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

• 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
• 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

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.

项目摘要/摘要 吗啡和大多数临床使用的阿片类镇痛药以及海洛因主要通过MU阿片类药物起作用 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的剪接变体。一种类型的 剪接变体是全长7跨膜（TM）C末端变体，除了 细胞内C末端尾端的序列。越来越多的证据支持药理 这些7TM C末端变体的重要性。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，内在化和内吞分选，以及 区域和细胞特异性表达。更重要的是，几种C末端变体的体内功能是 最近在具有两个近交小鼠背景的C末端截断小鼠模型中揭示。特别是外显子 7（E7）相关的C末端截断C57BL/6J菌株降低了吗啡的耐受性和奖励 改变物理依赖性，而E4相关的C末端截断加速了吗啡 耐受性和降低吗啡依赖性而不会影响吗啡奖励。在一起，这些研究 强调这些C末端剪接变体在介导MU的各种作用中的功能重要性 阿片类药物，并提供了令人信服的理由，以进一步探索C末端7TM剪接的分子机制 如本应用程序中所提出的那样，MU阿片类动作中的变体。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A的相互作用很重要 基础状态或对MU激动剂的响应，独特的蛋白质集导致其独特的信号 途径和功能。在此应用中，我们建议使用新开发的接近依赖性生物素 用工程的抗坏血酸过氧化物酶（APEX2）与串联质量标签（TMT）识别 蛋白质组学方法，以在两个基础状态下绘制这些瞬态或动态受体 - 蛋白质相互作用 响应于OPRM1-KD Be（2）C细胞和主要纹状体中不同MU激动剂的活化条件 源自OPRM1敲除小鼠的神经元。我们将比较两个与E7相关的C末端7TM变体 MMOR-1O和MMOR-1C具有独特的体外和体内药理学特征，具有E4相关 MMOR-1和截断版本ME1/2/3，缺少E3下游的其他C末端序列。我们 将使用A APEX2-TMT研究中选定候选者的身体和/或功能相互作用验证使用A 方法数量，例如纳米二进制技术（Nanobit）和RNAi。提出的研究 承诺揭示对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末端剪接变体，并提供适用于所有G蛋白耦合受体的一般方法。 大约有12％的非菲尔塔级GPCR具有替代C末端剪接变体的替代性GPCR，结果来自 该应用程序可能会产生非常广泛的影响。