Mapping mu agonist-induced receptor-protein interactions for OPRM1 7TM variants

绘制 OPRM1 7TM 变体 mu 激动剂诱导的受体-蛋白质相互作用图谱

基本信息

批准号：
9788403
负责人：
YING-XIAN PAN
金额：
$ 22.96万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-30 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9788403
关键词：
Affect Agonist Animals Biotin Brain C-terminal Cell model Cell physiology Cells Clinical Complex Corpus striatum structure Coupled Coupling Dependence Engineering Exons Future G-Protein-Coupled Receptors GTP-Binding Proteins Heroin Human In Vitro Inbred Mouse Knockout Mice Length Maps Mediating Methods Modeling Molecular Morphine Morphine Dependence Neurons Opioid Opioid Analgesics Peroxidases Pharmacology Phosphorylation Phosphotransferases Physical Dependence Physiological Play Proteomics RIPK1 gene RNA Interference RNA Splicing Receptor Gene Receptor Signaling Rewards Rodent Role Set protein Signal Pathway Signal Transduction Sorting - Cell Movement Stimulus Structure of thyroid parafollicular cell Tail Techniques Technology Testing Time Transfection Variant ascorbate in vivo insight knock-down mRNA Precursor morphine tolerance mouse model mu opioid receptors opioid use protein protein interaction receptor receptor function recruit response

项目摘要

Project Summary/Abstract Morphine and most clinically used opioid analgesics, as well as heroin, act primarily through mu opioid receptors. The single-copy mu opioid receptor gene (OPRM1) undergoes extensive alternative pre-mRNA splicing, generating an array of splice variants that are conserved from rodents to humans. One type of the splice variants are full-length 7-transmembrane (TM) C-terminal variants that are identical except for the sequences at the tip of the intracellular C-terminal tail. Increasing evidence supports the pharmacological importance of these 7TM C-terminal variants. Several in vitro cell models demonstrate functional differences in mu agonist-induced G protein coupling, phosphorylation, internalization and post-endocytic sorting, as well as region- and cell-specific expression. More importantly, in vivo functions of several C-terminal variants were recently revealed in C-terminal truncation mouse models with two inbred mouse background. Particularly, exon 7 (E7)-associated C-terminal truncation in C57BL/6J strain diminished morphine tolerance and reward without altering physical dependence, whereas the E4-associated C-terminal truncation accelerated morphine tolerance and reduced morphine dependence without affecting morphine reward. Together, these studies underscore the functional importance of these C-terminal splice variants in mediating the diverse actions of mu opioids, and provide a compelling rationale to further explore molecular mechanisms of C-terminal 7TM splice variants in mu opioid actions, as proposed in this application. We hypothesize that different C-terminal sequences of the Oprm1 full-length 7TM variants are important in determining interaction of a receptor with a unique set of proteins either at basal states or in response to mu agonists, leading to their distinct signaling pathways and functions. In this application we propose using newly developed proximity-dependent biotin identification with an engineered ascorbate peroxidase (APEX2) coupled with tandem mass tag (TMT) proteomics approach, to map these transient or dynamic receptor-protein interactions under both basal state and activated conditions in response to different mu agonists in OPRM1-KD Be(2)C cells and primary striatal neurons derived from Oprm1 knockout mice. We will compare two E7-associated C-terminal 7TM variants mMOR-1O and mMOR-1C that have unique in vitro and in vivo pharmacological profiles with E4-associated mMOR-1, and a truncated version, mE1/2/3, that lacks additional C-terminal sequences downstream of E3. We will validate physical and/or functional interactions of selected candidates from APEX2-TMT study using a number of approaches, such as NanoLuc Binary Technology (NanoBit) and RNAi. The proposed studies promise to reveal new insights into mu agonist-induced receptor-protein interactions, signaling and function of the C-terminal splice variants, and to provide a general approach applicable to all G-protein coupled receptors. With approximately 12% of non-olfactory GPCRs having alternative C-terminal splice variants, the results from this application may have a very broad impact.

项目概要/摘要吗啡和临床上使用的大多数阿片类镇痛药以及海洛因主要通过 mu 阿片类药物发挥作用受体。单拷贝 mu 阿片受体基因 (OPRM1) 经历广泛的替代前 mRNA 剪接，产生一系列从啮齿动物到人类都保守的剪接变体。其中一种类型是剪接变体是全长 7 次跨膜 (TM) C 端变体，除了序列位于细胞内C末端尾部的尖端。越来越多的证据支持药理学这些 7TM C 端变体的重要性。几种体外细胞模型显示出功能差异 mu 激动剂诱导的 G 蛋白偶联、磷酸化、内化和内吞后分选，以及区域和细胞特异性表达。更重要的是，几个 C 端变体的体内功能被最近揭示了具有两种近交小鼠背景的 C 端截断小鼠模型。特别是外显子 C57BL/6J 菌株中的 7 (E7) 相关 C 末端截断降低了吗啡耐受性和奖赏，但不影响吗啡耐受性和奖赏性。改变身体依赖性，而 E4 相关的 C 末端截断加速吗啡耐受性并减少吗啡依赖性而不影响吗啡奖励。这些研究共同强调这些C端剪接变体在介导mu的多种作用中的功能重要性阿片类药物，并为进一步探索 C 端 7TM 剪接的分子机制提供了令人信服的理由 mu阿片类药物作用的变体，如本申请中提出的。我们假设不同的C端 Oprm1 全长 7TM 变体的序列对于确定受体与受体的相互作用非常重要一组独特的蛋白质，要么处于基础状态，要么响应 mu 激动剂，导致其独特的信号传导途径和功能。在此应用中，我们建议使用新开发的邻近依赖性生物素使用工程抗坏血酸过氧化物酶 (APEX2) 结合串联质量标签 (TMT) 进行鉴定蛋白质组学方法，在两种基础状态下绘制这些瞬时或动态受体-蛋白质相互作用 OPRM1-KD Be(2)C 细胞和原代纹状体中不同 mu 激动剂的激活条件来自 Oprm1 敲除小鼠的神经元。我们将比较两个 E7 相关 C 端 7TM 变体 mMOR-1O 和 mMOR-1C 与 E4 相关，具有独特的体外和体内药理学特征 mMOR-1 和截短版本 mE1/2/3，缺少 E3 下游的额外 C 端序列。我们将使用 APEX2-TMT 研究验证选定候选人的身体和/或功能相互作用许多方法，例如 NanoLuc 二进制技术 (NanoBit) 和 RNAi。拟议的研究有望揭示 mu 激动剂诱导的受体-蛋白质相互作用、信号传导和功能的新见解 C 端剪接变体，并提供适用于所有 G 蛋白偶联受体的通用方法。大约 12% 的非嗅觉 GPCR 具有替代的 C 端剪接变体，结果来自这个应用可能会产生非常广泛的影响。