见解。 相关性（参见说明）： 该项目旨在识别和评估与穆阿片相互作用并调节穆阿片的蛋白质的功能 受体 (MOR)，细胞相关受体，介导吗啡的大部分镇痛作用 以及滥用药物。了解 GPR177（我们发现的一种新型相互作用蛋白）的功能， 可能为药物滥用和依赖性的病因学提供新的见解。 MOR相互作用蛋白 也可能代表治疗这些重要公共卫生问题的新治疗靶点。