The proteins interacting with C-terminal of μ receptor are identified by bacterial two-hybrid system from brain cDNA library in morphine-dependent rats

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• 作者：Peilan Zhou^a ; ^{zhoupeilan0502@sina.com" class="auth_mail" title="E-mail the corresponding author} ; Jiebing Jiang^a ; ^c ; ^{xiaoya.248@163.com" class="auth_mail" title="E-mail the corresponding author} ; Zhaoqi Dong^a ; ^{dongzq90@hotmail.com" class="auth_mail" title="E-mail the corresponding author} ; Hui Yan^a ; ^{yahhui74@aliyun.com" class="auth_mail" title="E-mail the corresponding author} ; Zhendong You^b ; ^{youzd630719@yahoo.com.cn" class="auth_mail" title="E-mail the corresponding author} ; Ruibin Su^a ; ^{ruibinsu@126.com" class="auth_mail" title="E-mail the corresponding author} ; Zehui Gong^a ; ^{gongzeh@126.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Bacterial two-hybrid ; CDNA library ; Nade ; Opioid addiction ; Screening
• 刊名：Life Sciences
• 出版年：2015
• 出版时间：15 December 2015
• 年：2015
• 卷：143
• 期：Complete
• 页码：156-167
• 全文大小：1392 K

文摘

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.

Main 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).

Key findings

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.

Significance

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.