title: The proteins interacting with C-terminal of μ receptor are identified by bacterial two-hybrid system from brain cDNA library in morphine-dependent rats.
abstract: OBJECTIVE
  Opioid addiction is associated with long-term adaptive changes in the brain that involve protein expression. The carboxyl-terminal of the μ opioid receptor (MOR-C) is important for receptor signal transduction under opioid treatment. However, the proteins that interact with MOR-C after chronic morphine exposure remain unknown. The brain cDNA library of chronic morphine treatment rats was screened using rat MOR-C to investigate the regulator of opioids dependence in the present study.
  
  
  METHODS
  The brain cDNA library from chronic morphine-dependent rats was constructed using the SMART (Switching Mechanism At 5' end of RNA Transcript) technique. Bacterial two-hybrid system was used to screening the rat MOR-C interacting proteins from the cDNA library. RT-qPCR and immunoblotting were used to determine the variation of MOR-C interacting proteins in rat brain after chronic morphine treatment. Column overlay assays, immunocytochemistry and coimmunoprecipitation were used to demonstrate the interaction of MOR-C and p75NTR-associated cell death executor (NADE).
  
  
  RESULTS
  21 positive proteins, including 19 known proteins were screened to interact with rat MOR-C. Expression of several of these proteins was altered in specific rat brain regions after chronic morphine treatment. Among these proteins, NADE was confirmed to interact with rat MOR-C by in vitro protein-protein binding and coimmunoprecipitation in Chinese hamster ovary (CHO) cells and rat brain with or without chronic morphine treatment.
  
  
  CONCLUSIONS
  Understanding the rat MOR-C interacting proteins and the proteins variation under chronic morphine treatment may be critical for determining the pathophysiological basis of opioid tolerance and addiction.
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China. Electronic address: zhoupeilan0502@sina.com.
authors.forename: Peilan
authors.initials: P
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China; College of Pharmaceutical Sciences, Guangxi Medical University, Nanning 530021, China. Electronic address: xiaoya.248@163.com.
authors.forename: Jiebing
authors.initials: J
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China. Electronic address: dongzq90@hotmail.com.
authors.forename: Zhaoqi
authors.initials: Z
authors.lastname: Dong
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China. Electronic address: yahhui74@aliyun.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Yan
authors.affiliation: Institute of Neuroscience and MOE Key Laboratory of Molecular Neurobiology, Second Military Medical University, Shanghai 200433, China. Electronic address: youzd630719@yahoo.com.cn.
authors.forename: Zhendong
authors.initials: Z
authors.lastname: You
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China. Electronic address: ruibinsu@126.com.
authors.forename: Ruibin
authors.initials: R
authors.lastname: Su
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China. Electronic address: gongzeh@126.com.
authors.forename: Zehui
authors.initials: Z
authors.lastname: Gong
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015723:Gene Library; D008297:Male; D009020:Morphine / Q000008:administration & dosage / Q000378:metabolism; D009021:Morphine Dependence / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D017450:Receptors, Opioid, mu; D009020:Morphine
keywords: Bacterial two-hybrid; CDNA library; Nade; Opioid addiction; Screening